Ubiquitination System: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Rita Xu and Version 1 by Vera Vozandychova.

Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host–pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. 

  • ubiquitination
  • deubiquitinating enzymes (DUBs)
  • effector protein
  • host–pathogen interaction

1. Introduction

Competition between host defense mechanisms and pathogens’ effective tools has been observed since ancient times. The immune system of the host organism is regulated by complex metabolic and signaling pathways, including, but not limited to, a network of post-translational modifications. Should any of these important pathways be impaired, such as by efficient mechanisms of pathogens, the immune balance is disrupted and pathogens may become more successful. During their development, pathogenic organisms have acquired several mechanisms by which they can specifically influence the immune response of the host and thus escape their defense mechanisms and prevent themselves from destruction. Better understanding how pathogens affect the host immune system will facilitate the development of more effective therapeutic agents against diseases caused by pathogenic microorganisms. This review will be devoted to one of the post-translational modifications, ubiquitination, in connection with host–pathogen interaction. Ubiquitination is a crucial mechanism in numerous cell processes, such as protein degradation, innate immune signaling, protein–protein interactions, and others. Many intracellular pathogens intervene through the ubiquitination system into the host’s innate immune response. Ubiquitination, in addition to acetylation and phosphorylation, is becoming another massively studied modification in connection with host–pathogen interactions. The importance of investigating this post-translational modification in the context of the host–pathogen interaction is constantly increasing, particularly due to the availability of new and sensitive approaches for analyzing ubiquitination.

2. Ubiquitin System

Ubiquitination is a significant, reversible post-translational modification managing numerous cellular processes. This modification mediates not only the degradation of proteins but also ensures proper protein function, protein–protein interaction, and subcellular localization [1].

More than 500 proteins possess the ability to recognize ubiquitin (Ub) and Ub-like molecules. These proteins participate in connecting Ub to a specific substrate or its removal from the target molecule. The conjugation and deconjugation of Ub are broadly diverse and complicated processes that regulate many cellular pathways. Deubiquitinating enzymes (DUBs) have recently been attracting greater attention because these proteins are interesting in their function, which is involved in many cellular pathways [2].

Human Ub is a small (8 kDa) and abundant protein consisting of 76 amino acids and comprising as much as 5% of total protein within a cell. This protein is highly conserved among eukaryotic organisms. Monoubiquitination, which is the connection of a single Ub molecule by its C-terminal glycine (G76) to the lysine residue of a substrate protein, influences protein localization, DNA repair, endocytosis, virus budding, and protein–protein interaction [3]. The Ub molecule contains seven lysine residues—K6, K11, K27, K29, K33, K48, and K63—and each of these can be used for the creation of poly-Ub chains by conjugation of Ub molecules in a process known as polyubiquitination. Another type, the M1-linked Ub chain, can be formed by linkage of G76 of the connecting Ub to the N-terminal methionine of a Ub already attached in place of one of the lysine residues. Accordingly, this differential type of Ub connection or creation of poly-Ub chains results in various functions of the modified proteins [4,5][4][5]. In general, the ubiquitination can have a degradative or non-degradative purpose. Poly-Ub chains with Ub molecules bound via K48 or K11 determine substrates for proteasomal degradation [6,7][6][7]. A Ub chain attached via K63 has a regulatory function in several cell processes as a non-degradative signal, including in DNA reparation, signalization, endocytosis, vesicle transportation, and progression of the cell cycle [3,8][3][8]. It has also been reported that K33-linked chains are implicated in intracellular trafficking [9], and, together with K6-linked Ub chains, they are involved in DNA repair [10,11][10][11]. Several studies report that K6-linked Ub chains are related to the autophagy process, mitophagy, and xenophagy [12,13][12][13]. K29-linked chains have been observed in a proteasome regulation role [14,15][14][15] and in other epigenetic regulation in connectivity with the deubiquitinase Trabid [16,17][16][17]. The linear M1 ubiquitination is an essential modification in nuclear factor κB (NF-κB) activation [18]. Although the ubiquitin system is being intensively studied, the functions of all types of ubiquitination are not fully understood. Partly, it is because the diverse attachment alternatives give rise to a huge number of a possible complex poly-Ub chain structure (Figure 1).

Microorganisms 09 00638 g001 550

Figure 1. The ubiquitination of substrates may vary in different constructions that play a role in various cell processes: protein interaction and localization (A), proteasomal degradation (B), NF-κB activation (C,D), DNA repair (C,E,F), lysosomal targeting (C), autophagy (E), intracellular trafficking (F) and epigenetic regulation (G), and many others still unknown functions (H). S—substrate, Ub—ubiquitin. Adapted from [5].

In addition to Ub itself, other important molecules play vital roles in the ubiquitination processes. These are ubiquitin-like (Ubl) molecules, such as small ubiquitin-related modifier (SUMO), bacterial protein ThiS, and neural precursor cell expressed, developmentally downregulated 8 (NEDD8). Similar to Ub, these are involved in various cellular processes, such as transcriptional regulation, DNA repair, apoptosis, or protein stability [19,20][19][20]. Pathogens often target these small molecules during infection because they are involved in critical signaling pathways within the host cell and because disruption of the ubiquitination process could be advantageous to the pathogens [21].

2.1. Ubiquitination Process

The ubiquitination process consists of a cascade of enzymatic reactions depending on three key enzymes: Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub-ligase (E3). As mentioned above, ubiquitination is a reversible process, thus similar to some other post-translational modifications, so DUBs are also essential counterparts to these enzymes in the whole process. The initial step involves ATP-dependent Ub activation consisting in acyl-adenylation on the Ub C-terminus. The following part of the process includes transferring Ub to the E2 cysteine active site. The third enzyme, Ub-ligase, is able to recognize the target protein and most typically forms an isopeptide bond between G76 of Ub and lysine of the target protein (Figure 2). The individual key enzymes involved in the ubiquitination system will be described briefly for better understanding of the ubiquitination process.

Microorganisms 09 00638 g002 550

Figure 2. Scheme of ubiquitin binding to the target protein [22]. Ub is activated by ATP-assisted acyl-adenylation, bound to the Ub-activating enzyme (E1), transferred to Ub-conjugating enzyme (E2), and attached to the target protein substrate (S) through Ub-ligase (E3). This cycle mechanism can be repeated for the creation of a polyubiquitin chain. ATP—adenosine triphosphate, AMP—adenosine monophosphate, PPi—pyrophosphate, Ub—ubiquitin. Adapted from [22].

2.2. Enzymes Participating in Ubiquitin System

Many different enzymes are included in the ubiquitin system. Surprisingly, the enzymes and components of the ubiquitination system comprise about 5% of the human proteome [5]. These include two Ub-activating enzymes [23], more than 50 Ub-conjugating enzymes, and several hundred Ub-ligases. The enormous number of different ubiquitination enzymes and varying complexity of Ub chains enable the formation of a subtle and complex regulation system tunable on many regulation levels and consisting of different products [24].

2.3. Ubiquitin Ligases

Ub-ligases comprise the biggest group of enzymes involved in the ubiquitination process. Their variability enables their modifying a wide variety of substrates. The primary function of Ub-ligases is transferring Ub to the target protein substrate [4,25][4][25]. The Ub ligation enzymes are divided into three families according to the domain each contains: Really Interesting New Gene (RING), homologous with E6-associated protein C-terminus (HECT), and RING-Between-RING (RBR) [26]. RING-type ligases work as scaffolds between the E2 enzyme and the target protein substrate and thus allow ubiquitination of the protein [27]. HECT-type ligases are different from RING-type ligases in the forming of a covalent thioester bond with Ub before its transfer to the substrate [28,29][28][29]. RBR-type ligases have both the RING and HECT domains, and their hybrid mechanism of action has been described [30,31,32][30][31][32].

2.4. Deubiquitinating Enzymes

The other enzymes playing a vital role in the ubiquitination system are DUBs, which are able to remove Ub from target substrates. DUBs are involved in many cellular processes and pathways, such as gene expression, apoptosis, cell cycle, DNA repair, and cytokine signaling [33]. DUBs were discovered in the group of metalloproteases, and mainly in the group of cysteine proteases. More than 90 DUBs have been identified and described in the human genome [2]. Cysteine DUBs are categorized into six subclasses according to the character of their ubiquitin-protease domains: Ub-specific proteases (USPs); Ub C-terminal hydrolases (UCHs); Machado–Joseph disease protein domain proteases (MJDs); ovarian tumor proteases (OTUs) [2]; a group of enzymes containing motif for interaction with the Ub-containing novel DUB family, known as MINDY [34]; and the newest family, ZUFSP (zinc finger with UFM1 specific peptidase) [35]. DUBs that were found in the metalloprotease group contain a domain that is referred to as JAMM (JAB1/MPN/Mov34 metalloenzyme) [2].

DUBs have widespread mechanisms of action for releasing Ub from the substrate. The Ub molecule is encoded by four genes (UBC, UBB, UBA52, and UBA80), and it is expressed as a linear chain of multiple Ub molecules. Subsequently, it has to be cut by DUBs. DUBs are essential for cleavage of polyubiquitin chains and complete removal of Ub chains from ubiquitinated proteins. These mechanisms lead to the maintenance of ubiquitin homeostasis in the cell, reverse Ub signaling, and correction of Ub-protein conjugates [36].

In contrast to other proteases, DUBs are generated as active enzymes. The catalytic activity of DUB cysteine proteases depends on two or three crucial amino acid residues forming a dyad or triad. These essential amino acids are from the group consisting of cysteine, histidine, aspartate, and asparagine. A variety of structures with the catalytic activity of DUBs has been reported [36]. Regulation of DUB activity is mediated by post-translational modifications, allosteric interactions, and subcellular localization. The catalytic triad of some DUBs has to be converted to active conformation by binding to a substrate, otherwise it remains inactive [2,36][2][36].

DUBs are able to recognize many ubiquitin-like molecules, isopeptides, and linear peptides, as well as various types of Ub linkage and chain structures. All DUB catalytic domains have a primary Ub-binding domain that encompasses interaction with the distal Ub in a poly-Ub chain. Moreover, each lysine residue in Ub molecule has a unique sequence that can be recognized by DUBs [36].

References

  1. Pickart, C.M.; Eddins, M.J. Ubiquitin: Structures, Functions, Mechanisms. Biochim. Biophys. Acta Mol. Cell Res. 2004, 1695, 55–72.
  2. Nijman, S.M.B.; Luna-Vargas, M.P.A.; Velds, A.; Brummelkamp, T.R.; Dirac, A.M.G.; Sixma, T.K.; Bernards, R. A Genomic and Functional Inventory of Deubiquitinating Enzymes. Cell 2005, 123, 773–786.
  3. Haglund, K.; Dikic, I. Ubiquitylation and Cell Signaling. EMBO J. 2005, 24, 3353–3359.
  4. Komander, D.; Rape, M. The Ubiquitin Code. Annu. Rev. Biochem. 2012, 81, 203–229.
  5. Park, C.-W.; Ryu, K.-Y. Cellular Ubiquitin Pool Dynamics and Homeostasis. BMB Rep. 2014, 47, 475–482.
  6. Thrower, J.S.; Hoffman, L.; Rechsteiner, M.; Pickart, C.M. Recognition of the Polyubiquitin Proteolytic Signal. EMBO J. 2000, 19, 94–102.
  7. Xu, P.; Duong, D.M.; Seyfried, N.T.; Cheng, D.; Xie, Y.; Robert, J.; Rush, J.; Hochstrasser, M.; Finley, D.; Peng, J. Quantitative Proteomics Reveals the Function of Unconventional Ubiquitin Chains in Proteasomal Degradation. Cell 2009, 137, 133–145.
  8. Huang, T.T.; D’Andrea, A.D. Regulation of DNA Repair by Ubiquitylation. Nat. Rev. Mol. Cell Biol. 2006, 7, 323–334.
  9. Yuan, W.-C.; Lee, Y.-R.; Lin, S.-Y.; Chang, L.-Y.; Tan, Y.P.; Hung, C.-C.; Kuo, J.-C.; Liu, C.-H.; Lin, M.-Y.; Xu, M.; et al. K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking. Mol. Cell 2014, 54, 586–600.
  10. Elia, A.E.H.; Boardman, A.P.; Wang, D.C.; Huttlin, E.L.; Everley, R.A.; Dephoure, N.; Zhou, C.; Koren, I.; Gygi, S.P.; Elledge, S.J. Quantitative Proteomic Atlas of Ubiquitination and Acetylation in the DNA Damage Response. Mol. Cell 2015, 59, 867–881.
  11. Morris, J.R.; Solomon, E. BRCA1: BARD1 Induces the Formation of Conjugated Ubiquitin Structures, Dependent on K6 of Ubiquitin, in Cells during DNA Replication and Repair. Hum. Mol. Genet. 2004, 13, 807–817.
  12. Manzanillo, P.S.; Ayres, J.S.; Watson, R.O.; Collins, A.C.; Souza, G.; Rae, C.S.; Schneider, D.S.; Nakamura, K.; Shiloh, M.U.; Cox, J.S. The Ubiquitin Ligase Parkin Mediates Resistance to Intracellular Pathogens. Nature 2013, 501, 512–516.
  13. Ordureau, A.; Sarraf, S.A.; Duda, D.M.; Heo, J.-M.; Jedrychowski, M.P.; Sviderskiy, V.O.; Olszewski, J.L.; Koerber, J.T.; Xie, T.; Beausoleil, S.A.; et al. Quantitative Proteomics Reveal a Feedforward Mechanism for Mitochondrial PARKIN Translocation and Ubiquitin Chain Synthesis. Mol. Cell 2014, 56, 360–375.
  14. Besche, H.C.; Sha, Z.; Kukushkin, N.V.; Peth, A.; Hock, E.-M.; Kim, W.; Gygi, S.; Gutierrez, J.A.; Liao, H.; Dick, L.; et al. Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates. EMBO J. 2014, 33, 1159–1176.
  15. Kim, W.; Bennett, E.J.; Huttlin, E.L.; Guo, A.; Li, J.; Possemato, A.; Sowa, M.E.; Rad, R.; Rush, J.; Comb, M.J.; et al. Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome. Mol. Cell 2011, 44, 325–340.
  16. Jin, J.; Xie, X.; Xiao, Y.; Hu, H.; Zou, Q.; Cheng, X.; Sun, S.-C. Epigenetic Regulation of the Expression of Il12 and Il23 and Autoimmune Inflammation by the Deubiquitinase Trabid. Nat. Immunol. 2016, 17, 259–268.
  17. Tran, H.; Hamada, F.; Schwarz-Romond, T.; Bienz, M. Trabid, a New Positive Regulator of Wnt-Induced Transcription with Preference for Binding and Cleaving K63-Linked Ubiquitin Chains. Genes Dev. 2008, 22, 528–542.
  18. Tokunaga, F.; Sakata, S.; Saeki, Y.; Satomi, Y.; Kirisako, T.; Kamei, K.; Nakagawa, T.; Kato, M.; Murata, S.; Yamaoka, S.; et al. Involvement of Linear Polyubiquitylation of NEMO in NF-KappaB Activation. Nat. Cell Biol. 2009, 11, 123–132.
  19. Mevissen, T.E.T.; Komander, D. Mechanisms of Deubiquitinase Specificity and Regulation. Annu. Rev. Biochem. 2017, 86, 159–192.
  20. Johnson, E.S. Protein Modification by SUMO. Annu. Rev. Biochem. 2004, 73, 355–382.
  21. Ribet, D.; Cossart, P. Ubiquitin, SUMO, and NEDD8: Key Targets of Bacterial Pathogens. Trends Cell Biol. 2018, 28, 926–940.
  22. Panasenko, O.O. Identification of Ubiquitinated Proteins. Mater. Methods 2014, 4, 827.
  23. Mulder, M.P.C.; Witting, K.; Berlin, I.; Pruneda, J.N.; Wu, K.-P.; Chang, J.-G.; Merkx, R.; Bialas, J.; Groettrup, M.; Vertegaal, A.C.O.; et al. A Cascading Activity-Based Probe Sequentially Targets E1–E2–E3 Ubiquitin Enzymes. Nat. Chem. Biol. 2016, 12, 523–530.
  24. Neutzner, M.; Neutzner, A. Enzymes of Ubiquitination and Deubiquitination. Essays Biochem. 2012, 52, 37–50.
  25. Ye, Y.; Rape, M. Building Ubiquitin Chains: E2 Enzymes at Work. Nat. Rev. Mol. Cell Biol. 2009, 10, 755–764.
  26. Ebner, P.; Versteeg, G.A.; Ikeda, F. Ubiquitin Enzymes in the Regulation of Immune Responses. Crit. Rev. Biochem. Mol. Biol. 2017, 52, 425–460.
  27. Metzger, M.B.; Pruneda, J.N.; Klevit, R.E.; Weissman, A.M. RING-Type E3 Ligases: Master Manipulators of E2 Ubiquitin-Conjugating Enzymes and Ubiquitination. Biochim. Biophys. Acta 2014, 1843, 47–60.
  28. Metzger, M.B.; Hristova, V.A.; Weissman, A.M. HECT and RING Finger Families of E3 Ubiquitin Ligases at a Glance. J. Cell Sci. 2012, 125, 531–537.
  29. Scheffner, M.; Kumar, S. Mammalian HECT Ubiquitin-Protein Ligases: Biological and Pathophysiological Aspects. Biochim. Biophys. Acta BBA Mol. Cell Res. 2014, 1843, 61–74.
  30. Smit, J.J.; Sixma, T.K. “Ubiquitylation: Mechanism and Functions” Review Series: RBR E3-Ligases at Work. EMBO Rep. 2014, 15, 142–154.
  31. Spratt, D.E.; Walden, H.; Shaw, G.S. RBR E3 Ubiquitin Ligases: New Structures, New Insights, New Questions. Biochem. J. 2014, 458, 421–437.
  32. Lechtenberg, B.C.; Rajput, A.; Sanishvili, R.; Dobaczewska, M.K.; Ware, C.F.; Mace, P.D.; Riedl, S.J. Structure of a HOIP/E2~ubiquitin Complex Reveals RBR E3 Ligase Mechanism and Regulation. Nature 2016, 529, 546–550.
  33. Wilkinson, K.D. DUBs at a Glance. J. Cell Sci. 2009, 122, 2325–2329.
  34. Abdul Rehman, S.A.; Kristariyanto, Y.A.; Choi, S.-Y.; Nkosi, P.J.; Weidlich, S.; Labib, K.; Hofmann, K.; Kulathu, Y. MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes. Mol. Cell 2016, 63, 146–155.
  35. Kwasna, D.; Abdul Rehman, S.A.; Natarajan, J.; Matthews, S.; Madden, R.; De Cesare, V.; Weidlich, S.; Virdee, S.; Ahel, I.; Gibbs-Seymour, I.; et al. Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability. Mol. Cell 2018, 70, 150–164.e6.
  36. Komander, D.; Clague, M.J.; Urbé, S. Breaking the Chains: Structure and Function of the Deubiquitinases. Nat. Rev. Mol. Cell Biol. 2009, 10, 550–563.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback