Preflagellin Peptidase

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Chapter 64

Preflagellin Peptidase DATABANKS MEROPS name: preflagellin peptidase MEROPS classification: clan AD, family A24, subfamily A24B, peptidase A24.016 Tertiary structure: Available Species distribution: order Methanococcales Reference sequence from: Methanococcus maripaludis

Name and History Archaea have a novel assortment of cell surface organelles [1 3], many constructed utilizing a bacterial type IV pilus model, i.e. composed of proteins initially synthesized with type IV pilin-like signal peptides (class 3 signal peptides [4]) processed by a specific signal peptidase homologous to the type IV prepilin peptidase (TFPP) of type IV pili systems widespread in bacteria [5]. Archaeal surface structures made with these types of subunits include, surprisingly, flagella [6,7], an archaeal-specific version of type IV pili [8,9], Iho fibers of Ignicoccus hospitalis [10] and a novel membrane structure of Sulfolobus solfataricus called the bindosome which is composed of sugar binding proteins [11]. The archaeal flagellum, the best studied of archaeal surface structures, is a novel motility apparatus [7] that is evolutionarily distinct from its bacterial namesake [6,12] and instead resembles bacterial type IV pili in many respects [6]. In bacteria, flagellins are synthesized without signal peptides and new flagellins are secreted through a type III secretion system located at the base, passing through the hollow flagella structure for assembly at the distal tip [13,14]. Archaeal flagellins share both N-terminal sequence homology to type IV pilins [15] and class 3 signal peptides, which are removed at the cytoplasmic membrane by a TFPP. This fact and the observation that archaeal flagella lack an internal channel large enough to allow passage of newly made flagellins [16,17] suggests strongly that the archaeal flagella are synthesized with incorporation of new flagellin subunits at the base [18]. Archaeal TFPPs that cleave the type IV pilin-like signal peptides from the structural proteins that comprise the archaeal surface structures have been named either

preflagellin peptidases (FlaK [19]) for their initial discovery in the processing of archaeal flagellins, the major subunits of archaeal flagella or as peptidases involved in the biogenesis of pilus-like proteins (PibD) for their role in processing an extended substrate range in addition to flagellins in certain archaea such as S. solfataricus [20] and Haloferax volcanii [21]. Archaeal preflagellin peptidases are members of an unusual clan of aspartic acid proteases that includes bacterial type IV prepilin peptidases (Chapter 63) and the eukaryotic protease, presenilin (Chapters 65, 66) [22,23]. Cleavage of the signal peptides occurs on the cytoplasmic side of the cytoplasmic membrane. TFPPs are widespread in Archaea, even among nonflagellated members, attesting to the expanded role outside of an involvement in flagella biosynthesis. In S. solfataricus, PibD processes not only flagellin, but also subunits that compose unusual UV inducible pili and sugar-binding proteins that are predicted to form a piluslike membrane structure, termed the bindosome [9,20,24]. In H. volcanii, a single enzyme (PibD) is also involved in processing both flagellins and other pilin-like proteins [21]. Unusually, two different TFPP-like enzymes are found in Methanococcus. In M. maripaludis, both pili and flagella are made from type IV pilin like proteins and each type of subunit has its own dedicated TFPP [25]. FlaK processes only flagellins and EppA processes only pilins, even though strong similarities in the signal peptides and cleavage sites are found in both proteins. Why M. maripaludis possesses two TFPPs, while other archaea utilize a single enzyme to cleave a variety of type IVpilin-like substrates, is currently unknown.

Activity and Specificity Typically, bacterial TFPPs are bifunctional enzymes: they remove the signal peptide and then methylate the resulting N-terminal amino acid (usually phenylalanine) [5]. The two activities are located in different domains of the enzyme. Recently, non-methylating TFPPs, lacking the N-terminal methylase domain, have been reported in bacteria [26]. Archaeal TFPPs have been shown to cleave type IV pilin-like signal peptides but they do not modify the N-terminal amino acid following signal peptide

Other Clans of Aspartic Peptidases | 64. Preflagellin Peptidase

removal [27]. No kinetic parameters of preflagellin peptidases have been determined.

Activity Assay: Substrate and Enzyme Sources for Assay In Vitro An in vitro assay for preflagellin peptidase activity was developed based on the bacterial prepilin peptidase assay [28 30]. The substrate was a preflagellin from Methanococcus voltae that was expressed in Escherichia coli. Since E. coli lacks the corresponding signal peptidase activity necessary to remove the signal peptide, the preflagellin form only exists in the E. coli cells, where it localizes to the membrane. These membrane preparations are used as substrate in the assay with membrane preparations of various methanococci as enzyme source. Processing of preflagellin to flagellin was followed by Western blot appearance of a faster migrating form (Figure 64.1). N-terminal sequencing of both processed and unprocessed flagellin demonstrated that correct processing had occurred [28]. Subsequently, identification of the putative preflagellin gene by bioinformatics led to overexpression of the suspected gene, later named flaK, in E. coli and the use of these E. coli membranes as source of enzyme in the in vitro assay [19,31]. Similar experiments identified PibD as a TFPP in S. solfataricus [20,32]. It was only recently that a purified histagged version of FlaK was used in a similar in vitro assay [23].

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while the 22 and 23 positions are typically charged amino acids, usually basic amino acids (lysine or arginine), although, especially in extreme halophiles, these positions can be held by acidic amino acids [34]. The 13 position in archaeal flagellins is always glycine. Site-directed mutagenesis studies aimed at investigating the importance of these conserved flagellin amino acids in the FlaK cleavage reaction were conducted, with processing of the mutant substrates assayed in vitro (Table 64.1). For M. voltae FlaB2, changing the 1 glycine to other amino acids tested (glutamic acid, phenylalanine, or arginine) resulted in a loss of signal peptide cleavage [33]. Poor partial processing was observed when alanine was substituted at this position, a finding also reported for bacterial prepilin peptidase [35]. When the 22 lysine was substituted, a complete loss in processing resulted, while changing the 23 lysine resulted in partial processing. Replacement of the invariant 13 glycine with

TABLE 64.1 Influence of site-directed amino acid changes in the FlaB2 flagellin on its ability to serve as a FlaK substrate. The signal peptide and N-terminal sequence of flagellin FlaB2 of Methanococcus voltae is shown below. Arrow indicates signal peptide cleavage site k MKIKEFMSNKKG

ASGIGTL *

Site-Directed Mutagenesis of Preflagellin Peptidase Substrates

Site-directed change

Cleavage of mutated substrate compared to wild-type

G-1A

Partial

The sequence conservation observed around the cleavage site of archaeal preflagellins suggested that these amino acids were likely important for cleavage by the preflagellin peptidase [33]. Examination of all archaeal flagellins demonstrated that the 21 position of the signal peptide (relative to the cleavage site) is almost always a glycine

G-1F

None

G-1E

None

G-1R

None

K-2E

None

K-2A

None

K-2R

Comparable

K-2N

None

K-3E

Partial

K-3A

Partial

F-7A

Partial

G 13V

None

Shortened signal peptide (LP10, 9, 8, 7, 5)

Comparable

Shortened signal peptide (LP6, 4, 3)

None

Flak mutant 1

2

3

4

Wild type 5

6

7

8

9

10 Preflagellin Flagellin

FIGURE 64.1 In vitro assay of FlaK activity. Membranes from M. maripaludis wild-type or flaK mutant cells, as source of preflagellin peptidase, were added to membranes of E. coli containing heterologously expressed methanogen preflagellin [28,29]. Samples were removed at various time points (0, 2, 10, 30 and 60 min) and appearance of the smaller, processed flagellin detected by Western blot. Arrows indicate the unprocessed and processed flagellin species. No processing occurs in the flaK mutant. Bars indicate position of molecular mass markers, 25 and 15 KDa.

*Amino acid numbering is relative to the cleavage site.

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valine led to no processing of the mutant preflagellin by FlaK. Since several archaeal flagellins were predicted to have extremely short signal peptides, studies were conducted to examine if signal peptide length influenced the proper processing of substrates by FlaK. M. voltae FlaB2 proteins with truncated signal peptides were generated, leaving the conserved amino acids around the cleavage site unaltered. All the mutant proteins with a signal peptide length shortened from 12 to five amino acids were processed significantly, except for a signal peptide length of 6 [33]. However, flagellins with a signal peptide length at or below four amino acids were not processed, suggesting that a minimal signal peptide length was crucial for FlaK activity. S. solfataricus PibD has a wider substrate range compared to FlaK, including natural substrates with much shorter signal peptides than those found on FlaK substrates [20]. When PibD was used as the TFPP in the in vitro assay with the heterologous methanococcal FlaB2 substrates with varied signal peptides, PibD was able to recognize and cleave not only the signal peptides cleaved by FlaK but also the shorter ones that were not processed by FlaK, indicating the inherent ability of PibD to accommodate a wider variety of substrates including ones with widely varying lengths in their signal peptides that would not be FlaK substrates. Attempts to complement a M. maripaludis flaK deletion strain with pibD were unsuccessful, for while the flagellins were processed, the cells could not assemble them into flagella on their surface [36]. In S. solfataricus, signal peptide mutants of preGlcS (the precursor form of glucose-binding protein), a natural substrate for PibD, were also constructed [20]. This study focused on the conserved residues around the cleavage site (Table 64.2). A large number of substitutions around the cleavage site in the preGlcS were tolerated without affecting the in vitro cleavage reaction [20]. These data, as well as the extended substrate range of PibD compared to FlaK and the ability of PibD to cleave signal peptides of widely varying lengths, all indicate that PibD is much more flexible in its ability to accept and process substrates.

Search for Other Potential Substrates With the realization in S. solfataricus that nonflagellar proteins, like sugar-binding proteins, were made with class 3 signal peptides, studies were undertaken to screen for proteins with class 3 signal peptides in other archaeal genomes. For this purpose, a PERL program called FlaFind was developed [25,37] which identified 388 proteins in 22 archaeal genomes with 102 of them annotated

TABLE 64.2 Influence of site-directed amino acid changes in the glucose binding protein on its ability to serve as a PibD substrate. The signal peptide and Nterminal sequence of glucose binding protein (preGlcS) of Sulfolobus solfataricus is shown below. The arrow indicates signal peptide cleavage site k MKRKYPYSLAKG

LTSTQIA *

Site-directed change

Cleavage of mutated substrate compared to wild-type

G-1A

Slight improvement

G-1L

None

G-1R

None

L 11I

Comparable

L 11F

Comparable

L 11R

None

L 11D

None

G-1A plus L 11I

Significant

K-2A

Comparable

K-2D

None

A-3K

Comparable

*Amino acid numbering is relative to the cleavage site.

with predicted functions, mainly flagellins and substratebinding proteins. FlaFind positives bear a structural similarity to type IV pilins and the co-localization of various FlaFind positives with genes involved in type IV pili assembly suggest that they are subunits of archaeal surface structures. When FlaFind positives were analyzed for sequence conservation, the highest sequence similarity was seen within the N-terminus, a region known to be essential for assembly of type IV pili [38]. An important discovery was the identification of 19 euryarchaeal proteins with a domain of unknown function (DUF361), containing the amino acid motif QXSXEXXXL, where Q is conserved at the 11 position relative to the predicted cleavage site. Several such genes were found in the same operon with a gene encoding a novel TFPP, called EppA, in M. maripaludis. The three DUF361-containing genes in this operon were subsequently all shown to be involved in pili formation [39]. The processing of the DUF361-containing proteins expressed in E. coli was possible only when EppA was co-expressed [25]. EppA processing did not extend to flagellin subunits, suggesting differences in the substrate specificity for FlaK and EppA, seemingly involving at

Other Clans of Aspartic Peptidases | 64. Preflagellin Peptidase

least the 11 position which in the pilins is glutamine. A further demonstration of this was done by replacing the amino acids from 22 to 12 in the EppA-dependent pilinlike proteins EpdA and EpdC with those of FlaB2 and vice versa. Due to the absence of the conserved glutamine at position 11, EppA was not able to cleave the modified EpdA (KGAS) and EpdC (KGAS), but could process the modified FlaB2 (RGQI). FlaK could still cleave the modified FlaB2 but not the modified pilins that had the flagellin amino acids at the cleavage site, indicating a broader cleavage site recognition for FlaK compared to EppA. It seems likely that this may include the universally conserved 13 glycine residue of flagellins [33].

Structural Chemistry Site-Directed Mutagenesis of Preflagellin Peptidases Typical bacterial TFPPs, like TcpJ of Vibrio cholerae, are predicted to have eight transmembrane domains (TMs) while archaeal TFPPs, such as FlaK and PibD, have six TMs which are similar to the C-terminus of bacterial TFPPs. EppA is unusual in having an extra four TMs. An unusual, large cytoplasmic loop exists between the last two TM domains in archaeal TFPPs that is missing in bacterial TFPPs [23,31,32]. The two catalytic aspartic acid residues in archaeal TFPPs are located between TM1 and 2 and between TM3 and 4, with the latter locating within a highly conserved GxGD motif (Table 64.3). In M. voltae FlaK, the two aspartic acid residues are Asp18 and Asp79. Site-directed mutations leading to Asp18Ala, Asp18Asn, Asp79Ala or Asp79Asn resulted in a loss of enzyme activity. A conservative substitution of

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Asp79Glu, however, was still active. In S. solfataricus PibD, the corresponding aspartic acid residues are Asp23 and Asp80. Consistent with the findings in FlaK, substituting either of these two aspartic acid residues with alanine inactivated the enzyme, while enzyme with a Asp80Glu conservative substitution remained partly active. Although several other conserved aspartic acid residues were also found by sequence alignment, aspartic acid to alanine substitutions of these in either FlaK or PibD had no affect on enzyme activity [31,32]. In M. maripaludis FlaK, the loss of activity was also observed in two aspartic acid mutations, Asp18Asn and Asp79Asn. Mutations of amino acids which are adjacent to the catalytic aspartic acid residues and in another conserved region near TM6 in the C-terminus had no significant effect on enzyme activity, except for two: a decrease of enzyme activity was observed in a Gly76Ala mutation and increased activity observed in a Pro208Ala mutation [23]. The site-directed amino acid changes and their effects on enzyme activity for the various archaeal TFPPs are summarized in Table 64.4.

Crystal Structure of FlaK Recently, the crystal structure of M. maripaludis FlaK has been solved [23]. Consistent with previous topology predictions [31,32], this archaeal TFPP indeed contains six TM α-helices and a soluble cytoplasmic loop between α5 and α6. The crystal structure indicated that the TM segments 4 and 6 are very short and TM6 may not be able to fully cross the membrane. Between α4 and α5 and the Cterminus, there are two amphipathic structures (including α4a and α6a), in which all the polar side chains face the hydrophilic extracellular side, whereas most of the

TABLE 64.3 Partial alignment of archaeal prepilin-like signal peptidase, along with sample bacterial prepilin peptidases, including the nonmethylating TadV, indicating sequences around the catalytically important aspartic acids (shown in bold) Bacteria EC_PppA

SAWLIAASVIDLDHQWLPDVFT

D143

LRKEALGMGDVLLFAALGGWVG

D208

PA_PilD

TWGLLAMSLIDADHQLLPDVLV

D149

TGKEGMGYGDFKLLAMLGAWGG

D213

AA_TadV

LLLLITLSVTDIRSRLISNRVV

D23

FSLHFIGAGDVKLVSVLMLAVP

D77

MM_EppA

FLLILTATYTDIKERIIPHFVI

D26

ILGVGMGGGDVKMFTALSPLFA

D88

MM_FlaK

VIGLLLASVQDFRSREIEDYIW

D18

MFLSGIGGGDGKILIGLGALVP

D79

HV_PibD

LPVLAWTAVRDVRTRRVPNVWY

D26

WRLGGFGGADAKALMVFAVLLP

D94

SS_PibD

IIMLIHTSILDLKYREVDPKIW

D23

YKLSLLGGADLFLNVILSLANA

D80

Archaea

EC, Escherichia coli; PA, Pseudomonas aeruginosa; AA, Aggegatibacter actinomycetemcomitans; MM, Methanococcus maripaludis; HV, Haloferax volcanii; SS, Sulfolobus solfataricus.

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TABLE 64.4 Effect of site-directed mutations in M. maripaludis FlaK on its cleavage activity using M. voltae FlaB2 as substrate* Site-directed change

Activity compared to wild-type substrate

D18N

None

R20A

Normal

R22A

Significantly reduced

E23A

Normal

E25A

Normal

D26A

Normal

E23A 1 E25A 1 D26A

Normal

W29A

Normal

G76A

Significant decrease

G77A

Normal

G78A

Normal

D79N

None

K81A

Normal

D184A 1 D185A

Normal

D184A 1 D185A 1 E186A 1 D187A

Normal

P201A

Normal

Q202A

Normal

I203A

Normal

P204A

Normal

L205A

Normal

I206A

Normal

I207A

Normal

P208A

Increased

*The original site-detected mutagenesis on FlaK was done on M. voltae FlaK. Here changes of D18A, D18N, D79A and D79N lead to no activity while D79E resulted in normal activity. Changes D186A (equivalent to D185 in M. maripaludis FlaK), D190A (equivalent to D187 in M. maripaludis FlaK) and D224A (equivalent to D221 in M. maripaludis FlaK) all resulted in normal activity. In S. solfataricus PibD, single mutations of D23A, D23E and D80A lead to an inactive enzyme while D80E restored part of the activity. Other single mutations including D30A, D157A, D173A, D180A, D187A, D188A and D207N all resulted in normal cleavage activity. However, a double mutation of D187A/D188A resulted in no activity.

nonpolar side chains interact with other helices or the lipid. Furthermore, a hydrogen bond between the conserved Asn120 (in α5) and Gly220 (in α6a) fixes a particular angle between α5 and α6a. These two characteristics make the protein tilted in the cytoplasmic membrane, so that the charged groups are properly positioned to avoid the hydrophobic lipid bilayer, leaving TM6 virtually perpendicular to the membrane.

According to the crystal structure (without substrate), the two catalytic aspartic acid residues (Asp18 and ˚ gap by the Asp79) are surprisingly uncoupled with a 12 A spatial relationship of the two helices. Preventing movement of Asp18 and Asp79 by cross-linking TM2 and TM6 left the enzyme inactive, implying conformation switches may occur during catalysis. The crystal structure of FlaK explained the unusual catalytic activity observed in mutant versions of FlaK harboring the Gly76Ala and Pro208Ala mutations (see above). In the case of Gly76Ala, considering that there is no obvious steric hindrance around this site, it was supposed that loss of backbone flexibility resulted in the large decrease of activity. The N-terminus of TM6 was shown from the crystal structure to be located near the active site. In the Pro208Ala mutation in the N-terminus which greatly increased enzyme activity, the packing between TM5 and TM6 might be altered by the substitution. If the conformational change needed for catalysis were to include movement of TM6, this altered packing of TM5 and TM6 might be responsible for the increased activity. The FlaK crystal structure revealed that the archaeal enzyme had an active site similar to that of the eukaryotic member of this protease clan, presenilin.

Purification Recently, purification of a His-tagged Se-Met version of FlaK used in crystallization studies was reported [23]. The protein was purified, following expression of a Histagged version in Escherichia coli, using metal affinity column chromatography after solubilization of the enzyme from the membrane with foscholine (final concentration of 1%), with a final purification through Sephadex 200 column. This procedure yielded 3 mg of FlaK from 1 liter of cells.

Biological Aspects Roles of Archaeal TFPPs in Surface Structures Deletion analysis has shown the essential role of TFPPs in Methanococcus surface structure synthesis. Deletion of flaK leads to nonflagellated but still piliated cells while deletion of eppA leads to nonpiliated cells that are still flagellated [31,40]. Double mutants, deleted for both flaK and eppA lack both surface structures (Figure 64.2) [40]. Cells lacking either or both surface appendages were unable to attach to a number of surfaces indicating a role for both pili and flagella in attachment in M. maripaludis [40]. Deletion of flaK allowed isolation of pili free from flagella contamination, leading to their structural identification as a unique prokaryotic pilus [8] and biochemical

Other Clans of Aspartic Peptidases | 64. Preflagellin Peptidase

(A)

(B)

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Related Peptidases Preflagellin peptidases are polytopic GxGD-type aspartyl proteases, which also include prepilin peptidases, signal peptide peptidases and presenilin [22,42]. The amino acid sequence homology among these proteases is poor and limited mainly to a few domains involved in catalysis, including the highly conserved GxGD motif [42].

(C)

(D)

Further Reading For further information on preflagellin peptidase, see Albers & Driessen [43], Albers et al. [44], Bardy et al. [45,46], Jarrell [47], Jarrell et al. [48,49], Lory & Strom [50] and Zolghadr et al. [51]. FIGURE 64.2 Electron micrographs of wild-type M. maripaludis cells (A) as well as cells deleted for eppA (B), flaK (C) and both eppA and flaK (D). Flagella (large arrow heads) as well as pili (thin arrows) are observed on wild-type cells while only flagella are seen on eppA cells, only pili (arrows) are found on flaK cells and neither appendage is seen on the double mutant. Cells are negatively stained with 2% phosphotungstic acid. Bar equal 200 nm. Courtesy of Meg Stark and James Chong.

identification of the major component, MMP1685, a small glycoprotein [39]. Deletion of pibD in H. volcanii prevented preflagellin processing leading to a non-swimming phenotype while at the same time eliminating the ability of H. volcanii cells to adhere to glass [21]. Since flagella in this organism are not involved in adherence, the disruption of glass adherence in pibD mutants suggested a role for a nonflagellar, type IV pilus-like structure in surface attachment in this halophile. Deletion of pibD has not been possible in S. solfataricus, suggesting PibD is required for processing of certain protein(s) critical for survival in this thermoacidophile [2].

Distinguishing Features Several traits distinguish archaeal preflagellin peptidases. First and foremost are the substrates which include archaeal flagellins, type IV pilins and sugar-binding proteins [27]. The closest relatives of preflagellin peptidases are bacterial prepilin peptidases which cleave signal peptides from type IV pilins but never from flagellins which in bacteria are never made as preproteins. Prepilin peptidases are bifunctional enzymes which remove the signal peptide and also methylate the resulting N-terminal amino acid [5]. Preflagellin peptidases lack this methylating capacity. Finally, the motifs recognized by prepilin peptidases and preflagellin peptidases, while similar, are different [41].

References [1] Albers, S.V., Meyer, B.H. (2011). The archaeal cell envelope. Nat. Rev. Microbiol. 9, 414 426. [2] Albers, S.V., Pohlschroder, M. (2009). Diversity of archaeal type IV pilin-like structures. Extremophiles 13, 403 410. [3] Ng, S.Y.M., Zolghadr, B., Driessen, A.J.M., Albers, S.V., Jarrell, K.F. (2008). Cell surface structures of archaea. J. Bacteriol. 190, 6039 6047. [4] Pohlschroder, M., Gimenez, M.I., Jarrell, K.F. (2005). Protein transport in archaea: sec and twin arginine translocation pathways. Curr. Opin. Microbiol. 8, 713 719. [5] Strom, M.S., Nunn, D.N., Lory, S. (1993). A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl. Acad. Sci. USA 90, 2404 2408. [6] Jarrell, K.F., VanDyke, D.J., Wu, J. (2009). Archaeal flagella and pili, in: Pili and Flagella: Current Research and Future Trend, Jarrell, K.F., ed., Norfolk, UK: Caister Academic Press, pp. 215 234. [7] Thomas, N.A., Bardy, S.L., Jarrell, K.F. (2001). The archaeal flagellum: a different kind of prokaryotic motility structure. FEMS Microbiol. Rev. 25, 147 174. [8] Wang, Y.A., Yu, X., Ng, S.Y.M., Jarrell, K.F., Egelman, E.H. (2008). The structure of an archaeal pilus. J. Mol. Biol. 381, 456 466. [9] Frols, S., Ajon, M., Wagner, M., Teichmann, D., Zolghadr, B., Folea, M., Boekema, E.J., Driessen, A.J., Schleper, C., Albers, S.V. (2008). UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation. Mol. Microbiol. 70, 938 952. [10] Muller, D.W., Meyer, C., Gurster, S., Kuper, U., Huber, H., Rachel, R., Wanner, G., Wirth, R., Bellack, A. (2009). The Iho670 fibers of Ignicoccus hospitalis: a new type of archaeal cell surface appendage. J. Bacteriol. 191, 6465 6468. [11] Zolghadr, B., Klingl, A., Rachel, R., Driessen, A.J., Albers, S.V. (2011). The bindosome is a structural component of the Sulfolobus solfataricus cell envelope. Extremophiles 15, 235 244. [12] Ng, S.Y., Chaban, B., Jarrell, K.F. (2006). Archaeal flagella, bacterial flagella and type IV pili: a comparison of genes and posttranslational modifications. J. Mol. Microbiol. Biotechnol. 11, 167 191.

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Ken F. Jarrell Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Email: [email protected]

Yan Ding Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Email: [email protected]

Divya B. Nair Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Email: [email protected] © 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00064-8

Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2

Lumen/extracellular APP

Presenilin

β Aβ D

D

γ ε Cytosol

AICD

FIGURE 65.1 Presenilin, the γ-secretase complex, and the proteolysis of APP to Aβ. Presenilin is processed into two pieces, an N-terminal fragment (NTF, dark portion) and a C-terminal fragment (CTF, light portion) that remain associated. Each fragment donates one aspartate essential for γ-secretase activity (arrows near these aspartates denote Nto C-terminal directionality of the protein sequence). APP is first cleaved in the extracellular domain by β-secretase, and the remnant is cleaved at least twice within the membrane (at sites γ and ε) by γ-secretase to produce the Aβ peptide of Alzheimer’s disease and the intracellular domain (AICD).