- Email: [email protected]
PUPylation: something old, something new, something borrowed, something Glu
Update Research Focus
PUPylation: something old, something new, something borrowed, something Glu George N. DeMartino Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390.9040, USA
Most eukaryotic proteins are degraded by the 26S proteasome as a consequence of their covalent modification with ubiquitin. Although the proteasome is found in some prokaryotes, ubiquitin is not, which indicates that substrates are targeted to prokaryotic proteasomes by a fundamentally different mechanism. A recent study has identified Pup (prokaryotic ubiquitin-like protein) as a mycobacterial protein that functions in a manner analogous to ubiquitin for proteasome-dependent proteolysis in prokaryotes.
The ubiquitin-proteasome system of protein degradation Protein degradation is a pervasive mechanism for regulating levels of constituent cellular proteins and the processes they mediate [1]. The degradation of most proteins in eukaryotic cells is catalyzed by the ubiquitin-proteasome system (UPS), which targets proteins for destruction by the 26S proteasome as a consequence of covalent modification by a polymer of ubiquitin, a highly conserved 8 000-dalton protein [2]. Ubiquitin conjugation occurs via an ‘isopeptide’ bond formed between the carboxyl of the C-terminal glycine of ubiquitin and the e-amino group of a target protein lysine and is catalyzed by the concerted action of three types of proteins: an ATP-dependent ubiquitin activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3 [3]. Multiple E2 and E3 proteins provide specificity for selective ubiquitylation of different protein targets. Protein modification by a single ubiquitin is a weak degradation signal, but isopeptide bond formation between C termini of additional ubiquitin moieties and resident lysines of the conjugated ubiquitin results in the tandem conjugation of ubiquitins to one another [4]. Such polyubiquitin ‘chains’ with four or more ubiquitins linked via lysine 48 are potent signals for degradation of their client proteins because they are targeted efficiently to the 26S proteasome [5]. The 26S proteasome is a 2 500 000-dalton protease complex composed of two functionally distinct subcomplexes: the 20S proteasome and PA700 (also known as the 19S regulatory particle) [6,7]. The 20S proteasome is a ‘self-compartmentalized’ protease, of which the catalytic sites are located within a central lumen of its cylindrical structure [8]. Substrates reach these sites after passing through reversibly gated narrow pores at the ends of the Corresponding author: DeMartino, G.N. ([email protected]).
cylinder [8,9]. PA700 (19S) is a 20-subunit ATPase that binds to the 20S proteasome at one or both ends of the 20S cylinder and regulates gate opening. It also provides other functions required for proteolysis including substrate binding (via the modifying polyubiquitin chain), unfolding (to enable substrate transit through the narrow entry pores), translocation (substrate transit through the open pores and to the degradation chamber) and deubiquitylation (to ease substrate translocation and to recycle ubiquitin). The overall process of 26S-proteasome-mediated protein degradation requires continuous ATP hydrolysis catalyzed by six AAA (ATPases associated with various cellular activities)-family PA700 subunits arranged as a heterohexameric ring that contact the ends of the 20S cylinder [10]. Proteasomes in non-eukaryotes 20S proteasomes are universally distributed and highly conserved among eukaryotic cells and are present in an architecturally similar, but genetically simpler, form in archaea and some but not all eubacteria [11,12]. However, neither PA700 nor ubiquitin has been identified in noneukaryotes leading to the assumption that novel ubiquitin-independent mechanisms are used for substrate targeting to and degradation by their proteasomes. Ubiquitin-independent mechanisms have been identified for substrate selection by other eubacterial proteases including ClpP, a non-proteasomal self-compartmentalized protease that associates with any of several homohexameric AAA protein complexes [13]. True 20S proteasomes in non-eukaryotic cells also can associate with homohexameric AAA complexes, the best characterized of which is PAN (proteasome activating nucleotidase) from Methanococcus janaschii [14]. Like PA700, PAN binds to the ends of the proteasome to promote proteolysis by inducing gate opening and substrate unfolding and translocation to sequestered catalytic sites, but the mechanism by which substrates are recognized remains unclear [15,16]. Pupylation: a novel covalent protein modification for proteolysis Darwin and colleagues [17,18] recently have defined features of the Mycobacterium tuberculosis (Mtb) proteasome, whose normal function is required for Mtb resistance to nitric oxide and related compounds produced by macrophages to inhibit bacterial growth. In screens for Mtb mutants that attenuate this resistance, they identified two proteins, Mpa and PafA, that mediate 155
Update the proteasome-dependent effect [19]. Mpa (mycobacterium proteasomal ATPase) is an AAA-family protein that forms a homohexameric ring in a manner similar to the rings formed by the AAA subunits of eukaryotic PA700 and other non-eukaryotic ATPase proteasome regulators [20]. Thus, Mpa also might bind to the proteasome. PafA (proteasome accessory factor A) is structurally related to g-glutamyl-cysteine synthetase-2, indicating that it is member of the carboxylate-amine ligase superfamily [21]. Both Mpa and PafA, like the proteasome itself, are required for the degradation of certain Mtb proteins such as FabD (malonyl co-A acyl carrier protein) [18]. In a two-hybrid screen for Mpa-interacting proteins, Darwin and colleagues [22] identified Rv2111c (subsequently termed ‘Pup’ for prokaryotic ubiquitin-like protein) and confirmed this interaction by biochemical analysis. Pup is a 6 900-dalton, 64-amino-acid protein that interacted reversibly with Mpa. Pup also interacted with FabD when tagged constructs of these proteins were expressed in Mycobacterium smegmatis, but this interaction had features of a covalent complex such as might be formed between a proteasome substrate and its modifying ubiquitin. Despite similar sizes, Pup and ubiquitin have no overall sequence similarity. Moreover, the Pup C terminus ends in Gly-Gly-Gln, whereas mature ubiquitin and other ubiquitin-like proteins have conserved and functionally essential C-terminal Gly-Gly residues that are generated by post-translational processing of ubiquitin from various precursors [23]. Mass spectral analysis of the FabD–Pup complex revealed that Pup was attached to FabD via an isopeptide bond between a C-terminal Glu of Pup and Lys173 of FabD. These results established the covalent nature of the interaction but indicated that the Pup C-terminal Gln was deamidated in the conjugated complex. Thus, Pup modification (Pupylation) is analogous to, but distinct from, features of ubiquitin modification. Several lines of evidence strongly indicate that pupylation is obligatory for FabD degradation. First, Pup-modified FabD accumulated in mpa mutants, presumably as a consequence of inhibited proteasomal degradation. Accumulation of ubiquitylated proteins is a hallmark of proteasome inhibition in eukaryotic cells. The lack of a discrete modified form of FabD indicates, but does not prove, that it undergoes multiple modifications, possibly representing poly-Pup chains. Second, Pup–FabD conjugates were not detected in pafA Mtb strains although unmodified FabD accumulated. These results provide additional support for a role of PafA in Pup- and proteasome-dependent protein degradation and in conjunction with proteomic analysis indicate a direct and possibly sufficient role for PafA in pupylation [21]. Third, the Lys173Ala FabD mutant was stabilized significantly. This result establishes the importance of a specific lysine as the site of substrate modification, as is the case for many ubiquitin-dependent substrates. The accumulation of multiple pupylated proteins in mpa Mtb cells indicates that Pup-dependent proteasomal degradation is not limited to FabD. In total, these experiments demonstrate that certain Mtb proteins are targeted for proteasomal degradation after covalent modification with the novel protein, Pup. 156
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Concluding remarks and future perspectives The discovery of Pup dispels the prevailing view that ubiquitin-like systems for protein modification and proteasome-dependent degradation are absent in non-eukaryotic cells. This seminal finding raises many questions analogous to those either previously answered or currently posed for ubiquitin and ubiquitin-like systems in eukaryotes. Although the first established function of ubiquitin was as a regulator of protein degradation, subsequent work has revealed its roles in a broad array of processes [24]. Does Pup share such functional diversity and mediate nonproteolytic processes? Emerging results indicate that both the number of ubiquitins conjugated to a protein and the type of ubiquitin–ubiquitin linkage might encode distinct functions. Does Pup form polymeric chains, and if so are they structurally and functionally diverse from one another and from mono-pupylation? Impressive progress has been achieved in defining the enzymology and mechanisms of ubiquitin conjugation. What proteins catalyze pupylation? Does PafA have a direct role in pupylation and is it sufficient for this process? Although the structural identification of PafA as a carboxylate-amine ligase indicates that PafA catalyzes ATP-dependent pupylation, direct experimental evidence will be required to confirm this speculation and to determine whether other proteins are involved. What is the distinction between isopeptide bond formation using Gly (for ubiquitin) and Glu (for Pup), and what is the importance of the apparent requirement for deamidation of Gln in this process? What features of target proteins dictate selection for modification by the pupylation machinery? Multiple non-AAA subunits of PA700 bind to polyubiquitin chains. What components of the prokaryotic proteasome recognize and bind to Pup? Eukaryotes contain numerous proteasomal and non-proteasomal proteins that remove ubiquitin from proteins and disassemble chains, and deubiquitylation is mechanistically linked to, and obligatory for, efficient proteolysis by the 26S proteasome [25]. Do corresponding de-pupylating proteins exist and what are their roles? Ubiquitin was the founding member of an eponymous protein family, the members of which feature common mechanisms of conjugation but utilize dedicated enzymatic machineries and mediate distinct biological processes [23,25] (Figure 1). Is there a family of Pup-like proteins and, if so, do the members exist in eukaryotes and in eubacteria? Ubiquitin-like proteins share only marginal similarities in primary structures but assume very similar tertiary structures. Thus, direct experimental evidence or deeper in silico analysis will be required to address this question. The UPS is the dominant system for protein degradation in eukaryotes and is utilized to regulate most aspects of cellular function. What is the relative role of Pup- and proteasome-dependent proteolysis in the regulation of cellular functions compared with other proteolytic systems? The answers to these and many other questions raised by this work will provide a new understanding of mechanisms for regulation of protein degradation in non-eukaryotic cells and for post-translational modifications that regulate cellular function.
Update
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Vol.34 No.4
Figure 1. General features and comparisons of mechanisms of ubiquitylation and pupylation. Ubiquitin (Ub) and Pup are covalently conjugated to e-amino groups of target proteins via isopeptide bonds with C-terminal carboxyl groups of glycine and glutamate, respectively. Features of pupylation that remain undetermined compared with corresponding features of ubiquitylation are denoted with question marks and include: the enzymatic machinery for Pup conjugation; the formation of poly-Pup chains; the mechanisms of proteasomal recognition; the de-pupylation of substrates. Ubiquitin conjugation is catalyzed by three proteins (E1, E2 and E3), whereas pupylation might require only PafA. Polyubiquitin chains are recognized by the 26S proteasome that degrades the client protein and recycles ubiquitin. The role of poly-pupylation in substrate targeting to the proteasome remains unclear.
Acknowledgements Work in our laboratory is supported by grants from the National Institutes of Health (DK 46181; www.nih.gov) and the Welch Foundation (I-500; www.welch1.org). I thank Thomas Gillette, Brajesh Kumar, Kim Orth, Sohini Mukherjee and anonymous reviewers for helpful comments.
References 1 Glickman, M.H. and Ciechanover, A. (2002) The ubiquitin-proteasome system: destruction for the sake of construction. Physiol. Rev. 82, 373– 428 2 Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 3 Pickart, C.M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 530–533 4 Hochstrasser, M. (2006) Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34 5 Thrower, J.S. et al. (2001) Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 6 Gillette, T.G. and DeMartino, G.N. (2007) Proteasomes: machines for all reasons. Cell 129, 659–662
7 Voges, D. et al. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015– 1068 8 Bochtler, M. et al. (1999) The proteasome. Annu. Rev. Biophys. Biomol. Struct. 28, 295–317 9 Groll, M. et al. (2000) A gated channel into the proteasome core particle. Nat. Struct. Biol. 11, 1062–1067 10 Pickart, C.M. and Cohen, R.E. (2004) Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 11 Groll, M. et al. (2005) Molecular machines for protein degradation. ChemBioChem 6, 222–256 12 Butler, S.M. et al. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553–562 13 Martin, A. et al. (2005) Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 14 Zwickl, P. et al. (1999) An archaebacterial ATPase, homologous to ATPases in the eukaryotic 26 S proteasome, activates protein breakdown by 20 S proteasomes. J. Biol. Chem. 274, 26008–26014 15 Benaroudj, N. et al. (2003) ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69–78 157
Update 16 Smith, D.M. et al. (2007) Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s a ring opens the gate for substrate entry. Mol. Cell 27, 731–744 17 Darwin, K.H. et al. (2003) The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 18 Pearce, M.J. et al. (2006) Identification of substrates of the Mycobacterium tuberculosis proteasome. EMBO J. 25, 5423–5432 19 Festa, R.A. et al. (2007) Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis. J. Bacteriol. 189, 3044–3050 20 Darwin, K.H. et al. (2005) Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue. Mol. Microbiol. 55, 561–571
Trends in Biochemical Sciences Vol.34 No.4 21 Iyer, L.M. et al. (2008) Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination. Biol. Direct 3, 45 22 Pearce, M.J. et al. (2008) Ubiquitin-like protein involved in the proteasome pathway of mycobacterium tuberculosis. Science 322, 1104–1107 23 Hochstrasser, M. (2000) All in the ubiquitin family. Science 289, 563– 564 24 Mukhopadhyay, D. and Riezman, H. (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315, 201–205 25 Amerik, A.Y. and Hochstrasser, M. (2004) Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta 1695, 189–207 0968-0004/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2008.12.005 Available online 11 March 2009
Research Focus
H2A.Z and DNA methylation: irreconcilable differences Michael S. Kobor1 and Matthew C. Lorincz2 1
Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada 2 Department of Medical Genetics, Life Sciences Institute, Room 5-507, University of British Columbia, 2350 Health Sciences Mall, Vancouver BC, V6T 1Z3, Canada
DNA methylation state and the composition of the nucleosome core particle influence chromatin structure and, in turn, transcriptional competence. Although it is clear that chromatin remodeling and covalent histone modifications regulate DNA methylation in plants and animals, the role of histone variants in directing DNA methylation, and vice versa, has not been addressed. A new genome-wide study in Arabidopsis thaliana reveals a broadly antagonistic relationship between H2A.Z occupancy and DNA methylation.
Nucleosomes, histone variants and DNA methylation In eukaryotes, DNA is intimately associated with the nucleosome core particle, which typically comprises the canonical histones H2A, H2B, H3 and H4. Several complexes with ATP-dependent chromatin-remodeling and/or histone-modifying activities act on these histones to orchestrate transcription [1,2]. Replacement of the core histones with variants of histones H2A or H3, which differ in amino acid sequence from the canonical histones [3] and are typically deposited by replication-independent chromatin assembly complexes [4], has emerged as another potential means of gene regulation. The histone variant H2A.Z is deposited in yeast by the Swi2/Snf2-related ATP-dependent nucleosome remodeling complex SWR1-C [5] and in plants and animals by related complexes [6–8]. In contrast to canonical H2A, H2A.Z is deposited predominantly at the 50 end of genes [7,9]; this is consistent with its proposed role in transcriptional regulation [6,7,10,11]. By contrast, DNA methylation, which commonly inhibits transcription, is excluded from the promoter regions of genes [12–14]. Surprisingly, although ATP-dependent chromatin remodeling complexes and covalent histone modifications are known to Corresponding author: Lorincz, M.C. ([email protected]).
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modulate DNA methylation in plants and/or animals [15– 22] (Table 1), the role of histone variants in regulating DNA methylation has not been addressed. Employing a series of elegant genome-wide analyses, the Zilberman and Henikoff laboratories now provide compelling evidence that in Arabidopsis thaliana, H2A.Z and DNA methylation not only occupy distinct genomic regions, but actually exclude one another from specific genomic neighborhoods [23]. H2A.Z enrichment inversely correlates with DNA methylation To survey the genome-wide distribution of H2A.Z, chromatin isolated from root tissue was treated with micrococcal nuclease, and mononucleosomes were subjected to chromatin immunoprecipitation (ChIP) with
Glossary Bisulfite sequencing: a sequencing-based method for determining DNA methylation status at single nucleotide resolution. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Associated changes in DNA sequence that depend on the methylation status of cytosine residues are revealed by PCR of the genomic region of interest followed by sequencing of the amplification products. Combined bisulfite restriction analysis (COBRA): a restriction endonucleasebased method for determining DNA methylation status at specific restriction sites. Methylation-dependent sequence differences are introduced into the genomic DNA by sodium bisulfite treatment and then PCR amplified. Specific restriction sites harboring unmethylated cytosines are lost, whereas those harboring methylated cytosines are retained. Digestion of the amplification product of interest with specific restriction endonucleases followed by electrophoresis reveals the initial methylation status of the restriction site surveyed. Methyl-DNA immunoprecipitation (meDIP): an immunoprecipitation-based method for determining DNA methylation status at a genome-wide level. Genomic DNA is sheared by sonication or digested with a restriction endonucleases, denatured and immunoprecipitated using a monoclonal antibody specific for 5-methylcytosine. Immunoprecipitated DNA is purified and hybridized to a DNA microarray, revealing those regions in the genome that are enriched for DNA methylation.
PUPylation: something old, something new, something borrowed, something Glu George N. DeMartino Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390.9040, USA
Most eukaryotic proteins are degraded by the 26S proteasome as a consequence of their covalent modification with ubiquitin. Although the proteasome is found in some prokaryotes, ubiquitin is not, which indicates that substrates are targeted to prokaryotic proteasomes by a fundamentally different mechanism. A recent study has identified Pup (prokaryotic ubiquitin-like protein) as a mycobacterial protein that functions in a manner analogous to ubiquitin for proteasome-dependent proteolysis in prokaryotes.
The ubiquitin-proteasome system of protein degradation Protein degradation is a pervasive mechanism for regulating levels of constituent cellular proteins and the processes they mediate [1]. The degradation of most proteins in eukaryotic cells is catalyzed by the ubiquitin-proteasome system (UPS), which targets proteins for destruction by the 26S proteasome as a consequence of covalent modification by a polymer of ubiquitin, a highly conserved 8 000-dalton protein [2]. Ubiquitin conjugation occurs via an ‘isopeptide’ bond formed between the carboxyl of the C-terminal glycine of ubiquitin and the e-amino group of a target protein lysine and is catalyzed by the concerted action of three types of proteins: an ATP-dependent ubiquitin activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3 [3]. Multiple E2 and E3 proteins provide specificity for selective ubiquitylation of different protein targets. Protein modification by a single ubiquitin is a weak degradation signal, but isopeptide bond formation between C termini of additional ubiquitin moieties and resident lysines of the conjugated ubiquitin results in the tandem conjugation of ubiquitins to one another [4]. Such polyubiquitin ‘chains’ with four or more ubiquitins linked via lysine 48 are potent signals for degradation of their client proteins because they are targeted efficiently to the 26S proteasome [5]. The 26S proteasome is a 2 500 000-dalton protease complex composed of two functionally distinct subcomplexes: the 20S proteasome and PA700 (also known as the 19S regulatory particle) [6,7]. The 20S proteasome is a ‘self-compartmentalized’ protease, of which the catalytic sites are located within a central lumen of its cylindrical structure [8]. Substrates reach these sites after passing through reversibly gated narrow pores at the ends of the Corresponding author: DeMartino, G.N. ([email protected]).
cylinder [8,9]. PA700 (19S) is a 20-subunit ATPase that binds to the 20S proteasome at one or both ends of the 20S cylinder and regulates gate opening. It also provides other functions required for proteolysis including substrate binding (via the modifying polyubiquitin chain), unfolding (to enable substrate transit through the narrow entry pores), translocation (substrate transit through the open pores and to the degradation chamber) and deubiquitylation (to ease substrate translocation and to recycle ubiquitin). The overall process of 26S-proteasome-mediated protein degradation requires continuous ATP hydrolysis catalyzed by six AAA (ATPases associated with various cellular activities)-family PA700 subunits arranged as a heterohexameric ring that contact the ends of the 20S cylinder [10]. Proteasomes in non-eukaryotes 20S proteasomes are universally distributed and highly conserved among eukaryotic cells and are present in an architecturally similar, but genetically simpler, form in archaea and some but not all eubacteria [11,12]. However, neither PA700 nor ubiquitin has been identified in noneukaryotes leading to the assumption that novel ubiquitin-independent mechanisms are used for substrate targeting to and degradation by their proteasomes. Ubiquitin-independent mechanisms have been identified for substrate selection by other eubacterial proteases including ClpP, a non-proteasomal self-compartmentalized protease that associates with any of several homohexameric AAA protein complexes [13]. True 20S proteasomes in non-eukaryotic cells also can associate with homohexameric AAA complexes, the best characterized of which is PAN (proteasome activating nucleotidase) from Methanococcus janaschii [14]. Like PA700, PAN binds to the ends of the proteasome to promote proteolysis by inducing gate opening and substrate unfolding and translocation to sequestered catalytic sites, but the mechanism by which substrates are recognized remains unclear [15,16]. Pupylation: a novel covalent protein modification for proteolysis Darwin and colleagues [17,18] recently have defined features of the Mycobacterium tuberculosis (Mtb) proteasome, whose normal function is required for Mtb resistance to nitric oxide and related compounds produced by macrophages to inhibit bacterial growth. In screens for Mtb mutants that attenuate this resistance, they identified two proteins, Mpa and PafA, that mediate 155
Update the proteasome-dependent effect [19]. Mpa (mycobacterium proteasomal ATPase) is an AAA-family protein that forms a homohexameric ring in a manner similar to the rings formed by the AAA subunits of eukaryotic PA700 and other non-eukaryotic ATPase proteasome regulators [20]. Thus, Mpa also might bind to the proteasome. PafA (proteasome accessory factor A) is structurally related to g-glutamyl-cysteine synthetase-2, indicating that it is member of the carboxylate-amine ligase superfamily [21]. Both Mpa and PafA, like the proteasome itself, are required for the degradation of certain Mtb proteins such as FabD (malonyl co-A acyl carrier protein) [18]. In a two-hybrid screen for Mpa-interacting proteins, Darwin and colleagues [22] identified Rv2111c (subsequently termed ‘Pup’ for prokaryotic ubiquitin-like protein) and confirmed this interaction by biochemical analysis. Pup is a 6 900-dalton, 64-amino-acid protein that interacted reversibly with Mpa. Pup also interacted with FabD when tagged constructs of these proteins were expressed in Mycobacterium smegmatis, but this interaction had features of a covalent complex such as might be formed between a proteasome substrate and its modifying ubiquitin. Despite similar sizes, Pup and ubiquitin have no overall sequence similarity. Moreover, the Pup C terminus ends in Gly-Gly-Gln, whereas mature ubiquitin and other ubiquitin-like proteins have conserved and functionally essential C-terminal Gly-Gly residues that are generated by post-translational processing of ubiquitin from various precursors [23]. Mass spectral analysis of the FabD–Pup complex revealed that Pup was attached to FabD via an isopeptide bond between a C-terminal Glu of Pup and Lys173 of FabD. These results established the covalent nature of the interaction but indicated that the Pup C-terminal Gln was deamidated in the conjugated complex. Thus, Pup modification (Pupylation) is analogous to, but distinct from, features of ubiquitin modification. Several lines of evidence strongly indicate that pupylation is obligatory for FabD degradation. First, Pup-modified FabD accumulated in mpa mutants, presumably as a consequence of inhibited proteasomal degradation. Accumulation of ubiquitylated proteins is a hallmark of proteasome inhibition in eukaryotic cells. The lack of a discrete modified form of FabD indicates, but does not prove, that it undergoes multiple modifications, possibly representing poly-Pup chains. Second, Pup–FabD conjugates were not detected in pafA Mtb strains although unmodified FabD accumulated. These results provide additional support for a role of PafA in Pup- and proteasome-dependent protein degradation and in conjunction with proteomic analysis indicate a direct and possibly sufficient role for PafA in pupylation [21]. Third, the Lys173Ala FabD mutant was stabilized significantly. This result establishes the importance of a specific lysine as the site of substrate modification, as is the case for many ubiquitin-dependent substrates. The accumulation of multiple pupylated proteins in mpa Mtb cells indicates that Pup-dependent proteasomal degradation is not limited to FabD. In total, these experiments demonstrate that certain Mtb proteins are targeted for proteasomal degradation after covalent modification with the novel protein, Pup. 156
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Concluding remarks and future perspectives The discovery of Pup dispels the prevailing view that ubiquitin-like systems for protein modification and proteasome-dependent degradation are absent in non-eukaryotic cells. This seminal finding raises many questions analogous to those either previously answered or currently posed for ubiquitin and ubiquitin-like systems in eukaryotes. Although the first established function of ubiquitin was as a regulator of protein degradation, subsequent work has revealed its roles in a broad array of processes [24]. Does Pup share such functional diversity and mediate nonproteolytic processes? Emerging results indicate that both the number of ubiquitins conjugated to a protein and the type of ubiquitin–ubiquitin linkage might encode distinct functions. Does Pup form polymeric chains, and if so are they structurally and functionally diverse from one another and from mono-pupylation? Impressive progress has been achieved in defining the enzymology and mechanisms of ubiquitin conjugation. What proteins catalyze pupylation? Does PafA have a direct role in pupylation and is it sufficient for this process? Although the structural identification of PafA as a carboxylate-amine ligase indicates that PafA catalyzes ATP-dependent pupylation, direct experimental evidence will be required to confirm this speculation and to determine whether other proteins are involved. What is the distinction between isopeptide bond formation using Gly (for ubiquitin) and Glu (for Pup), and what is the importance of the apparent requirement for deamidation of Gln in this process? What features of target proteins dictate selection for modification by the pupylation machinery? Multiple non-AAA subunits of PA700 bind to polyubiquitin chains. What components of the prokaryotic proteasome recognize and bind to Pup? Eukaryotes contain numerous proteasomal and non-proteasomal proteins that remove ubiquitin from proteins and disassemble chains, and deubiquitylation is mechanistically linked to, and obligatory for, efficient proteolysis by the 26S proteasome [25]. Do corresponding de-pupylating proteins exist and what are their roles? Ubiquitin was the founding member of an eponymous protein family, the members of which feature common mechanisms of conjugation but utilize dedicated enzymatic machineries and mediate distinct biological processes [23,25] (Figure 1). Is there a family of Pup-like proteins and, if so, do the members exist in eukaryotes and in eubacteria? Ubiquitin-like proteins share only marginal similarities in primary structures but assume very similar tertiary structures. Thus, direct experimental evidence or deeper in silico analysis will be required to address this question. The UPS is the dominant system for protein degradation in eukaryotes and is utilized to regulate most aspects of cellular function. What is the relative role of Pup- and proteasome-dependent proteolysis in the regulation of cellular functions compared with other proteolytic systems? The answers to these and many other questions raised by this work will provide a new understanding of mechanisms for regulation of protein degradation in non-eukaryotic cells and for post-translational modifications that regulate cellular function.
Update
Trends in Biochemical Sciences
Vol.34 No.4
Figure 1. General features and comparisons of mechanisms of ubiquitylation and pupylation. Ubiquitin (Ub) and Pup are covalently conjugated to e-amino groups of target proteins via isopeptide bonds with C-terminal carboxyl groups of glycine and glutamate, respectively. Features of pupylation that remain undetermined compared with corresponding features of ubiquitylation are denoted with question marks and include: the enzymatic machinery for Pup conjugation; the formation of poly-Pup chains; the mechanisms of proteasomal recognition; the de-pupylation of substrates. Ubiquitin conjugation is catalyzed by three proteins (E1, E2 and E3), whereas pupylation might require only PafA. Polyubiquitin chains are recognized by the 26S proteasome that degrades the client protein and recycles ubiquitin. The role of poly-pupylation in substrate targeting to the proteasome remains unclear.
Acknowledgements Work in our laboratory is supported by grants from the National Institutes of Health (DK 46181; www.nih.gov) and the Welch Foundation (I-500; www.welch1.org). I thank Thomas Gillette, Brajesh Kumar, Kim Orth, Sohini Mukherjee and anonymous reviewers for helpful comments.
References 1 Glickman, M.H. and Ciechanover, A. (2002) The ubiquitin-proteasome system: destruction for the sake of construction. Physiol. Rev. 82, 373– 428 2 Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 3 Pickart, C.M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 530–533 4 Hochstrasser, M. (2006) Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34 5 Thrower, J.S. et al. (2001) Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 6 Gillette, T.G. and DeMartino, G.N. (2007) Proteasomes: machines for all reasons. Cell 129, 659–662
7 Voges, D. et al. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015– 1068 8 Bochtler, M. et al. (1999) The proteasome. Annu. Rev. Biophys. Biomol. Struct. 28, 295–317 9 Groll, M. et al. (2000) A gated channel into the proteasome core particle. Nat. Struct. Biol. 11, 1062–1067 10 Pickart, C.M. and Cohen, R.E. (2004) Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5, 177–187 11 Groll, M. et al. (2005) Molecular machines for protein degradation. ChemBioChem 6, 222–256 12 Butler, S.M. et al. (2006) Self-compartmentalized bacterial proteases and pathogenesis. Mol. Microbiol. 60, 553–562 13 Martin, A. et al. (2005) Rebuilt AAA+ motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 14 Zwickl, P. et al. (1999) An archaebacterial ATPase, homologous to ATPases in the eukaryotic 26 S proteasome, activates protein breakdown by 20 S proteasomes. J. Biol. Chem. 274, 26008–26014 15 Benaroudj, N. et al. (2003) ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69–78 157
Update 16 Smith, D.M. et al. (2007) Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s a ring opens the gate for substrate entry. Mol. Cell 27, 731–744 17 Darwin, K.H. et al. (2003) The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 18 Pearce, M.J. et al. (2006) Identification of substrates of the Mycobacterium tuberculosis proteasome. EMBO J. 25, 5423–5432 19 Festa, R.A. et al. (2007) Characterization of the proteasome accessory factor (paf) operon in Mycobacterium tuberculosis. J. Bacteriol. 189, 3044–3050 20 Darwin, K.H. et al. (2005) Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue. Mol. Microbiol. 55, 561–571
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Research Focus
H2A.Z and DNA methylation: irreconcilable differences Michael S. Kobor1 and Matthew C. Lorincz2 1
Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada 2 Department of Medical Genetics, Life Sciences Institute, Room 5-507, University of British Columbia, 2350 Health Sciences Mall, Vancouver BC, V6T 1Z3, Canada
DNA methylation state and the composition of the nucleosome core particle influence chromatin structure and, in turn, transcriptional competence. Although it is clear that chromatin remodeling and covalent histone modifications regulate DNA methylation in plants and animals, the role of histone variants in directing DNA methylation, and vice versa, has not been addressed. A new genome-wide study in Arabidopsis thaliana reveals a broadly antagonistic relationship between H2A.Z occupancy and DNA methylation.
Nucleosomes, histone variants and DNA methylation In eukaryotes, DNA is intimately associated with the nucleosome core particle, which typically comprises the canonical histones H2A, H2B, H3 and H4. Several complexes with ATP-dependent chromatin-remodeling and/or histone-modifying activities act on these histones to orchestrate transcription [1,2]. Replacement of the core histones with variants of histones H2A or H3, which differ in amino acid sequence from the canonical histones [3] and are typically deposited by replication-independent chromatin assembly complexes [4], has emerged as another potential means of gene regulation. The histone variant H2A.Z is deposited in yeast by the Swi2/Snf2-related ATP-dependent nucleosome remodeling complex SWR1-C [5] and in plants and animals by related complexes [6–8]. In contrast to canonical H2A, H2A.Z is deposited predominantly at the 50 end of genes [7,9]; this is consistent with its proposed role in transcriptional regulation [6,7,10,11]. By contrast, DNA methylation, which commonly inhibits transcription, is excluded from the promoter regions of genes [12–14]. Surprisingly, although ATP-dependent chromatin remodeling complexes and covalent histone modifications are known to Corresponding author: Lorincz, M.C. ([email protected]).
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modulate DNA methylation in plants and/or animals [15– 22] (Table 1), the role of histone variants in regulating DNA methylation has not been addressed. Employing a series of elegant genome-wide analyses, the Zilberman and Henikoff laboratories now provide compelling evidence that in Arabidopsis thaliana, H2A.Z and DNA methylation not only occupy distinct genomic regions, but actually exclude one another from specific genomic neighborhoods [23]. H2A.Z enrichment inversely correlates with DNA methylation To survey the genome-wide distribution of H2A.Z, chromatin isolated from root tissue was treated with micrococcal nuclease, and mononucleosomes were subjected to chromatin immunoprecipitation (ChIP) with
Glossary Bisulfite sequencing: a sequencing-based method for determining DNA methylation status at single nucleotide resolution. Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Associated changes in DNA sequence that depend on the methylation status of cytosine residues are revealed by PCR of the genomic region of interest followed by sequencing of the amplification products. Combined bisulfite restriction analysis (COBRA): a restriction endonucleasebased method for determining DNA methylation status at specific restriction sites. Methylation-dependent sequence differences are introduced into the genomic DNA by sodium bisulfite treatment and then PCR amplified. Specific restriction sites harboring unmethylated cytosines are lost, whereas those harboring methylated cytosines are retained. Digestion of the amplification product of interest with specific restriction endonucleases followed by electrophoresis reveals the initial methylation status of the restriction site surveyed. Methyl-DNA immunoprecipitation (meDIP): an immunoprecipitation-based method for determining DNA methylation status at a genome-wide level. Genomic DNA is sheared by sonication or digested with a restriction endonucleases, denatured and immunoprecipitated using a monoclonal antibody specific for 5-methylcytosine. Immunoprecipitated DNA is purified and hybridized to a DNA microarray, revealing those regions in the genome that are enriched for DNA methylation.