Structure and regulation of acetyl-CoA carboxylase genes of metazoa

Biochimica et Biophysica Acta 1733 (2005) 1 – 28 http://www.elsevier.com/locate/bba

Review

Structure and regulation of acetyl-CoA carboxylase genes of metazoa Michael C. Barber*, Nigel T. Price, Maureen T. Travers Hannah Research Institute, Ayr, KA6 5HL, Scotland, United Kingdom Received 9 August 2004; received in revised form 2 November 2004; accepted 1 December 2004 Available online 24 December 2004

Abstract Acetyl-CoA carboxylase (ACC) plays a fundamental role in fatty acid metabolism. The reaction product, malonyl-CoA, is both an intermediate in the de novo synthesis of long-chain fatty acids and also a substrate for distinct fatty acyl-CoA elongation enzymes. In metazoans, which have evolved energy storage tissues to fuel locomotion and to survive periods of starvation, energy charge sensing at the level of the individual cell plays a role in fuel selection and metabolic orchestration between tissues. In mammals, and probably other metazoans, ACC forms a component of an energy sensor with malonyl-CoA, acting as a signal to reciprocally control the mitochondrial transport step of long-chain fatty acid oxidation through the inhibition of carnitine palmitoyltransferase I (CPT I). To reflect this pivotal role in cell function, ACC is subject to complex regulation. Higher metazoan evolution is associated with the duplication of an ancestral ACC gene, and with organismal complexity, there is an increasing diversity of transcripts from the ACC paralogues with the potential for the existence of several isozymes. This review focuses on the structure of ACC genes and the putative individual roles of their gene products in fatty acid metabolism, taking an evolutionary viewpoint provided by data in genome databases. D 2004 Elsevier B.V. All rights reserved. Keywords: Gene structure; Transcription; AMP-activated protein kinase; Lipogenesis; h-oxidation; Malonyl-CoA; Biotin; Insulin resistance; Membrane targeting; Alternative splicing; Pseudogene

1. Introduction In mammals, acetyl-CoA carboxylase (ACC: EC 6.4.1.2) catalyses the first committed step in the fatty acid biosynthetic pathway, the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA. Malonyl-CoA is a substrate for fatty acid synthase (FAS: EC 2.3.1.85), which, through seven rounds of condensation with an acyl-CoA acceptor, catalyses the synthesis of palmitic acid (C16:0) [1] and also for distinct elongases in the pathway of very longchain fatty acyl-CoA synthesis (NC22:0) [2]. The pathway for the de novo synthesis of long-chain fatty acids from glucose and other metabolic precursors plays a pivotal role in the function of cells. Fatty acids are key building blocks for the phospholipid components of cell membranes and are

* Corresponding author. Tel.: +44 1292 674032; fax: +44 1292 674003. E-mail address: [email protected] (M.C. Barber). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.12.001

determinants of intracellular communication, in the form of lipid second messengers [3], and fatty acyl moieties of proteins that modify their location and function [4]. Not surprisingly, disruption of this fundamental pathway in most prokaryotes and eukaryotes results in impaired cell survival and loss of organismal viability [5–8]. In mammals, a second well defined function of malonylCoA, levels of which reflect the energy-state of the cell, is to act as an inhibitor of carnitine palmitoyltransferase I (CPT I) (EC 2.3.1.7), the rate limiting enzyme for the import of fatty acids into the mitochondria for h-oxidation [9,10]. In key tissues, the regulation of the rate of h-oxidation plays a major role in orchestrating whole body metabolic adaptations to changes in nutrient availability and to fuel locomotion. Detailed consideration of the role of malonylCoA in energy sensing pathways is reviewed elsewhere [11,12]. Functional divergence in the roles of malonyl-CoA within specialised cells is reflected by mammals possessing paralogous genes encoding ACC activity, ACC-a or ACC1 (Gene name ACACA), and ACC-h or ACC2 (ACACB), that

2

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28

arose through duplication of an ancestral ACC gene, probably between 590 and 440 million years ago. Genes homologous to mammalian CPT I are present within the genomes of nematodes and flies, but not in those of bacteria, unicellular eukaryotes or plants, indicating that the emergence of this gene is fundamental to the physiology of metazoa. Although Drosophila CPT I is inhibited by malonyl-CoA [13], there is only one gene for ACC in this species [14], and this has to presumably generate malonylCoA for both the regulation of h-oxidation and for fatty acid synthesis, and prompts the question of how a single gene for ACC regulates both pathways. Furthermore, with increasing organismal complexity, there is an increasing diversity of transcripts, generated by multiple promoters and alternative splicing, from both of the mammalian paralogues, with potential for several isozymes and regulating malonyl-CoA concentration at a variety of levels. This review will discuss data on the evolution of ACC genes in the animal kingdom, using information derived from recently published work and also that extracted from public access genome databases and aims to extend and integrate previous reviews on the transcriptional [15,16] and short-term regulation [17,18] of ACC. The emergence of conserved networks that regulate the expression of the gene(s) and the activity of the encoded enzyme(s) will be highlighted and discussed.

2. Evolution of the structure of mammalian ACC 2.1. The biotin carboxylase gene family ACC is a member of a family of enzymes that catalyse the intermolecular transfer of carboxyl groups via the transient formation of a carboxyphosphate intermediate covalently linked to a prosthetic biotin moiety. The reaction sequence for ACC is illustrated in Fig. 1. All biotin-dependent enzymes share a similar catalytic mechanism that involves two partial reactions: (1) carboxylation of the biotin moiety of the biotin carboxyl carrier protein (BCCP) and (2) carboxyl transfer from biotin to the acceptor molecule. Two classes of carboxytransferase (CT) are present in nature, the acetyl-CoA/propionyl-CoA CT (ACCT) of ACC and propionyl-CoA carboxylase (PCC), and the pyruvate/oxaloacetate CT (PycB) present in pyruvate carboxylase and oxaloacetate decarboxylase [19,20]. Utilisation of the three components of this basic reaction in different combinations throughout evolution has resulted in the participation of ATP + HCO3- + BCCP

BC

BCCP.CO2- + acetyl CoA

CT

BCCP.CO2- + ADP + Pi BCCP + malonyl-CoA

Fig. 1. Reaction sequence for ACC. Biotin carboxylase (BC) catalyses the ATP-dependent phosphorylation of bicarbonate and formation of carboxyphosphate-BCCP. Acetyl-CoA carboxytransferase (ACCT) catalyses the transfer of the carboxyl group from carboxybiotin-BCCP to an acetyl-CoA acceptor, resulting in malonyl-CoA.

biotin-containing enzymes in a wide variety of metabolic pathways [20]. The selection of the three components into functional enzymatic entities has also favoured co-regulation of expression and domain fusions into one or two polypeptides [20,21]. Lateral gene transfer of biotin-dependent enzymes between prokaryotes and eukaryotes has probably occurred by mitochondrial and chloroplast endosymbiosis, prior to transfer into the nuclear genome [19–21]. Plants also have two forms of ACC. A cytosolic ACC, involved in secondary metabolite biosynthesis and fatty acylCoA elongation, possesses a similar domain structure to that of animal ACC [22,23]. However, the exon organisation of the multifunctional ACC of plants is different from that of animal ACC and suggests that multifunctional ACCs have evolved independently in the two phyla. The second form resides in the chloroplast, where de novo fatty acid synthesis occurs. In dicotyledonous and most monocotyledonous plants, the catalytic components of the plastid ACC are encoded by separate genes, as in bacteria. The Gramineae have a nuclear-encoded homomeric plastid ACC, with a plastid-targeting transit peptide [24]. The domain order in multifunctional ACCs (BC: BCCP:ACCT, Fig. 2) is the same as that in PCCs, however, in the latter, only BC and BCCP are fused in the a-subunit, while the CT domain remains as a separate polypeptide, the h-subunit. Some bacterial ACCs have this two component PCC-like structure [25], and this situation may also exist in eukaryotes. The nematode Turbatrix aceti appears to have a cytosolic PCC-like two subunit enzyme as the sole source of ACC activity [26]. 2.2. Functional domains within ACC and their structures At present the structural data for ACC, prokaryotic or eukaryotic, is limited. Crystal structures are available for the BC and BCCP subunits of E. coli ACC, [20,21], while the only ACCT domain structure determined is that for yeast [27]. The BC subunit of E. coli ACC contains an ATPbinding pocket, and the residues which interact with ATP are found to be conserved in human ACC-a [20]. Because of the paucity of structural data, the detailed reaction mechanism and many aspects of the regulation of ACC remain at least partially obscure. The ACCT domain of multifunctional ACCs has arisen from the fusion of the bacterial AccD (h-CT) and AccA (aCT) genes, which themselves are distantly related paralogues [20]. This is further revealed in the yeast CT domain structure, which consists of two intimately associated sub-domains [27]. These form similar backbone folds, despite their primary sequences showing little conservation. The yeast CT domain also forms a dimer, as is found for the E. coli subunits (a2h2) [28]. The structure of the complex of CT shows the enzyme’s active site to be at the dimer interface. The residues involved are generally well conserved between yeast, human and wheat ACCT [27], suggesting that the three adopt similar structures.

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28 BIOTIN CARBOXYLASE 1 1

2

BC

2

3-13

CARBOXYL TRANSFERASE 3

BCCP

14 -16 17 -18

P P

P 27

β

27

28

4

ACCT

5

19 -39

40 -52

52 -54

P

B

α

3

P 29

30

31

32

29

30

31

32

33

34

34

Fig. 2. Functional regions of mammalian ACC. Boxes are drawn approximately to scale. Region 1 corresponds to the size for ACC-a: that for ACC-h is almost 3 times this size. Numbers within boxes refer to protein coding exon numbers, and those above correspond to the five regions of the molecule discussed within the text (Section 2.4.2). It should be noted that exon numbers differ from those used in Figs. 3 and 4. The site of biotin modification (B) within the BCCP domain and regions modified by phosphorylation (P) are shown. Differences between ACC-a and -h within region 4 are shown in the lower part of the figure. Shading of E28 in ACC-a denotes an alternatively spliced exon. E33 may also be alternatively spliced in chicken ACC-a (Section 3.1.3).

While in the other biotin-dependent enzymes the BCCP domain is the carboxyl terminal (or solitary) domain of a polypeptide [19,20], with the receptor site for biotinylation approximately 35 residues from the terminus, in the multifunctional ACCs, this domain is towards the centre of the primary sequence, the biotinyl-lysine being residue 786 in human ACC-a (Fig. 2). The fact that biotin protein ligases (BPLs) from many different organisms can modify the BCCP domain from several different substrate proteins [29] suggests that this structure, a tightly folded anti-parallel betabarrel [21], is highly conserved, irrespective of location. Little is known about the regions of BCCP that interact with the BC or CT domains, even in the multimeric enzymes. 2.3. Comparison of ACC gene structure and protein sequences ACC genes in various eukaryotic species can be identified on the basis of the presence of the various catalytic domains. It is apparent that a large proportion of the ACC molecule constitutes poorly understood noncatalytic regions. Many species have two ACC genes, however it is clear that in species more distantly related to mammals these genes do not have the same functional division attributed to mammalian ACC-a and -h. 2.3.1. Sequences for ACC genes from complex metazoa Complete gene sequences for both ACC isoforms from human, rat and mouse are available, together with limited data for other mammals (Table 1). As the structure of a complete mammalian ACC-h gene has not been previously described, we present a brief comparison with that of the ACC-a gene. Almost complete genome sequences are also available for the domestic chicken, and two fish species, zebrafish and pufferfish (Fugu). We found that these species also have two ACC genes which are closely related (orthologous) to

mammalian ACC-a and -h. The chicken ACC-h gene has not been previously characterized [30]. The chicken ACCs are highly similar in primary sequence to their mammalian counterparts. The two pairs of fish ACC-a and -h protein sequences show 93% and 81% identity, respectively. While the fish ACC-a sequences are closely related to their mammalian counterparts (N87% identity), fish ACC-h sequences appear equally related to both mammalian ACC-a and -h (approx. 73% identity in each case). Their N-terminal sequences and gene structure (Section 2.4.5), however, clearly distinguish them as ACC-h genes. To obtain these novel sequences, as with those for the lower eukaryotes (Section 2.5), exon positions within genomic sequence were identified by comparison to the known mammalian ACC sequences (not shown). In some cases, these exons were absent from, or differed from, those in computer predicted transcripts. Also, gaps currently present in some of the genomic sequence assemblies resulted in a small number of gaps in some of our derived cDNA sequences, however in all cases the sequences were sufficiently complete to allow meaningful comparison of intron positions and sequence conservation. The properties of the gene-derived sequences are summarised in Table 1, together with a summary of previously published data. Importantly, the availability of the mammalian gene sequences allowed us to identify errant cDNA sequences within the public databases (Table 1), with differences not being accountable solely to single nucleotide polymorphisms. Three in particular warrant further brief comment. 2.3.2. Errant mammalian ACC sequences A chimaeric sequence containing the 5V-end/N-terminus of human ACC-a linked to the 3V-end/C-terminus of ACC-h has been commented on in the literature [31], but still remains in the database as if representing a single cDNA. The amino acid sequence of rat ACC-h [32] differs from the genome derived sequence at 89 positions (not shown). Also,

4

Table 1 Properties of ACC genes and their protein products Protein

cDNA

Gene name(s) Chromosomal localisation

Gene size (protein Unigene coding exons only)

References

Human ACC-a

NP_942133 AAC50139a X68968a XP_109883b Note 2 Not transcribed/translated

NM_198836 U19822a CAA48770a XM_109883b Note 2 Not transcribed

ACACA ACC1, HGNC:84 Acaca

17q21/17q12 cM 54 (Note 1)

2346 (265 kDa)

245 kb

Hs.449863

[31,45,48,57]

11 C cM

54

2345 (265 kDa)

206 kb

Mm. 31374

Acaca-psi

12 A1.1 cM

(Intronless)

(8 kb)

–

This paper

Acaca

10q26 cM

54

(Not transcribed/ translated) 2345 (265 kDa)

NP_071529

NM_022193

195 kb

[46,49]

X80045 AF175308 (535 nt only) NM_174224 NM_205505

ACACA ACACA ACACA ACACA

11 12p13–p12 cM 19q13–q14 cM 19

?? ?? ?? 52 (Note 3)

2346 (265 kDa) ?? 2346 (265 kDa) 2324 (263 kDa)

?? ?? ?? N91.5 kb

Note 4b Note 4b AJ575592 NM_001093a XM_132282b Note 5 NM_053922a Note 4b

ACACA ACACA ACACB

17 ?? 12q24.1 cM

53 52 52

2352 (265 kDa) 2349 (265 kDa) 2458 (277kDa)

23.2 kb 102.5 kb 129 kb

Acacb

5 F cM

52

2450 (276 kDa)

N86.7 kb

Mm.81793

Rat ACC-h Chicken ACC-h

Note 4b Note 4b CAE01471 NP_001084a XP_132282b Note 5 NP_446374a Note 4b

Rn.44372 Rn.122519 Ssc.16018 Bt.4735 Gga.1480 Gga.11412 – No entry Hs.234898

Sheep ACC-a Pig ACC-a Cattle ACC-a Chicken ACC-a

CAA56352 AAF22966 (partial) NP_776649 NP_990836

Fugu ACC-a Zebrafish ACC-a Human ACC-h

Acacb ACACB

12q16 cM 15

52 Note 6 52 Note 7

[32] This paper

Note 4b Note 4b

Note 4b Note 4b

ACACB ACACB

2 5

52 52 Note 7

N105 kb Note 6 N22.5kb Note 7 N15 kb N47 kb Note 7

Rn.44359 No entry

Fugu ACC-h Zebrafish ACC-h

– No entry

This paper This paper

Sea Squirt ACC Fly ACC-a

Note 8 CG11198-PB NP_724636 CG11198-PA NP_610342 XP_314071b XP_314071b NP_493922 Note 10

AK114664 (1721 nt only) CG11198-RB CG11198 NM_165581 Note 9 CG11198-RA CG11198 NM_136498 Note 9 XM_314071b Note 9 XM_314071b Note 9 NM_061521 W09B6.1a, pod-2, YK6887, 2B441

z48 Note 8 12

2455 (276 kDa) 2379 (270 kDa) Note 7 2408 (271 kDa) 2120 (240 kDa) Note 7 z2324 (261 kDa) 2323 (262 kDa)

z25.8 kb 11.7 kb

No entry Dm.11366

This Paper

13

2482 (279 kDa)

11.7 kb

Dm.11366

9 10 8

2323 (261 kDa) 2480 (278 kDa) 2054 (231 kDa)

15.5 kb 15.5 kb 8.8 kb

Aga.19167 Aga.19167 Cel.17810

Mouse ACC-a Mouse ACC-a PSEUDOGENE Rat ACC-a

Mouse ACC-h

Fly ACC-h Mosquito ACC-a Mosquito ACC-h C. elegans ACC1

2R ( ) 44A1–44A2 cM 2R ( ) 44A1–44A2 cM 2L 2L II; 12.77 cM

Protein coding Amino acids exons (predicted size)

[242,243] [244] [62] [30,59,72] This paper This paper [33,38,245,246]

[41]

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28

Gene

C. elegans ACC1 variant C. elegans ACC2

AAR12979 Note 11 NP_503072 Note 12 C. briggsae ACC1 ENSCBRP-00000010661 C. briggsae ACC2 Note 4b Yeast ACC1 cytosolic Q00955 CAA96294 Yeast HFA1 NP_013934 mitochondrial Note 13

AF025469 (CDs only)

W09B6.1b

II;

NM_070671

4S243 T28F3.5 CBG07699 CBG00376 ACC1, FAS3 YNR016c, HFA1, YMR207C

ENSCBRT-00000010661 Note 4b Z71631 NC_001145

12.77 cM

6

813 (91 kDa)

8.8 kb

–

IV; +17.23 cM

18

z1657 (z187 kDa) 6.5 kb

Cel.12266

[42]

?? ?? XIV; 661371-654670 XIII; 683563-677642

12 10 1

2048 (229 kDa) 1658 (187 kDa) 2233 (250 kDa)

7.2 kb 7.6 kb 6.7 kb

– – –

[6,247]

1

z2273 (z260 kDa) 6.8 kb

–

[44,248]

Notes 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13.

Human ACACA has been mapped to chromosome 17q21 [45] or 17q12 [31] by different groups. This predicted transcript/translate is truncated at the 5Vend/N-terminus. Chicken ACACA may contain two additional alternatively spliced exons (Section 2.4.5), adding a possible 8+15 amino acids to the protein. Sequences for fish ACC-a and -h, chicken ACC-h and C. briggsae ACC1 and 2 were derived from genome data available in ENSEMBL using predictions based on homology to mammalian sequences (not shown). None of these predicted sequences are currently represented completely by ENSEMBL predictions. The current prediction for mouse ACC-h lacks 3 exons and has 1 extra exon, plus one difference in intron/exon boundary compared to our prediction (not shown). The currently available rat genome data has a gap in the region containing protein coding E50. For chicken and zebrafish ACACB, there are 3 and 5 putative exons, respectively, covered by gaps in the current assembly—the number of 52 exons assumes the presence of these exons, and that none of the associated introns have been lost. Numbers of amino acids given also assumes that these exons are the same size as those in related sequences. Zebrafish ACC-h probably lacks a hydrophobic segment at the extreme N-terminus (Section 2.4.3). Partial 3Vsequence is available at the Institute of Genome Research (TIGR): TC35582. Our assignment of N-terminus encoding exons is preliminary. The putative ACC-a and -h are generated from alternatively spliced transcripts of the same gene in these insects. It is likely that both insects generate additional N-terminal variants though use of two or more promoters (data not shown). The possible existence of a second form with an extended N-terminus, due to alternative splicing at the 5Vend, was reported [41]. This entry describes a variant lacking 2 adjacent internal protein coding exons which encode the C-terminal end of the BCCP domain and all of the ACCT domain. C. elegans ACC2 lacks residues encoding part of the CT domain. Comparison to C. briggsae ACC2 sequence suggests the presence of errors in the gene sequence. The reference sequence lacks z150 amino acids upstream of the first Met. Translation is thought to initiate from a non-AUG codon to give a protein with a mitochondrial import sequence [44].

a These sequences contain what are almost certainly sequencing and/or other errors (Section 2.3.2). CAA48770/X68968 [57] contains the 5Vend/N-terminus of ACC-a linked to the 3Vend/C-terminus of ACC-h. U19822/AAC50139 [31], NM_001093/NP_001084 [33] and NM_053922/NP_446374 [32] contain several apparent errors in nucleotide/protein sequence compared to the gene derived sequences (39 differences, 141 differences with 41 gaps and the presence of an intron, and 89 differences with 5 gaps, respectively). b Theoretical gene-derived predictions are available, but differ from our derived sequences which are based on homology to validated ACC sequences.

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28

Where possible, accession numbers for curated reference sequences are given. In the case of human ACC-h, the reference sequence contains likely errors. For C. briggsae ACC2, ENSEMBL [240] transcript/ peptide numbers are given. Where variant transcripts exist, the accession number for the predominant transcript is given, and the number of residues is derived from this form. It should be noted that some of the references, particularly Unigene numbers [241], are liable to future change, but should still provide entry points to the updated data. Some gene sizes are given as Nx kb as they contains gaps of unknown size in the assembly.

5

6

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28

an entry for human liver ACC-h [33] differs from the genome derived amino acid sequence at 141 positions, with 41 gaps required in alignments of the two versions of the protein sequence. The sequence also contains an intron, which would disrupt the reading frame of the protein were it not for apparent sequencing errors. This sequence is considered further below (Section 3.2.3), as the two exons adjacent to this intron are reported to be alternatively spliced by the same authors [33]. Authors from this group have recently deposited a cDNA sequence for human ACC-h from the heart (Accession no. AY382667) that is in accord with that of the genomic data. The GenBank curated reference sequences for rat and human ACC-h are currently generated from the entries that differ significantly from genome derived sequences (Table 1). 2.3.3. Mouse Acaca pseudogene Our database searches revealed the presence of a previously undocumented Acaca pseudogene, which we named Acaca-psi, on mouse chromosome 12 (Table 1). This is present in all genotypes sequenced. The gene is intronless and includes a poly-(A) region, suggesting that it may have arisen through a reverse transcriptase-mediated retrotransposition event. As is common for such processed pseudogenes, it is 5V-truncated relative to Acaca transcripts (starting at Acaca protein coding E7), presumably due to the reverse transcriptase abortively dissociating from the RNA template. There is no evidence, or expectation, that this gene is transcribed. It is very highly conserved (99% identity to the Acaca transcript, with 13 gaps in the alignment, 8 of which are in the Acaca 3Vuntranslated region) with only 2 frame-shifts relative to Acaca, and with no selection pressure on function, this suggests a very recent origin. This is supported by the fact that we could not detect a similar pseudogene in the rat or any other mammalian genome. Interestingly, Acaca-psi can be seen to be derived from a transcript which lacked the alternatively spliced protein coding exon (E)28 described in Section 2.4.5. 2.4. Functional domains within vertebrate ACC 2.4.1. Catalytic regions The distribution of the different catalytic domains throughout the primary structure of the mammalian ACCs and the relationship to exon structure is shown in Fig. 2. The ACCs from chicken and fish have an identical structure, but in the latter, there are fewer exons due to intron loss (Section 2.4.5). 2.4.2. Non-catalytic regions The catalytic domains actually account for less than half of the mammalian ACC molecule. The remainder can be assigned to five regions (Fig. 2): 1)

The amino terminus. This is approximately 75 and 139– 218 residues long in ACC-a and -h, respectively, and

2)

3)

4)

5)

constitutes the region of greatest difference between the two. However, although not previously recognised, a small part of this region is actually conserved between ACC-a and -h (see Section 2.4.4). The amino terminus includes sites known to be phosphorylated in ACC-a, and potential phosphorylation sites in ACC-h (Section 4). The extreme N-terminus of ACC-h is hydrophobic, with the remainder of the region being rich in hydroxylated amino acids and prolines. This region has been shown to be responsible for targeting ACC-h to mitochondria [34] (Section 4.4.1). A region containing a regulatory site phosphorylated by AMP-activated protein kinase (AMPK) (Ser-79 and Ser-218 in rat ACC-a and -h, respectively1). A relatively highly conserved region of approximately 110 amino acids flanked by the BC and BCCP domains. An approximately 800 amino acid region separating the BCCP and ACCT domains. This region is again highly conserved, with distinctive differences between ACC-a and -h, as discussed below. It also contains phosphorylation sites at Ser-1200 and -1215 in rat ACC-a1 which are potentially involved in regulation (Section 4.3.1), however neither is conserved in ACC-h. And finally, the C-terminus, whose residues and length are highly conserved between species.

The additional presence of region 4 linking the BCBCCP domains with the ACCT domain is the main gross structural difference between multifunctional ACCs and the PCCs, where the ACCT domain is found within a separate polypeptide. The roles of these five regions, despite constituting the majority of the molecule, can only be partially explained by our current understanding of ACC. Obviously, some of the extra sequence, particularly between the BC and BCCP domains (region 3), may act simply as a scaffold to link the catalytic domains together, although the high degree of sequence conservation suggests an additional role(s). The multimeric bacterial forms of ACC clearly do not require such large amounts of additional sequence to maintain structure or for catalytic activity. The central 800 amino acid region (4) probably has function(s) beyond that of a scaffold, as evidenced by the presence of the potential regulatory phosphorylation site in ACC-a. 2.4.3. Functional differences between mammalian ACC-a and -b resulting from differences in their N-termini The two major ACC isozymes differ most in region 1, the N-terminus, with mammalian ACC-h having an approx-

1 The position of phosphorylation sites within the primary sequence of ACC from different species differs slightly due to small differences in sequence length, for example Ser-222 in human ACC-h is equivalent to Ser-218 in rat ACC-h.

M.C. Barber et al. / Biochimica et Biophysica Acta 1733 (2005) 1–28

imately 140 amino acid extension relative to ACC-a. Downstream of this the two show approximately 75% identity. Key observations by Wakil and colleagues provide good evidence that the 280 kDa protein derived from ACACB plays a significant role, as predicted by its expression pattern, in the regulation of mitochondrial h-oxidation. ACC-h is targeted to mitochondria, whereas ACC-a is cytosolic [34]. This suggests that these isozymes generate distinct cellular pools of malonyl-CoA with diverse metabolic fates. Further evidence for this idea has arisen from the generation of Acacb null mice [35]. These mice display elevated rates of fatty acid oxidation in muscle and liver, and are lean, despite being hyperphagic [35], and demonstrate resistance to diet-induced obesity [36]. In the muscle, elevated h-oxidation is associated with marked reduction in malonyl-CoA levels, whereas in liver this occurs in the absence of changes in total hepatic malonyl-CoA levels, indicating that ACC-h must exist in a unique cellular compartment that is not accessible to malonyl-CoA generated by ACC-a. These mutant mice also demonstrate that, unlike Acaca, Acacb is not an essential gene. The molecular basis for the targeting of ACC-h to the outer mitochondrial membrane has yet to be resolved, but is likely to involve the 25 amino acid N-terminal hydrophobic region, absent in ACC-a. Using a consensus of secondary structure prediction methods [37], residues 24 (i.e. beyond the hydrophobic terminus) to 150 of human ACC-h are predicted to form a random coil. The chicken and fish ACC sequences are highly similar to those of mammals, including the conservation of the phosphorylation site in region 2, although a gap in the chicken ACACB currently prevents the comparison of this region. For ACC-h, most inter-species variation is seen within region 1. Like the mammalian protein, the predicted chicken and Fugu proteins have hydrophobic stretches at their extreme N-termini and thus are presumed to be mitochondrially targeted. The zebrafish protein may lack this hydrophobic terminus, however this region of the gene is adjacent to a gap in the current genome assembly, and thus the situation is presently unclear. The length of region 1 (the coding portion of protein coding E1) in mammals (218 residues in humans) is longer than that of Fugu (173 residues) and chicken (approx. 139). This may perhaps be the result of an accumulation of insertions in the relatively poorly conserved unstructured central portion of this region in the mammalian progenitor, or perhaps might reflect convergent evolution for the N-terminal portion of the exon. 2.4.4. Sequence similarity between the N-termini of ACC-a and -b When ACC-h was first cloned, the derived sequence of the first protein coding exon (region 1) was reported to have no homology to that of ACC-a [33,38]. Careful alignment, however, reveals a conserved serine and acidic residue-rich region, which includes a known in vivo phosphorylation site

7

in ACC-a (Ser-29; Section 4.3.1). This similarity extends to the chicken and fish ACCs (Fig. 3). This may provide clues to the evolution of the two genes post duplication, suggesting that both first protein coding exons were derived from the ancestral ACC gene. 2.4.5. Exon numbers and intron positions As the ACC genes contain varying or unknown numbers of 5Vleader exons, we refer to the first protein coding exon as E1 throughout this section for ease of comparison. With the exception of the C-terminal end of the ACCT domain, which lies within E52, the boundaries between the various catalytic and non-catalytic regions of mammalian ACC discussed above are delimited by introns (Fig. 2). Mammalian ACACA and ACACB have 54 and 52 protein coding exons, respectively. Intron positions are exactly conserved in the two genes, and hence ACACA has two additional novel exons and associated introns (rather than just extra introns dividing exons also present in ACACB). These differences are within region 4 (Fig. 2). Protein coding E28 encodes eight amino acids and has been shown to be alternatively spliced [39] (Section 3.1.3). The presence or absence of this exon inluences the phosphorylation of residues within E29, as discussed in Section 4.3.1.2. A second exon (E33) encoding 15 amino acids is also present exclusively in ACC-a (Fig. 2). The protein coding sequences of both of these exons are highly conserved between mammalian species. These orthologue-specific gene differences are also conserved in the two fish species, suggesting that the differences arose shortly after the gene duplication, giving rise to the two ACAC genes, but prior to speciation. The cloned chicken ACC-a lacks both of these a-specific exons, however the gene has the potential to encode both in alternatively spliced transcripts, as discussed in Section 3.1.3. All the ACACB exon boundaries are found to be conserved between fish and mammals, where sequence coverage is available (not shown; Table 1). For ACACA, there are two exceptions. Mammalian intron 15 is absent in

Human/Rat/Mouse Chicken Fugu Zebrafish Human Mouse/Rat Chicken Fugu Zebrafish

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Fig. 3. Region of sequence similarity within the N-terminal regions of ACC-a and -h. Sequence similarity between a region encoded by the first protein coding exons of ACC-a and -h. This similarity is conserved across mammals, fish and chicken. The numbering of residues in zebrafish ACC-h is provisional (see text). Ser residues that are known to be phosphorylated in mammalian ACC-a, at least in vitro, are shown. Conserved Ser residues are highlighted.

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both pufferfish and zebrafish, while intron 24 is also absent in zebrafish. These must therefore represent examples of intron loss in the fish genomes, as it is highly unlikely that both mammalian ACAC genes have gained introns in exactly the same place. Such precise intron loss without changing the flanking coding regions has been documented previously, whereas intron gain appears not to occur in mammals [40]. As mammalian ACACA and ACACB have 52 common exons, and these same exons are found in the fish genes (although some fish ACACA introns were subsequently lost), it can be assumed that these were present in the ancestral gene prior to duplication. More sequence variation is seen amongst ACC-h family members than with ACC-a, suggesting more rapid evolution due to slightly less functional constraint. Much of this variation is due to sequence differences within the ACC-h N-termini. This contrasts with the fact that ACACB is a much more compact gene than ACACA (Section 3). 2.5. ACC gene number and structure in metazoa In addition to mammals, birds and fish, other classes of metazoa also have two ACC genes (Table 1). However, in these cases, it is clear from the subcellular compartmentalisation of their protein products that these genes are not functionally equivalent to ACACA and ACACB. As well as the presumed cytosolic form of ACC [41], which is referred to as ACC1 (C. elegans W09B6) the nematode worms C. elegans and C. briggsae, both have a second gene encoding a predicted second ACC (ACC2, Table 1), although some databases label it as a carbamoyl phosphate synthase. C. elegans ACC2 (T28F3.5) is predicted to be intra-mitochondrial [42]. This may be the equivalent of yeast HFA1p, a paralog of Acc1, which is targeted to an intra-mitochondrial location and provides malonyl-CoA for the synthesis of lipoic acid, an essential cofactor for respiratory enzymes. However, this awaits experimental verification. C. elegans ACC2 lacks residues encoding part of the ACCT domain, which would be predicted to render it non-functional. However, comparison to the C. briggsae ACC2 sequence suggests that there are possibly errors present in the C. elegans gene sequence, and thus the situation awaits clarification. Conversely, other classes of metazoa only appear to have one ACC gene. As fruitfly has a highly malonyl-CoA sensitive CPT I [13], we had expected to find a second ACC gene encoding a mitochondrial outer membrane targeted ACC, as in mammals. Although only one ACC gene is present in the D. melanogaster genome, further analysis reveals that this gene generates two transcripts through alternative splicing, the longer of which encodes an ACC with an extended N-terminus with similarities to that of mammalian ACC-h. The N-termini of the long and short forms of Drosophila ACC target GFP to the mitochondrion and cytosol, respectively, thus generating the presumed

functional equivalents of mammalian ACC-a and -h from a single gene (unpublished observations). Ciona intestinalis has only a single ACC gene (Table 1), and of all the invertebrate sequences analysed, the predicted protein sequence of its product is the closet relative to mammalian ACC, showing approximately 62% identity. 2.6. Functional regions in invertebrate ACC 2.6.1. Catalytic regions The novel sequences listed in Table 1 were identified through homology to mammalian ACCs and the presence of structural BCCP and the two catalytic domains. ACC2 from both Caenorhabditis species lacks approximately 75 residues from the C-terminal half of the aligned ACCT domains, although this may reflect an error in the gene sequence, as discussed above. A consensus across all BCCPs of (A/I/V)-M-K-(L/V/M/ A/T) has been observed to be the biotinylation signature sequence, where the lysine residue is the site of biotin attachment [22]. Assuming that all the sequences we analysed are indeed biotinylated, the consensus needs to be widened. In all four Caenorhabditis ACCs, the sequence starts Ser-Met-Lys, and in C. briggsae ACC2, the gene derived sequence is Ser-Met-Lys-Ile. 2.6.2. Non-catalytic regions Of the five non-catalytic regions identified in mammalian ACC, differences are seen in ACCs from less closely related species, particularly in region 4 and N-terminal regions 1 and 2. Yeast ACC1 and the Caenorhabditis ACCs have shorter N-termini upstream of the BC domain and they lack the direct equivalents of the mammalian phosphorylation site in region 2, as does sea squirt (C. intestinalis) ACC. Drosophila ACC is a target for phosphorylation by AMPK on a serine residue within region 2 equivalent to Ser-79 of rat ACC-a [14]. This residue is conserved within a similar context in the mosquito ACC (not shown). Although worms also have AMPK [43], this site is not conserved in worm ACC1 or 2 and is also absent in the sea squirt ACC. C. elegans ACC1 gene was also reported to exist in a variant form with an extended N-terminus due to alternative splicing at the 5V-end [41]. Thus the existence of multiple isoforms is not restricted to complex metazoans. It is not known whether this form of ACC1 is non-cytosolic. The N-terminal end of region 4 is poorly conserved in yeast ACC and is partially absent from the worm ACC1s (not shown). A larger region of the C-terminal end of this region is also absent in the worm ACC2s. Indeed Caenorhabditis ACCs show a lower level of sequence homology than the other ACCs throughout this region, which is also true to a lesser extent throughout the remainder of their sequence. In fact the yeast ACC1 is more similar to the mammalian sequences than are those from the worm.

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2.6.3. Exon numbers and intron positions All the metazoan ACC sequences examined have at least some intron positions conserved with those in the mammalian genes, suggesting that they all have a common ancestor. There is strong evidence for both intron loss and gain in ACAC genes from the two insects, based on the observation that nine introns are present in conserved positions compared to mammals in one or both of the insect genes, while only two of these are found in both species. Each insect species also has one intron in non-conserved positions that is not found in the other insect species gene (not shown). Likewise, the worm ACC genes have three exons in common with each other and mammalian genes, suggesting a common ancestor. Again there has been subsequent intron loss and gain. The insect, worm and sea squirt ACAC genes most resemble mammalian ACACB in that they lack the two ACACA specific exons described above. However C. intestinalis ACC has an additional unrelated 57 bp exon (confirmed by an EST) in a position corresponding to mammalian protein coding E28. The intron defining the start of the BC domain is conserved in mammalian, fish, insect, sea squirt and worm ACC genes (with the exception of Drosophila, which probably recently lost this intron as discussed above) and is the only intron that is common to all of these species. The intron immediately upstream, separating E1 (region 1, Fig. 2) and E2 (region 2), is absolutely conserved in all of these genes, with the exception of those from nematodes. All the C. elegans and C. briggsae ACCs lack the consensus phosphorylation site in region 2, as does yeast (whose gene is intronless). Thus it can be reasoned that evolution has added two further levels of regulatory adaptation to the Nterminus of the more dbasicT ACC gene products—the constant E2 which contains the regulatory phosphorylation site (region 2), and the variable upstream exon(s) (E1, except in insects where additional introns are present) encoding region 1. As the regulatory phosphorylation site is right at the N-terminal end of E2, phosphorylation is almost certainly influenced by adjacent residues within E1 (Section 4.3.1.3), and thus the two regions (1 and 2) cannot just be considered as independent modules. The variation in the N-terminus is seen not only between paralogous genes (e.g. mammalian ACACA and ACACB), but also from alternative splicing and/or alternative promoter usage within a single gene, as discussed in greater detail in Section 3 for the mammalian genes, and thus consideration of evolutionary origins must address both protein function and regulation, and transcriptional regulatory advantages. Currently, only the difference between the dnormalT forms of mammalian ACC-a and -h has a clear functional role, that of differential targeting to cytosol versus the mitochondrial outer membrane. Parallels can be drawn with other organisms. The two yeast ACC genes are closely related apart from their Ntermini—Acc1p is cytosolic while signals in the N-terminus of Hfa1p target it to the mitochondrial lumen [44]. Similarly,

9

in Arabidopsis the two tandemly repeated genes ACC1 and ACC2 encode near identical proteins, with the relatively extended N-terminus of ACC2 acting as a plastid-targeting transit peptide, while ACC1 is cytosolic [22]. It is also probable that the extended N-terminus of the splice variant of insect ACC has the same functional role as that of mammalian ACC-h, however their sequences are insufficiently similar to support a common origin for the exons encoding them. Our current understanding of the role of this N-terminus and the residues involved in targeting is however very limited and it may be that this region is subject to little functional constraint other than targeting to mitochondria.

3. Organisation, transcript diversity and tissue-specific regulation of ACC genes 3.1. ACC-a 3.1.1. Organisation of ACACA The 265 kDa isozyme of ACC is encoded by ACACA, located on human chromosome 17q12-21 [31,45]. The mouse orthologue is located on chromosome 11 within a region of synteny with human chromosome 17 (Table 1). FASN, the gene for fatty acid synthase, is also present in this large region of synteny among mammals, although the linkage is not conserved in chicken [30]. cDNAs for this isozyme, referred to as ACC-a [46], ACC1 [31], or ACC265 [47], have been cloned from a number of species (see Table 1). Human ACACA spans 330 kbp and is composed of 58 principal exons, with E5 to E58 encoding the 265 kDa protein [48]. A protein-encoding gene annotated as FLJ39647 in the human genome is located within the 43 kbp intron 1 of mammalian ACACA (Fig. 4). FLJ39647 is probably a low abundance transcript in most tissues, judging from the presence of very few spliced expressed sequence tags (ESTs) in databases. 3.1.2. Transcript diversity The organisation of the regulatory regions of ACACA in mammals is consistent with the emergence of four promoters, PI, PIA (previously annotated as rat ACC PI [49]), PII, and PIII, in a mammalian progenitor, that operate in a species-specific fashion in modern species (Fig. 4) [48,50]. PII is a CpG island promoter that is functionally conserved in all mammalian species. CpG islands are features of housekeeping-type genes in which the CpG dinucleotide is observed at a higher frequency (N10 CpG/200 bp) compared to bulk DNA (2.5 CpG/200 bp). In line with this, PII transcripts, which encode 265 kDa ACC-a derived from translation from E5, are ubiquitously expressed, albeit at variable levels [50–53]. PIA, located 12.5 kbp upstream of PII in rat Acaca, also encodes the 265 kDa ACC-a. P1A is a tissue-specific promoter operating principally in white adipose tissue of

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Fig. 4. The ACC-a gene. (A) The distribution of exons and regulatory regions, PI, PII, and PIII of human ACACA relative to the divergently orientated TADA2L and FLJ39647 hypothetical gene within intron 1 of ACACA. PIA denotes a region of homology with ACACA of ruminants and rodents that functions as a promoter in these mammalian lineages. (B) Species context of alternative promoter usage and exon splicing of hypothetical ACC-a isozymes. PIA transcripts of ruminants and rodents initiate translation within E5 (not shown). P denotes the location of AMPK phosphorylation site encoded by E6.

rodents and ruminants [52,54,55]. Although sequence within intron 1 of human ACACA shows homology to the leader exon of transcripts derived from the PIA of rodent and ruminant Acaca, and to the upstream flanking sequence, this region to date has not been demonstrated to function as a promoter in human cells [48]. PI, adjacent to E1, and located approximately 50 kbp upstream of PII, is also a CpG island promoter that forms the 5V-boundary of ACACA with the divergently oriented gene, TADA2L. The intergenic region is approximately 100 bp. TADA2L encodes ADA2a, a component of a transcriptional adapter complex involved in chromatin remodelling [56]. This gene pairing is conserved in all mammalian species studied [50]. Transcripts derived from PI were originally identified in human adipose tissue [57], where they were thought to be functionally equivalent, but not phylogenetically related, to the adipose-restricted PIA transcripts. This now appears not to be the case, as in all mammalian species studied, these transcripts are most highly expressed in the brain. The shared promoter operates asymmetrically, as in contrast to ACC-a transcripts, TADA2L transcripts are ubiquitously expressed [50,58]. The molecular basis for the action of the shared promoter is

not yet known. It is apparent that PI transcripts that include the 47 nt alternatively spliced E4 (Section 4.1) encode a 37 amino acid extension to the 265 kDa ACC-a that is initiated within E1 [50], giving an ACC-a isoform with a predicted molecular mass of 270 kDa. The primary 11 amino acids of this extended sequence are hydrophobic and may facilitate targeting of this isoform to specific membrane compartments. The physiological basis for the predominately CNSrestricted expression of the PI transcript is not yet known. TADA2L is also divergently oriented with respect to ACACA on chicken chromosome 19. Currently it is not clear if the two genes are adjacent, as the gene CR390154 (unrelated to FLJ39647) is located between the single characterized promoter of chicken ACACA [59] and the first exon of TADA2L. Human [48,60] and ruminant ACACA [61–64] also transcribe mRNA from a promoter, PIII, downstream of E5. PIII results in the synthesis of an isoform in which the 75 amino acid N-terminus encoded by E5 is replaced by 17 residues encoded by E5A, generating a 259 kDa ACC-a. Sequence with high homology to E5A and upstream flanking sequence is present between E5 and E6 in rat and mouse Acaca. However these species do not express

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mRNA encoding an equivalent ACC-a isoform, indicating that the functionality conferred by E5A has become redundant at some point in the rodent lineage [60]. 3.1.3. Alternatively spliced variants Transcripts derived from the ACACA promoters are also subject to alternative exon splicing, mainly in the 5V UTR (untranslated region) [48,55,65] (Fig. 2), but also within the coding region [39,66,67]. The 47 nt E4 is alternatively spliced in transcripts derived from promoters PI, PIA, and PII. A 47 nt sequence homologous to E4 is also present in chicken ACACA and appears to be included in all transcripts derived from the single characterized promoter [59]. The exclusion of E4 from transcripts derived from PI terminates the open-reading frame (ORF) initiated in E1, and if (re)-initiation occurs downstream in E5, this would result in the synthesis of the 265 kDa ACC-a. However as E4 is alternatively spliced in transcripts derived from PIA and PII, it may have additional functions. In bovine and human ACACA, the utilisation of weak splice sites located within E4 results in the inclusion of truncated sequences encoded by this exon into a minority of transcripts [48,62]. An additional 61 bp exon, E3, is also included at low incidence in transcripts derived from the PII of rat Acaca in lactating mammary gland [65]. Similarly, E3 (not phylogenetically related to E3 of rat Acaca) of bovine ACACA is also included at low incidence in transcripts derived from PII [62]. Transcripts derived from human ACACA demonstrate an increased level of complexity not observed in other species (Fig. 2), though much of the diversity is represented by low abundance transcripts [48]. Although the majority of transcripts derived from PI are of the type E[1/4/5], a minority include additional alternative spliced exons, E1A, E1B and E1C, located upstream of E2. Inclusion of these exons in any combination would terminate the ORF extension encoded by E1. E1C is homologous to the leader exon of transcripts derived from the PIA of ruminant and rodent ACACA. Other exons with weak splice sites are also represented by E3 and EV5A (Fig. 2, [48]). E5B, a 111 bp exon located downstream of E5A, is incorporated into transcripts derived from PI, PII and PIII [48,60]. This acts to terminate the ORF initiated in E1 (PI transcripts), E5 (PII transcripts), or E5A (PIII transcripts), such that translation could be initiated or re-initiated in E6. This would generate a 257 kDa isoform lacking the key AMPK phosphorylation motif encoded by E6. The inclusion of E5B in transcripts where translation is initiated in E5 results in the synthesis of a 77 residue protein when such templates are assessed in vitro [48]. The physiological significance of the inclusion of E5B in these transcripts is unknown, as such a protein has yet to be demonstrated to occur in vivo, and if translation was not re-initiated in E6, such transcripts might become susceptible to nonsense-mediated mRNA decay [68]. A variant ACC-a transcript lacking 24 nucleotides due to the alternative splicing of E33 (protein coding E28: Fig. 2)

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has been described in rat [39], mice [69] and sheep [66]. Levels of transcript lacking E33 vary between tissues, being lowest in ovine spleen, lung, muscle, heart, adipose tissue and brain (10–20%) to up to 80% of total ACC-a transcript in the ovine and rodent mammary gland during lactation [66], with exclusively E33-containing transcripts in the developing mouse brain [70]. There is evidence from mouse that such splicing may be regulated by the nutritional changes accompanying weaning [69]. The inclusion or exclusion of these eight amino acids influences the phosphorylation of Ser-1200 (see Section 4.3.1.2). The chicken liver ACACA cDNA also lacks the equivalent of E33, although the recently available chicken genome sequence reveals the potential of chicken ACACA to encode such an exon (not shown). Here it should be noted that the use of a synthetic peptide based on the sequence of rat ACC-a, and containing the C-terminal end of the alternatively spliced exon, has wrongly been reported as deriving from chicken [71]. Although not previously commented on, perhaps because the rat [46] and chicken [72] ACC-a cDNA sequences were first published in the same year, the exon equivalent to mammalian E38 (protein coding E33) is also absent from the chicken cDNA. Again the chicken ACACA gene has the potential to encode this 45 nt exon (not shown), and thus it is possible that both protein coding E28 and E33 are subject to alternative splicing in chicken. Our analyses show both of these exons to be ACCa specific, as discussed in Section 2.3.5 (Fig. 2). There are no reports of alternative splicing of protein coding E33 in mammals. 3.2. ACC-b ACACB, located on human chromosome 12q24 [33], encodes the 280 kDa isozyme of ACC, referred to as ACCh [38], ACC2 [33], or ACC280 [47]. Prior to the mid-1990s a number of groups had provided evidence, either based on streptavidin reactivity to covalently bound biotin or through the use of specific antisera, for at least two isozymes of ACC, principally of 265 and 280 kDa [47,73]. Brownsey et al. [74], however, provided peptide sequence data showing that these two proteins were likely derived from separate genes. Inadvertently, Ha et al. [57] postulated an ORF for human ACC mRNA that was subsequently demonstrated [33,38] to be a chimera of the ACC-a and -h genes. ACC-h mRNA is principally expressed in oxidative tissues such as skeletal muscle and heart, and has been postulated to generate malonyl-CoA for the regulation of fatty acid oxidation [33,38], which is a feature of the metabolism of these tissues. It is also the predominant isozyme in rat brown adipose tissue [47]. ACACB is transcribed from multiple promoters in a tissue-specific fashion. 3.2.1. Organisation of ACACB Alignment of the human ACC-h mRNA with the human and mouse genomic sequences indicates that the coding

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mammary gland and preputial gland. An exception to this is adipose tissue, in which PI transcripts are predominant (unpublished observations). A recent paper [76] has confirmed the higher abundance of PII compared to PI transcripts in rat liver. The existence of a third ACACB promoter, PIII, is suggested by a cDNA (Accession no CA392203) isolated from a human retinal pigment epithelium/choroid library in which E3, encoding the mitochondrially targeting Nterminus, is replaced by a unique exon that encodes a 15 amino acid variant N-terminus. The novel, approximately 240 bp, exon E4 is located 14.6 kbp downstream of E3 and 12 kbp upstream of E5, which encodes the putative regulatory phosphorylation motifs of ACC-h (Fig. 5). A 285 bp sequence demonstrating 66.1% identity with human E4 is also present at the homologous position of mouse Acacb, and the putative variant 15 amino acid N-terminus is conserved at 11/15 positions between human and mouse. A high proportion of mouse ACC-h mRNA is transcribed from PIII in brain, adipose tissue and testis (unpublished observations), indicating that non-mitochondrially targeted ACC-h isoforms may play a significant role in the generation of malonyl-CoA in these tissues. Furthermore, alternative splicing of E3 is evident in transcripts derived from PI and PII in human tissues (unpublished observations) and may have the potential to direct the synthesis of an ACC-h isoform that, in addition to lacking the mitochon-

region comprises 52 principal exons. The first 218 amino acids of the 280 kDa ACC-h are encoded within a single exon, E3 (Fig. 5). The exon structure is conserved in the two species, and downstream of E3 is conserved with mammalian ACACA, with the exceptions discussed in Section 2.3.5. The ACACB transcriptional unit is contained within a 150 kbp region, whereas the exons of mouse Acacb span approximately 105 kbp. 3.2.2. Transcript diversity Transcripts encoding the 280 kDa ACC-h derive from two promoters, PI and PII [75], the primary exons, E1 and E2 (Fig. 5) (1a and 1b in [75]), encompassing divergent 5V UTRs, being separated by 14.5 kbp. PII transcripts are predominant in human muscle. As PII was inactive in hepatoma cells, Lee et al. [75] postulated that the liver might express ACC-h mRNA from a promoter distinct from PII. Consequently they demonstrated that Alexander and HepG2 cells express ACC-h mRNA from PI, and not PII, and concluded that PII was a muscle-type promoter, and PI, a potential hepatic promoter. No tissue expression data was presented to support this assertion. Exons homologous to human E1 and E2 are present in mouse Acacb. PI transcripts in mouse are almost exclusively expressed in cardiac and skeletal muscles, whereas PII transcripts are most highly expressed in liver and other tissues, with high coincident levels of fatty acid synthesis, for example lactating

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Fig. 5. The ACC-h gene. (A) Orientation of human ACACB relative to the flanking genes UNG (uracil N-glycosylase) and FOXN4 (forkhead transcription factor). The distribution of exons and regulatory regions, PI, PII, and PIII of ACACB are shown. (B) Effect of alternative promoter usage and exon splicing on the translation and mitochondrial targeting of hypothetical ACC-h isozymes. P denotes the residue within ACC-h phosphorylated by AMPK.

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drial targeting sequence, is also devoid of the N-terminal phosphorylation motifs. Whether malonyl-CoA generated from non-mitochondrially targeted isoforms of ACC-h is functionally distinct from that generated by ACC-a is an open question. 3.2.3. Re-assessment of a reported human ACC-b splice variant It has been reported that two isoforms of ACC-h occur in human liver due to the inclusion or exclusion of a sequence of 303 nt/101 amino acids [33]. Comparison to the gene sequence shows this 303 nt region to in fact consist of E25 and E26 (protein coding E22 and E23), together with intron (I)25. Thus the longer form of ACC-h described, purported to represent the majority ACC-h transcript, contains an intron of 89 nt, the retention of which would cause a frameshift based on the genomic sequence. The presence of this intron may have arisen from artifactual priming of cDNA synthesis by oligo (dT) in the adenosine-rich region present within I28, as depicted in the authors’ figure [33], and therefore probably represents a partially-spliced RNA. We have sequenced such a cDNA and found it to lack all other introns except I25 (unpublished results). The shorter form reported therefore lacks two exons covering 214 (not 303) nt, relative to the correctly spliced transcript, which introduces an in-frame termination codon in E29 (protein coding E24), and would therefore be likely to be removed by nonsense-mediated mRNA decay [77]. The upstream PCR primer used in [33] contains a 3Vmismatch to the genederived sequence, and may have resulted in biased amplification of this likely rare mRNA species. 3.3. Mechanisms for hormonal, nutrient, and tissue-specific regulation of ACC genes in mammals The activity of ACC and the rate of fatty acid synthesis in key tissues fluctuate rapidly in response to various physiological factors [78,79]. Controls on ACC activity are exerted acutely, through metabolite activators and inhibitors, and reversible protein phosphorylation (Section 4). Long-term regulation is achieved through transcriptional and post-transcriptional mechanisms. ACC-a transcript abundance is co-ordinated with that of mRNAs encoding other enzymes of the lipogenic pathway, e.g. FAS and ATPcitrate lyase, and to some extent with mRNA components of related pathways of glycolysis, cholesterol ester and phospholipids synthesis [80–82]. Despite an incomplete picture, it is becoming increasingly apparent that basic regulatory networks that regulate the activity of ACC, both acutely and in the longer-term, are conserved from the earliest metazoans and have been refined throughout evolution with increasing complexity. 3.3.1. Regulation of ACACA ACC-a is most abundant in tissues involved in the partitioning and storage of energy derived from the

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metabolism of excess carbohydrate [54,83]. ACC-a is however also ubiquitously expressed as all cells synthesise lipid for membrane replenishment and intracellular processes [51–53]. Acaca, similar to Fasn, is an essential gene for embryonic development in mice [7]. Mammary expression of ACC-a is subject to developmental cues co-incident with the partitioning of metabolic potential to the gland and the synthesis of other components of milk [84,85]. 3.3.1.1. Nutritional regulation. The serum concentration of key humoral factors communicates nutritional status [86]. Increases in the insulin:glucagon ratio, glucose and T3, and suppression in the levels of free fatty acids either precede or parallel the induction of the rate of hepatic fatty acid synthesis in animals fed carbohydrate after a period of starvation [87–90]. Adipokines, such as leptin [91] and adiponectin [92], report the level of body fat stores to the liver and limit hepatic triglyceride accumulation, achieved, in part, by suppressing the expression of ACC-a mRNA. The hepatic response of Acaca to carbohydrate feeding is markedly impaired in streptozotocin-diabetic rats, indicating that insulin is performing a modulating role [93,94]. Hepatic ACC-a mRNA accumulation in carbohydrate-fed diabetic rats is also modulated by T3 status [93]. Carbohydrate feeding dramatically induces PIA transcripts in rat liver [94]. These transcripts are principally expressed in adipose tissue and are barely detectable in the liver of fed animals [94]. PII transcripts also increase, though the magnitude of their elevation is disputed [51,94]. PIA transcripts are markedly repressed, relative to PII transcripts, upon subsequent food-restriction, mediated, at least in part, by a marked decline in their stability [94]. In the absence of carbohydrate feeding, insulin does not activate PII or PIA in liver when administered to streptozotocin-diabetic rats, but markedly induces PIA, but not PII, in the adipose tissue of the same animals [94], highlighting potential differences in the regulation of PIA in the these tissues. 3.3.1.2. Molecular mechanisms. The action of convergent pathways, induced by glucose and insulin, to recruit respectively a carbohydrate-responsive factor (ChoRF) and sterol responsive element binding protein (SREBP)-1c to distinct elements, is proposed as a unifying mechanism for the induction of lipogenic genes in response to carbohydrate re-feeding in liver [95]. Carbohydrate responsive-elements (ChoREs) have been identified in a number of mammalian lipogenic genes and are composed of two E-box (bind members of basic helix-loop-helix (bHLH) class of transcription factor) type motifs separated by 5–9 bp that function co-operatively [95–97]. A ChoRE (5VCATGTG(N)7CGTG-3V) has been identified in the rat PIA promoter at -122 relative to the transcriptional start site (tss) [96]. This element is present in one or two copies in the proximal region of PIA in all mammalian species studied so far. The deletion of the upstream E-box in ovine PIA

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impairs, but does not abolish, regulation by insulin in transfected adipocytes [52]. Conversely, although 1 kbp flanking PIA of rat Acaca is potently glucose-responsive when transfected into rat hepatocytes, this construct is not responsive to insulin or SREBP-1c over-expression [96], which has a modest effect on endogenous transcripts [97]. SREBP-1c responsive elements could therefore be located at a distance beyond the proximal promoter. The PIA promoter of rats is responsive to T3 [98] though the mediating elements have yet to be identified. A similar rat PIA construct was not induced by glucose in 30A5 adipocytes [99]. Synergistic signalling between glucose and insulin increases PII activity in these cells and is dependent on two Sp1 motifs [99,100] that flank two SRE-half sites that support a response to cholesterol depletion in CV-1 cells [101] and the action of insulin, mediated by increased SREBP-1c activity, in MIN6 mouse pancreatic h-cells [102]. Adipocyte cell lines, such as 30A5 cells, express low levels of PIA transcripts [103] compared to adipose tissue [52] and therefore it is possible that the PIA promoter is subject to transcriptional repression in these cells that impairs the response to glucose (see Section 3.3.1.3). A candidate for ChoRF has emerged in the form of carbohydrate-response element binding protein (ChREBP) [104,105], previously identified as WBSCR14, a gene located on human chromosome 7 within a region deleted in the neurological condition Williams-Beurren syndrome [106]. ChREBP (also known as MondoB) and its paralogue, MondoA, are a class of bHLH protein, present within the genomes of worms and flies (Section 3.4) that undergo cytoplasmic-nuclear shuttling [107]. The nuclear import of ChREBP and its DNA binding activity are negatively regulated by phosphorylation by PKA and AMPK in response to glucose deprivation [108,109]. Conversely, glucose metabolism promotes its de-phosphorylation by a xylulose-5-phosphate-activated PP (protein phosphatase) 2A [110] that is similar or identical to the PP2A that activates fructose-6-P 2 kinase/Fru-2,6 P2ase [111] and thereby co-ordinates glycolytic flux with transcriptional regulation of fat synthesis [112]. Consistent with this, ChREBP null mice demonstrate an impaired hepatic induction of ACC-a mRNA in response to high levels of dietary carbohydrate despite normal levels of SREBP-1 [113]. Chicken ACACA, like the mammalian PIA promoter, demonstrates dual regulation by carbohydrate and additional humoral factors. To date a single promoter, encompassed within a CpG island, has been demonstrated to be responsive to the synergistic action of glucose and T3 [114]. The T3 responsive region at approximately -100 is composed of a DR4 (direct repeat separated by 4 nucleotides) TRE (thyroid hormone response element) with an adjacent SRE [115,116]. T3 activation is associated with increased levels of SREBP-1 mRNA in chick embryo hepatocytes (CEH) and increased binding of the transcription factor to the SRE, that is stabilised by a direct

interaction with bound TR (thyroid receptor) [117]. Insulin accelerates the effect of T3 by rapidly increasing the processing of the SREBP-1 precursor [117]. By contrast, SREBP-1c acts in concert with the heterotrimeric transcription factor NF-Y to enhance T3 regulation of rat S14 gene transcription in the liver, but this interaction spans 2.3 kb and is probably not facilitated by direct interaction between SREBP-1 and TR [118]. There is no TRE-related element in the vicinity of the two SRE half sites in mammalian PII, which correlates with the lack of T3 responsiveness of rodent PII in vivo [98]. However, human PII reporter constructs are potently responsive to T3, as well as SREBP-1a [48], suggesting that this may occur through a TRE-independent mechanism. The mechanism of glucose regulation of the chicken ACACA promoter is presently unknown. The effects of glucose in CEHs are independent of SREBP-1 levels [117] and are related to the accumulation of intermediates of the pentose phosphate pathway, such as xylulose 5-phosphate [114], suggesting the involvement of a ChREBP-related factor. A second ACACA promoter has been suggested to be nutritionally responsive in chicken liver though its location within the gene has not been determined [119]. 3.3.1.3. Promoter PIA: species-specific expression in mammals. The rodent PIA promoter functions to mediate rapid disposal of serum glucose in key tissues characterized by a substantial synthetic capacity in triglyceride. Characteristics of the PIA transcript that may support this function include differential regulation of transcript stability [94] and potential enhanced translatability [120] compared to PII transcripts. Such regulatory flexibility is not apparent in the chicken ACC-a transcript whose levels appear to be controlled principally by transcription rate [121]. Ovine PIA demonstrates significant sequence identity to the rodent promoter in a 200 bp region upstream of the tss, including conservation of the TATA transcriptional initiator element, C/EBP (CAAT/enhancer binding protein) binding-site and E-box motifs that are targets of ChREBP, and generates transcripts that are restricted to adipose tissue and liver [52]. Although bovine PIA demonstrates 93% identity with the ovine homologue in a 2.6 kbp region upstream of the tss, unexpectedly the TATA-initiator is absent. This is associated with a considerably less stringent expression profile [62]. The evolutionary driver of this altered expression profile and its physiological consequences are unknown. A feature of the evolutionary history of the PIA promoter in mammals is the propagation of lineage-specific repetitive elements, which exert a repressive influence on promoter activity, within the region immediately upstream of the proximal promoter. A [CA]28 repeat microsatellite located between -271 and -216 in the flanking region of rat PIA acts as repressor of promoter activity, which is relieved by the deletion of the microsatellite or ectopic expression of C/EBP [103]. Similarly, a distal region of a bovidae specific Art2 retrotransposon element, located between -762 and -1297,

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together with a more proximal element, exerts a repressive influence on transfected bovine PIA constructs [62]. The insulin responsiveness of ovine PIA reporter constructs is modified by the activity of the repressor upstream of the proximal promoter. The differentiation of preadipocytes is associated with the attenuation of the repressor activity and induction of endogenous PIA transcripts [52], suggesting that the interaction of the insulin response motifs with the repressor may play a role in the tissue-specificity of PIA. 3.3.1.4. Mammary gland expression. ACC-a mRNA is transcribed exclusively from PII in the rodent mammary gland and is increased several fold at the onset of lactation [54]. In ruminants, PIII transcripts, which are not expressed in rodents, demonstrate the greatest induction (15 to 28fold) in mammary gland with lactation, contributing up to 30% of total ACC-a transcripts [61,63]. No other tissue has been found to express PIII transcripts at this level. By contrast, PII transcripts are only induced modestly (2 to 3fold). PIA transcripts are also induced in bovine mammary epithelium [122]. Key regulatory regions of each ovine promoter are defined by modification of chromatin structure in distal and proximal locations with lactation. Binding motifs for STAT5 (signal transducer and activator of transcription), a mediator of prolactin signalling [123], are present in the distal regions; the STAT5 motif in bovine PIII mediates the induction of reporter constructs by prolactin in transfected HC11 mammary cells [63]. The proximal region of PIII recruits SREBP-1 in lactation. Conserved SRE motifs are also present in PII [64] (Section 3.3.1.2). The target of SREBP-1 action in the proximal region of ovine PIII, an E-box motif 5V-CACCTG-3V, is not conserved in the homologous region of human PIII [48,60]. This is corroborated by human PIII reporter constructs not being induced by the ectopic expression of SREBP-1a, whereas PII is activated by the transcription factor [48]. These observations could suggest that PIII is not induced in breast tissue during lactation in humans, or induction is achieved by an SREBP-1 independent mechanism. 3.3.1.5. Expression in tissues with a low capacity for nutritionally-regulated fatty acid synthesis. ACC-a mRNA is ubiquitously expressed, albeit at variable levels, in animal tissues, reflecting a requirement for lipids for cellular structures, secretory functions and intra-cellular signalling. Appreciable levels of ACC-a mRNA are present in the lung, adrenal gland, pancreatic h-cells and regions of the brain [124–126]. Expression in these tissues, which is principally derived from PII, is not subject to nutritional regulation. In several human and ruminant tissues, PIII transcripts are also expressed at a low level [48,60]. Pancreatic h-cells express little FAS mRNA, indicating that the metabolic rate of malonyl-CoA is not primarily linked to the synthesis of fatty acids [124]. Although the increase in malonyl-CoA that precedes insulin secretion is associated with an elevation in cellular fatty acyl-CoA,

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coincident with the inhibition of h-oxidation, it is apparent that h-cells express low levels of ACC-h [124,127], questioning the function of the high ACC-a:FAS ratio in these cells. By contrast, chronic hyperglyceamia and hyperinsulinaemia promotes the induction of ACC-a and FAS mRNA by an SREBP-1c dependent mechanism that results in ectopic accumulation of triglyceride in h-cells [102]. In the developing brain, malonyl-CoA is utilised for both the de novo synthesis of palmitate [128], and for the elongation of essential fatty acids for membrane phospholipids [129]; the expression of ACC-a is linked to brain growth [70]. The expression of ACC-a in the peripheral nerve appears to be regulated by SREBP-2 to co-ordinate fatty acid and cholesterol synthesis for myelination [69]. 3.3.2. Regulation of ACACB ACC-h expression is subject to tissue-specific regulation. In the liver, ACC-h mRNA is repressed upon starvation and induced on re-feeding a carbohydrate meal, similar to the expression of ACC-a transcripts [76]. This reflects the intrahepatic partitioning of fatty acids towards h-oxidation in the starved-state and towards triglyceride synthesis and secretion in the fed-state [130]. In rat liver, PII-derived transcripts form the principal component of ACC-h mRNA and are more sensitive to nutritional regulation than are PI transcripts. The induction of PII transcripts is associated with the recruitment of SREBP-1 to a region of the proximal promoter defined by two SRE-type motifs at -62 and -44 that appear to function co-operatively. SREBP-1 is not recruited to the proximal region of PI. The investigation of the molecular determinants of PI activity has been hampered by the finding that reporter constructs are inactive in cells, even in those that synthesise PI transcripts, upon transient transfection [75,76]. By contrast, although starvation results in the reduction in the tissue concentration of malonyl-CoA and accompanying increases in the rate of h-oxidation in skeletal muscle, these modulations are achieved by changes in the catalytic activity of ACC-h without changes in enzyme abundance [131,132]. ACC-h is induced in response to the differentiation of H9C2 myoblasts into myotubes [133] consistent with its tissue distribution in animals [33,38,133]. Several E-box elements in the proximal region of PII of human ACACB are targets of muscle-specific transcription factors that could explain the expression of these transcripts in human muscle [75]. Extensive data on the tissue expression of ACC-h transcripts is currently lacking to provide a framework for evaluating the regulation of these promoters in different tissues and physiological contexts. 3.4. Regulation of ACAC in less complex metazoans The evolution of complex metazoans is thought to have arisen as a result of several phases of segmental or complete

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duplication of the blueprint for multicellular organisms established in the ancestors of modern-day protosomes (arthropods, nematodes), resulting in gene families with functional specialisation [134]. For example, 35 out of 38 vertebrate classes of bHLH proteins have protosome orthologues [135]. Consistent with the conservation of ancestral pathways, the expression of ACC in these organisms is regulated by orthologues of key regulators of mammalian lipogenic genes and is central to linking strategies for withstanding nutrient deprivation with developmental progression. The expression of ACC in protosomes is regulated by an ancestral SREBP, present as a single gene in these organisms, that functions to link, at least in Drosophila [136], lipid synthesis with the phosphatidylethanolamine content of the endoplasmic reticulum by negative feedback. RNAi against the C. elegans orthologue of SREBP (Y47D38.7) or ACC1 (W09B6), but not ACC2 (T28F3.5), produces pale skinny worms that arrest as early larvae [8,137], that may be due, in part, to an impairment in cell fate determination [41]. The starvation of early-stage fruitfly larvae impairs their development and results in death. Their survival is enhanced by maintenance on sugar and is associated with the induction of ACC, although the developmental program is not restored. These metabolic adaptations are likely mediated by a transcription network centred around Bigmax (CG3350) [138]. Bigmax has a ubiquitous distribution in Drosophila, whereas its potential dimerisation partner, dmondo (CG18362), an orthologue of ChREBP, is expressed dynamically during embryogenesis and at a low level in the fat body [139]. Thus, dmondo could play a role, in addition to SREBP, in the synthesis of fat in this species.

4. Post-translational regulation While ACC activity in mammals is known to be regulated chronically (hours–days) through changes in abundance, due to alterations at the level of transcription and mRNA stability (Section 3), it is also subject to acute regulation (minute–hours) by post-translational mechanisms. The two main isozymes of ACC, ACC-a and ACC-h, are subject to acute regulation both via allosteric activators and inhibitors, and reversible phosphorylation. The net activity in a cell or tissue is therefore a result of the interaction between the levels of the various allosteric effectors and the activities of specific protein kinases and phosphatases. Net activity is also a result of the amount not only of the major isozymes, but also of their variants, some of which may have altered regulatory characteristics. This section will review data from both in vitro and in vivo experiments relating to the mechanisms of the acute regulation of mammalian ACC and discuss this in terms of the conservation of the structure, and possible physiological role, of the various members of this enzyme family in a number of species.

Biotin has non-specific effects on the whole family of biotin-dependent enzymes [140]. Although not reviewed here, there may be cases where biotin also has specific effect(s) on ACC at the level of transcription and/ or translation, possibly in an isoform-specific manner [140–147]. 4.1. Subunit composition of the active form of ACC The involvement of ACC in the biosynthesis of fatty acids in mammals was recognised by Wakil in 1958 [148], and the enzyme was purified [149–151] and characterized over the next three decades [152,153]. Purified ACC separated into two major fractions on sucrose density gradients: a lower molecular weight fraction (approx. 500 kDa) protomer, subsequently found under strong denaturing conditions to be a dimer of two subunits of approximately 250 kDa [154,155], and a higher molecular weight (N1 million Da) polymeric form [153,156]. Other than the identification of the two catalytic domains however [16,46] (Section 2.3.1), little is known about the molecular structure of the subunits or how they interact. Citrate, a previously identified activator of ACC activity, was found to increase the proportion of the enzyme in the polymeric form, suggesting that this was the active form [153,156,157]. Evidence for the existence of ACC polymers in vivo was however quite limited and, as pointed out previously by Hardie [17], could as easily be explained by dimer formation as by formation of a polymer [156,158]. The development of rapid-quench methods for measuring enzyme activity and stopped-flow light scattering technology to measure the rate of polymerisation in the early 1980s demonstrated that activation actually preceded polymerisation [159], which was therefore incidental to enzyme activation, and indicated that polymerisation was probably an in vitro phenomenon. The lack of definitive evidence however on the absence, or presence, of the polymers in vivo has led to continued speculation on possible function of these structures e.g. as scaffolds for the association of other lipogenic enzymes. The potential for an increased complexity of this system arose with the discovery of the second mammalian gene for ACC-h in 1996 (Section 3.2). A number of tissues were found to contain varying ratios of both isozymes on SDSPAGE, and the fact that immunoprecipitations with antibodies to either isozyme co-precipitated the other form suggested the existence of heterodimers [47,74,160]. However as this methodology involves tissue-disruption, an alternative interpretation is that these assays detect polymers containing different homodimers rather than heterodimers. The characterisation of an additional two isoforms of ACCa, and a number of others indicated by the presence of mRNAs, some with potentially different kinetics (Section 3.1.3), would also make the total number of possible heterodimers quite substantial. The overall kinetics of ACC in a given tissue would therefore be the result of the

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amount of each type of subunit and the proportion of each type of heterodimer, which could vary from one tissue, and physiological state, to another. Some evidence suggests that this may be the case [47], but at present the transcript, protein and kinetic analysis has not been carried out in enough tissues to make it clear if this occurs. The existence of heterodimers would also appear to defeat the object of the recently characterized differential subcellular targeting of ACC-a (cytoplasm) and -h (mitochondrial outer membrane) by their N-terminal sequences. A more thorough study with a range of antibodies, and possibly full length GFP constructs, may help to resolve this question. 4.2. Allosteric regulation 4.2.1. Citrate The TCA cycle intermediates, citrate and isocitrate, were found to activate ACC in vitro and stimulate lipogenesis in vivo [161,162]. Citrate therefore acts as a classical feedforward activator of ACC, being the immediate precursor of the substrate, acetyl-CoA. Citrate appears to activate both half reactions (Fig. 1), probably via the induction of a conformational change [163,164]. Citrate activation elicits a change in the V max of the enzyme, but has no effect on the K m for acetyl-CoA [153,156]. Citrate prevents the binding of inhibitory long-chain acyl-CoAs (LCACoA) to ACC and dissociates bound LCACoA from the enzyme in the presence of BSA or other LCACoA-acceptor [165] (see Section 4.2.4). In initial isolates of ACC from avian liver, the K act citrate was approximately 2 mM [159], although values reported by different groups for rat were quite variable [47,166,167]. The association of citrate with ACC appears to be very tight, the K d being approximately 2 AM [153]. The discrepancy between this and the K act citrate may be explained by the fact the ACC reaction also requires ATP which chelates citrate, thus reducing free citrate to levels approximating the K d. When it became apparent that there were two isozymes of ACC, attempts were made to assess possible differences in their kinetics using enzyme purified from tissues in which one isozyme predominated. Witters group found that in heart and skeletal muscle, where ACC-h predominates, the K act citrate was approximately 2 mM, compared with 0.65 mM for ACC in white adipose tissue, which contained mainly ACC-a [47]. Values obtained by different groups for this parameter for ACC isoforms were again not very consistent [168–170], possibly due to the numerous factors which can alter this, e.g. the degree of phosphorylation, associated allosteric effectors, or indeed the confounding effects of other isoforms. Given this an update of the kinetic analyses may be warranted, and the Acacb null mouse may prove invaluable here. A number of studies however suggest that ACC-h may not be regulated to the same degree as ACC-a by phosphorylation, but that citrate, possibly malate, and substrate levels may be more important [168,171]. However there is now some evidence in skeletal muscle that

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the activation of AMPK can supersede regulation by citrate and inactivate ACC [172,173]. The regions of the ACC molecule that bind citrate are unknown, however comparison of ACC sequences from oleaginous (Candida 107; citrate activated) versus non-oleaginous (C. utilis; citrateinsensitive) Candida species [174], or S. cerevisiae (citrateindependent [175]), should provide clues. 4.2.2. Glutamate Glutamate is also an allosteric activator of ACC [176], possibly due to its structural similarity to citrate, although the K act for glutamate was much higher (15–20 mM) than for citrate. This could still however be of physiological relevance as cellular concentrations of glutamate have been found to range from 5 to 30 mM. Glutamate levels are influenced by glutamine levels via the enzyme glutaminase, and this regulation could provide a link between amino acid metabolism and the metabolism of fatty acids and carbohydrates. 4.2.3. Malonyl-CoA Malonyl-CoA, the product of the reaction catalysed by ACC, acts as a competitive inhibitor with a K i of approximately 5–10 AM and can result in an increase in the K act in vitro [153,159]. In vivo this could serve to prevent the complete inhibition of fatty acid oxidation by limiting the production of malonyl-CoA, however the K i is relatively high compared to the concentration found in e.g. muscle cells. Relatively high local concentrations of malonyl-CoA could however exist in the vicinity of ACC. In the case of tissues with low lipogenic capacity, where ACC-h generally predominates, malonyl-CoA is thought to be degraded by malonyl-CoA decarboxylase, although the role of this enzyme is not without controversy, particularly with respect to its subcellular localisation [177] and regulation by AMPK [178]. 4.2.4. Long-chain acyl-CoAs LCACoAs, as products of the lipogenic pathway, have been considered as classical feed-back inhibitors of ACC, although very few studies have been performed at relevant physiological concentrations in the presence of the appropriate acyl-CoA-buffering proteins. The K i of ACC for LCACoA (esters of saturated fatty acids with 16–20 carbons being most effective) is 5.5 nM [179]. Under normal physiological conditions intracellular free acyl-CoA concentration is in the range 0.1–200 nM [180]. Cytoplasmic LCACoAs are bound by acyl-CoA binding protein (ACBP), and if the cytosolic ACBP/LCACoA ratio stays below 1, the free LCACoA concentration is estimated to be in the range 2–10 nM [180,181]. The fact that liver fatty acid synthesis proceeds despite the low K i value suggests that concentration of free cytosolic LCACoA is below 5.5 nM under these conditions [180]. Thus LCACoAs are likely to be a physiological regulator of ACC [180].

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LCACoAs are also reported to activate AMPKK [182,183], thus activating AMPK (Section 4.3), potentially amplifying their effect on ACC. These mechanisms could be of importance during fasting when plasma free fatty acids, and cellular LCACoAs, are increased. 4.3. Regulation by phosphorylation/dephosphorylation Since the original observation that purified ACC contained phosphate [150], and that the incubation of crude enzyme preparations from rat liver with Mg.ATP led to inactivation [184], a large body of literature has built up describing the mechanism of regulation of ACC by reversible phosphorylation. Most of this refers to ACC-a, however some recent data addresses ACC-h. Two types of phosphorylation site have been identified, those which have a direct effect on enzyme activity and those which apparently do not. The following sections review the in vitro and in vivo data on the phosphorylation of the family of ACCs in relation to their primary structure, conservation of phosphorylation sites, and possible role in the cell. 4.3.1. Phosphorylation of ACC-a 4.3.1.1. In vitro. A number of kinases phosphorylate ACCa on specific serine residues in vitro. Seven sites were identified by peptide sequence analysis [167,185–187], however only phosphorylation of some of these, by PKA and AMPK, appear to have a direct effect on activity. PKA phosphorylated both Ser-77 and -1200 of rat liver ACC-a, leading to an inactivation of the enzyme due to a small decrease in V max (15–30%) and an approximately two-fold increase in the K act citrate. The incubation of ACC from rat tissues and cells with AMPK resulted in the phosphorylation of Ser-79, -1200 and -1215, and inactivation, in this case associated with a decrease in V max of approximately 90% and a five-fold increase in the K act citrate [167]. Interestingly prior phosphorylation by AMPK decreased the extent of phosphorylation of Ser-77 by PKA, and similarly prior phosphorylation with PKA prevented the phosphorylation of Ser -79, but not -1215, by AMPK. In the later case the subsequent phosphorylation of Ser-1215 by AMPK did not further alter the kinetics beyond that caused by PKA, indicating that Ser-1215 was not involved in the direct regulation of ACC activity. Limited proteolysis of ACC, which removes approximately 200 N-terminal residues, including Ser-77 and -79, reversed the effects of phosphorylation by both PKA and AMPK, implying that these sites, and not Ser-1200, were responsible for mediating the effects of these kinases, although the mechanism of this process remains unknown [167]. Data from Ha et al. [188], who determined the effect of PKA and AMPK on expressed ACC in which they had mutated the four important serine residues, alone and in combinations, agreed with Hardie’s data in terms of indicating that Ser-79, and not -1200 or -1215, mediated

the effect of AMPK, however it also suggested that the effect of PKA was mediated through Ser-1200 and not Ser77 [167]. In this experiment however both AMPK and PKA caused similar small changes in V max and the K act citrate, in contrast to the large effects observed due to phosphorylation by AMPK in vitro or in vivo. The reasons for these discrepancies are unclear, but could possibly reflect conformational changes due to the mutations. The significance of these differences was however put in perspective when the phosphorylation status of the enzyme in response to the activation of these two kinases in vivo was determined. 4.3.1.2. In vivo. The activation of PKA by glucagon and adrenaline in isolated adipocytes and hepatocytes resulted in the phosphorylation of Ser-79 and -1200, sites phosphorylated in vitro by AMPK, and not Ser-77, as was expected from its in vitro phosphorylation by PKA [189,190]. This was found to be due to these hormones acting directly through PKA [190], and implied an indirect activation of AMPK. Recently Birnbaum et al. [119] have shown that increased levels of cAMP in 3T3-L1 adipocytes results in the activation of AMPK and an increase in lipolysis. Here, it could be envisaged that an increase in lipolysis, via PKA activation of hormone-sensitive lipase (HSL), could increase the level of LCACoAs and activate AMPK-kinase (AMPKK), leading to the phosphorylation and inactivation of ACC. This mechanism however cannot explain activation of AMPK in isolated hepatocytes, which do not express HSL. That the effects of AMPK and PKA are mediated through ACC-a Ser-79 or its equivalent in other species is reflected by the high degree of conservation of this residue and its sequence context, not only in ACC-a from mammals, chicken and fish, but also in their ACC-h, as well as in insects, which have only one ACC gene. Ser-77, whose phosphorylation blocks Ser-79 phosphorylation, is also is conserved in all of these species. In adipocytes the phosphorylation of Ser-79 in response to adrenaline and glucagon is accompanied by the phosphorylation of Ser-1200 [190], presumably by AMPK, although as this residue is also a PKA target in vitro, this is uncertain. The sequence contexts of Ser-1200, and Ser1215, do not fit the consensus motif for an AMPK site well [191,192], however neither do the sequences of a number of other known substrates of this enzyme, e.g. eNOS and HMG-CoA reductase. In this respect Hardie et al. noted that both Ser-1200 and -1215 were phosphorylated at a slower rate by AMPK in vitro than was Ser-79 [167]. The relatively loose consensus sequence for PKA, (R/K)1–2(X)1–2(S/T) [193], is met by the sequence surrounding Ser-1200. ACC-a is a poor substrate relative to other PKA targets [194], and this may be due to the negative influence of Phe-1201 [195]. The significance of Ser-1200 and -1215 in relation to function is unclear. They exist within a highly conserved region of mammalian and fish ACC-a (Section 2.3.2) but

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are not conserved in ACC-h, or in insect or worm ACC. Kong et al. [39] identified an isoform of ACC-a which lacked E33 (protein coding E28) (Section 3.1.1), resulting in a deletion of eight amino acids, from positions -11 to -4 relative to Ser-1200. The inclusion of this sequence in in vitro expressed protein fragments inhibits PKA phosphorylation of Ser-1200, despite there being no apparent change in the specificity determinants for phosphorylation by PKA, implying that other elements must be involved. The ability of AMPK to phosphorylate Ser-1200 in these protein fragments has not been tested. Although chicken ACC-a contains the equivalent of Ser-1200, and thus may be a good PKA substrate, it is not clear whether the E28 equivalent is included in any transcripts (Section 3.1.1). Most mammalian tissues express predominately transcripts for the longer isoform, the exception being lactating mammary gland [196]. The shorter form would, extrapolating from the data of Kim’s group [39], produce an ACC-a more susceptible to phosphorylation and therefore more dependent on allosteric effectors, e.g. citrate and, possibly glutamate, which can reach high concentrations in mammary epithelial cells during lactation [196], and could thus provide a mechanism to link the production of milk fat and protein. However Hardie et al.’s data [190], suggesting that Ser-1200 may be phosphorylated by AMPK in vivo, and that, irrespective of the kinase involved, the phosphorylation of this site has no direct effect on activity, would make this explanation untenable. It is possible that the phosphorylation of Ser1200 could result in a conformational change enabling activation or de-activation, however structural data to elucidate this is lacking. The regulatory importance of this region is supported by the fact that of all the mammalian phosphorylation sites, only that equivalent to Ser-1215 (Ser1157) is conserved in the yeast cytosolic ACC1. ACC1 is known to be regulated by phosphorylation by Snf1p, a yeast AMPK [197], and a synthetic peptide containing Ser-1157 proved a good substrate for AMPK in vitro [71]. This residue is also conserved in Ciona ACC, as is the Ser-77, but not the Ser-79 equivalent. Insulin action results in the activation of ACC in adipocytes [186], hepatocytes [198], and intact liver [199], and this is associated with a decrease in the total phosphate content of the enzyme and an inactivation of AMPK [200]. Insulin also however increased phosphorylation of Ser-29 [185], and an unidentified site in a proteolytic peptide termed the I-peptide [201]. Phosphorylation of the I-peptide survived purification of the enzyme, however the effect of insulin did not [202–204], implying that phosphorylation at this site has no direct effect on enzyme activity, and that loss of the insulin effect could be attributable to loss of a low molecular weight activator. A 130 kDa dactivatorT protein which can increase the activity of ACC in a phosphorylation-independent manner has since been identified by Denton’s group [205], who also isolated a kinase which appears to phosphorylate the I-peptide relatively specifically [205]. The mechanism of action,

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and physiological relevance, of both of these proteins awaits their further characterization. Insulin causes an increase in ACC-a phosphorylation on Ser-29 [185], a site phosphorylated in vitro by casein kinase II, and in 3T3-L1 adipocytes and H4IIE hepatoma cells the activity of this kinase increases in response to insulin [206]. As with the I-peptide however phosphorylation at this site appears to have no direct effect on ACC activity. Interestingly the Ser-29 residue, in addition to being conserved in ACC-a, is also conserved in ACC-h, and in a context that would suggest it could remain a substrate for casein kinase II (Fig. 3). The relevance of this in relation to possible regulation by insulin is however somewhat obscure, as ACC in heart and skeletal muscle, where ACC-h predominates, appears not to be regulated by fasting/re-feeding, unlike ACC-a [131]. Hypotonic swelling of isolated hepatocytes mimics the metabolic effects of insulin on ACC activity and may also act through changes in phosphorylation [207]. 4.3.1.3. Phosphorylation of isoforms of ACC-a. Three main isoforms of ACC-a have been characterized: the major 265 kDa isoform, an isoform with an alternative, shorter, Nterminus (E5A) [60], and the isoform resulting from the exclusion of E33 (D24) [39]. The D24 splice variant also exists in the context of E5A, as well as the 265 kDa form (unpublished observation). A number of other isoforms with alternative N-terminal sequences are predicted from transcripts characterized in mammalian species. Differences in the coding sequences of some of these may impact on the regulation of their activity or subcellular localization. The transcripts described in Section 3.1.2 could potentially give rise to at least four N-terminal variants of mammalian ACC-a. Due to the relatively small differences in molecular weight between these forms, and their possible low abundance, based on transcript levels, they cannot easily be distinguished by gel electrophoresis (Fig. 4). The 265 kDa isoform appears, again on the basis of transcript abundance, to be the major isoform and is therefore the best characterized (see above). In the E5A variant [60] the 75 amino acids encoded by E5 in the 265 kDa isoform are replaced by 17 amino acids encoded by E5A, thus removing Ser-23, -25 and -29 which are phosphorylated in vivo. Apart from the possible involvement of Ser-29 in the effect of insulin, these residues have no known role and therefore the effect of their removal is unknown. This alternative N-terminal sequence also modifies the AMPK consensus motif around Ser-79 to a less ideal fit, and could therefore possibly allow this isoform to remain active in situations in which the 265 kDa isoform was inhibited. The relatively hydrophilic E5A N-terminus is unlikely to target this isoform to a membrane, though it may interact with other cellular components. A number of transcripts have a sequence which would result in the first in-frame AUG being found in E6. If this isoform is translated it would not only have lost the three

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most N-terminal Ser residues, as in E5A, but also the context enabling the phosphorylation of Ser-79 by AMPK. This could result in a constitutively active form of ACC-a, which even if expressed at low level, could be highly functionally significant. In addition, depending on the exons following it, translation could occur from an AUG in E1, for example in the context of E[1/4/5/6 etc.] and even if cryptic splice sites in E5 or E4 are used, generating shorter sequences termed ED5 and ED4 [48], these maintain the reading frame. This longer isoform obviously contains the cluster of phosphorylation sites found in the N-terminus of the 265 kDa isoform, plus additional Ser residues which, although not in consensus motifs for AMPK or PKA, could potentially be substrates for other kinases. This relatively hydrophobic extended N-terminus could also interact with other molecules, though at present its existence as a protein in vivo remains to be confirmed. 4.3.2. Phosphorylation of ACC-b ACC-h has been shown to be phosphorylated in vivo and in vitro by AMPK and PKA [74,160]. The sequence context around Ser-29, -77, and -79 of ACC-a are conserved in ACC-h in a number of species, including fish, and although Ser-23 is also conserved its sequence context is slightly different (Fig. 3). Ser-25, -1200, and -1215 are not conserved, however the N-terminal extension of ACC-h contains numerous other Ser/Thr residues with potential for involvement in the regulation of its activity. The conservation of both Ser-77 and -79 and the surrounding sequence in ACC-h, together with the fact that an anti-phospo(Ser-79)-ACC-a antibody also recognises ACC-h [208], suggests that this isozyme may be regulated by AMPK in a similar manner to ACC-a. Winder et al. [172] have also shown that incubation of ACC from skeletal muscle, which is predominantly ACC-h, with AMPK in vitro decreases the V max and increases the K act citrate of the enzyme. ACC-h appears to be a better substrate for PKA in vitro than ACC-a, being phosphorylated by this kinase on multiple sites, however this had no direct effect on enzyme activity [172,209]. One proteolytic peptide was phosphorylated by both PKA and AMPK, reminiscent of the peptide containing Ser-77 and -79 which, as stated above, is conserved in ACC-h. The activation of PKA in intact cardiac myocytes using the h-adrenergic agonist isoprenaline results in the phosphorylation of the same tryptic peptides as were phosphorylated by PKA in vitro, in contrast to the situation found for ACC-a. Again, no changes in kinetic parameters were observed, however with ACC-h it has been hypothesised that phosphorylation may alter the association of the enzyme with mitochondria (Section 4.4.1). Unlike that from skeletal muscle, ACC from heart tissue is reported to be inactivated by PKA [172,210]. ACC-h in these tissues has also been reported to be of different size: 272 kDa in muscle, and 280 kDa in heart. It is clear further work is needed to explain these

discrepancies and to more fully examine the role of phosphorylation in the regulation of this isozyme. 4.3.2.1. Phosphorylation of isoforms of ACC-b. Compared to ACC-a, little has been done on characterizing transcripts from the ACACB. When ACC-h was first cloned [33] a transcript encoding an isoform lacking 100 amino acids was described, however the availability of the gene sequence now suggests this to have been an artefact (Section 3.2.3). Two other transcripts which could result in isoforms of ACC-h have been characterized. One, identified in a human EST database, originates from PIII, and has now been found to be expressed in tissues and cell lines from a number of species (unpublished data). If translated this would give rise to an isoform of ACC-h in a similar manner in which the E5A variant of ACC-a is generated; the N-terminal 218 residues of the 280 kDa isoform, encoded by E3, would be replaced by a 15 amino acid sequence encoded by E4. This would, as in ACC-a, alter the sequence adjacent to the Ser220 and -222 and could therefore affect the regulation of this isoform. In addition transcripts from PI and PII in which E3 has been spliced-out have their first AUG in E5, and therefore, like the ACC-a isoform which starts in E6, would lack all Ser/Thr residues N-terminal to Ser-222, which itself is no longer in an appropriate context to be phosphorylated by AMPK, and could therefore be constitutively active. Replacement of the N-terminus of the 280 kDa isoform, containing the hydrophobic region thought to target the protein to mitochondria, with a 15 amino acid sequence which is relatively hydrophilic, or indeed with no alternative sequence, makes it unlikely that these shorter isoforms would also target to mitochondria. The existence of nonmitochondrially associated ACC-h isoforms, together with the existence of several potential isoforms of ACC-a, suggests that the regulation of ACC may be even more complex than previously imagined. 4.3.3. Dephosphorylation of ACC Treatment of purified ACC with phosphatase leads to increased activity [211,212], and in vivo activation in response to insulin or glucose is associated with a decrease in total phosphate content of the enzyme [127,213,214], although increased phosphorylation at specific sites (see above) [185,186]. Net dephosphorylation is therefore associated with a change in the balance of activity of specific kinases and phosphatases, although some groups also postulate the presence of an allosteric effector mediating the effects of insulin [202–204]. Using inhibitors, it was determined that the protein Ser/ Thr phosphatase involved in the in vivo regulation of ACC was of the PP2A class [215,216]. A glutamate/Mg2+ dependent phosphatase responsible for the activation of ACC was then identified in the liver. Activation by glutamate resulted in dephosphorylation of Ser-79 of ACC-a, but not of other substrates of PP2A, e.g. phosphorylase [217,218]. PP2A enzymes consist of a common catalytic subunit and a

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regulatory subunit, and can sometimes also acquire a third subunit, as in cardiac and skeletal muscle. The effects of glutamate appear to be mediated through the regulatory subunit, whereas Mg2+ is thought to have a different mechanism. All the multimeric forms of PP2A are stimulated to some degree by glutamate and Mg2+ [217,218]. Glutamate/Mg2+ dependent ACC phosphatases have now been identified in liver [217,218], heart and white adipose tissue [176], and pancreatic islets [219]. It is possible that the same subtype of PP2A is involved in the activation of ACC in all of these tissues, and therefore of both isozymes. Phosphorylation of the I-peptide and Ser-29 do not appear to play a major role in the activation of ACC by insulin. Recently it has been shown that the transcription factor ChREBP, involved in mediating the effects of glucose on gene expression, is activated by a PP2A subtype, A/By/ C, which is in turn activated by the glucose metabolite xyulose 5-phosphate, produced by the pentose phosphate pathway [110]. The P1A promoter of ACC-a is a target of ChREBP [96], and this therefore represents a mechanism for the chronic regulation of ACC activity. As insulin potentiates glucose uptake and flux and therefore activation of this phosphatase, it may be possible that this phosphatase could also play a part in the acute regulation of ACC. 4.4. Interactions of mammalian ACCs with other proteins and lipids As discussed above (Section 2.3.2) large regions of ACC sequence not directly involved in catalytic function are highly conserved. These regions may be involved in as yet uncharacterized interactions with different cellular structures and proteins. 4.4.1. The interaction of ACC-b with mitochondria The association of ACC-h with mitochondria is assumed to involve the 25 hydrophobic residues at the extreme Nterminus [33,34,38], although this has yet to be verified experimentally. Although this region may span the mitochondrial outer membrane, there is no charged residue near the N-terminus which would serve to anchor it and its association could equally involve protein–protein interactions. Kim [38] has postulated that positive charges introduced by phosphorylation downstream of this may reversibly tether the protein to negatively charged membrane phospholipids. ACC-h has also been found in the cytosol, e.g. in liver [47], however it is not clear whether this is due to dissociation from mitochondria during purification, reflects a phosphorylation-regulated association with mitochondria, or indeed one of the potential isoforms which lack some of the phosphorylation motifs. 4.4.2. Association of ACC with other cellular components ACC has been found associated with a number of proteins. AMPK was originally identified as a contaminant of ACC preparations [74,210,220]. This kinase consists of

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three subunits, a/h/g. In mammals, each subunit is encoded by two or three genes (a1, a2, h1, h2, g1, g2, g3) [221]. The a2-isoform was identified as being tightly bound to ACC in rat heart and muscle [173,210], however this does not exclude the involvement of the a1-isoform in phosphorylation. Indeed others have suggested that the a1-isoform may be associated with ACC in liver [71]. ACC association may be mediated indirectly through one or both of the unidentified non-catalytic h- and g-subunits of AMPK [210]. Protein phosphatases were also originally thought to associate tightly with ACC, maintaining their association during purification [222,223]. However this was not the case and identification of the ACC phosphatases was carried out in other ways (Section 4.3.3) [216–218]. Another surprising partner for ACC is the predominately nuclear-localised protein BRCA1, mutations in which are associated with an increased susceptibility to breast and ovarian cancer [224]. This protein was found to interact with ACC-a in vitro through its BRCT domains, which occur in a number of proteins and bind phosphoproteins [225]. BRCT domains harbouring mutations which occur in patients disrupted the association with ACC. The relevance of this interaction is unclear, however BRCA1 has recently been shown to undergo nuclear-cytoplasmic shuttling [226]. Obviously there is now a need to demonstrate that this association occurs in vivo. A small fraction of ACC has been found associated with the cytoskeleton in hepatocytes [227]. Cytoskeletal components have also been proposed to regulate hepatic CPT I in a malonyl-CoA independent manner [228], and the localised production of malonyl-CoA from a cytoskeletal pool of ACC obviously has implications that warrant further investigation [229]. In oligodendrocytes approximately 10% of the cytoplasmic ACC activity, assumed to be ACC-a, is associated with the myelin fraction [230], some with the intra-lamellar cytoplasmic spaces, and a second more firmly attached fraction which is resistant to extraction with high salt. This group suggests that as a proportion of FAS, and other lipogenic enzymes, are also found in this salt-resistant fraction, that this is a site of myelin lipid synthesis. It is possible that a specific ACC could associate and form the core of a lipid generating enzyme structure, but a characterization of the ACC isoforms in these different fractions is necessary, particularly in light of the potential for nonmitochondrial ACC-h. Two recent proteomic analyses of lipid droplet associated proteins have identified ACC-a, though not a specific isoform [231,232], and an inactive form of ACC has been reported in bovine milk-fat globule membrane [233]. It is not yet clear whether the ACC is associated directly with the phospholipid monolayer surrounding the lipid droplets [234], or interacts with other droplet membrane proteins. With respect to function this may appear an obvious location for lipid synthesizing enzymes, though neither analysis identified the presence of FAS. It is of note that the

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N-terminus of the bnormalQ 265 kDa form of ACC-a does not appear to target GFP to any intracellular membrane [34]. The origins and functions of the ACC in the lipid droplet and milk-fat globule membrane are therefore still obscure. ACC was not found among lipid droplet associated proteins of yeast [235], possibly suggesting that its role is specific to the workings of multicellular organisms.

5. Concluding remarks The evolution of vertebrates, especially mammals, is accompanied by increasing complexity in the structure and regulation of two paralogous ACC genes that maintain distinct cellular pools of malonyl-CoA that direct the reciprocal regulation of fatty acid synthesis and h-oxidation in tissues. The use of multiple promoters and alternative exon splicing in both ACC genes generates an array of mRNA isoforms and the potential for isozymic variants that may allow increased flexibility for controlling malonyl-CoA levels in particular cells or directing the synthesis of the metabolite in distinct cellular compartments. This is particularly evident in humans. It is apparent that multiple isozymic variants of ACC are also encoded in the genomes of invertebrates. As invertebrates also encode CPT-I [13] this may indicate that the functional specialisation of both mammalian ACC genes are potentially operable in these simpler organisms. Thus consideration of the function of the invertebrate ACC isozymes in terms of tissue-expression, mutant phenotypes, subcellular location of isozymes, interacting proteins and coexpressed genes may yield valuable insight into the distinct roles of the ACC genes in the physiology of mammals. An increasing awareness of the regulatory importance of malonyl-CoA in cellular processes that become dysregulated in insulin resistance, obesity and type-2 diabetes [236,237] has heralded a renewed interest in mammalian ACC. Insight gained from the Acacb null mouse [35] and recent experiments using isozyme non-selective inhibitors of ACC that target the carboxytransferase domain [238,239] have demonstrated the utility of targeting ACC isozymes in the treatment of the metabolic syndrome. A drive for more selective ACC inhibitors is likely to herald new efforts in the area of structure-function studies of ACC isozymes, which to date have lagged behind those of other important proteins. Further insight may also be gained by in-depth phylogenetic analysis of the domain structure of ACC enzymes, which at present is limited by the availability of complete ACC gene sequences, especially those from dintermediateT organisms such as the sea squirt.

Acknowledgements Research in the author’s laboratories is supported by the Scottish Executive.

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