Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.     

An improved SUMmOn‐based methodology for the identification of ubiquitin and ubiquitin‐like protein conjugation sites identifies novel ubiquitin‐like protein chain linkages

Ubiquitin (Ub) and the ubiquitin‐like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half‐life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis‐database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine‐rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin‐related modifier (SUMO) chain (SUMO‐2 K42, SUMO‐3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.     

Detection of modification by ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins (Ubls) are conjugated to many target proteins either as monomeric units or as polymeric chains. There are at least 12 members of the ubiquitin family in the human genome and their conjugation dramatically alters the properties of the modified protein. The presence of highly active proteases that specifically deconjugate Ubls often means that, in the cell, the steady state level of modified protein is low. Detection of protein species modified by Ubls can therefore represent a significant challenge. Here, we describe methods that have been developed to allow detection of Ubl modified proteins both in vivo and in vitro.     

An Atg10-like E2 enzyme is essential for cell cycle progression but not autophagy in Schizosaccharomyces pombe

Many proteins involved in autophagy have been identified in the yeast Saccharomyces cerevisiae. For example, Atg3 and Atg10 are two E2 enzymes that facilitate the conjugation of the ubiquitin-like proteins (Ubls) Atg8 and Atg12, respectively. Here, we describe the identification and characterization of the predicted Atg10 homolog (SpAtg10) of the evolutionarily distant Schizosaccharomyces pombe. Unexpectedly, SpAtg10 is not essential for autophagy. Instead, we find that SpAtg10 is essential for normal cell cycle progression, and for responses to various stress conditions that perturb the cell cycle, independently of Atg12 conjugation. Taken together, our data indicate that autophagic Ubl conjugation pathways differ between eukaryotes and, furthermore, that enzymes such as Atg10 may have additional functions in controlling key cellular processes such as cell cycle progression. Atg10-related proteins are found from yeast to humans, and, thus, this study has implications for understanding the functions of this protein family in Ubl conjugation in eukaryotes.     

The basis for selective E1-E2 interactions in the ISG15 conjugation system

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.     

A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.     

Structures of proteases for ubiqutin and ubiquitin-like modifiers

Post-translational modifiers can alter the function of proteins in many different ways. The conjugation of ubiquitin (Ub) and ubiqutin-like modifiers (Ubls) to proteins has been shown to be especially crucial in regulating a variety of cellular processes including the cell cycle, growth control, quality control, localization and many more. It is a highly dynamic process and involves a number of enzymes called E1, E2 and E3. Ub and Ubls are removed from the target proteins by deubiquitinating enzymes (DUBs) or Ubl-specific proteases (ULPs), thereby deconjugation can act as an additional level of control over the ubiquitin-conjugation system. In addition, DUBs and ULPs are responsible for activating Ub and Ubls from their inactive corresponding precursor forms. Here we review recent progress in molecular details of these deconjugating enzymes of Ubls.     

Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes

Ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are conjugated to their targets by specific cascades involving three classes of enzymes, E1, E2, and E3. Each E1 adenylates the C terminus of its cognate Ubl, forms a E1 approximately Ubl thioester intermediate, and ultimately generates a thioester-linked E2 approximately Ubl product. We have determined the crystal structure of yeast Uba1, revealing a modular architecture with individual domains primarily mediating these specific activities. The negatively charged C-terminal ubiquitin-fold domain (UFD) is primed for binding of E2s and recognizes their positively charged first alpha helix via electrostatic interactions. In addition, a mobile loop from the domain harboring the E1 catalytic cysteine contributes to E2 binding. Significant, experimentally observed motions in the UFD around a hinge in the linker connecting this domain to the rest of the enzyme suggest a conformation-dependent mechanism for the transthioesterification function of Uba1; however, this mechanism clearly differs from that of other E1 enzymes.     

Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.     

Crystal structure of ubiquitin-like small archaeal modifier protein 1 (SAMP1) from Haloferax volcanii

The ubiquitin-like (Ubl) system has been shown to be ubiquitous in all three kingdoms of life following the very recent characterization of ubiquitin-like small archaeal modifier proteins (SAMP1 and 2) from Haloferax volcanii. The ubiquitin (Ub) and Ubl molecules in eukaryotes have been studied extensively and their cellular functions are well established. Biochemical and structural data pertaining to prokaryotic Ubl protein (Pup) continue to be reported. In contrast to eukaryotes and prokaryotes, no structural information on the archaeal Ubl molecule is available. Here we determined the crystal structure of SAMP1 at 1.55 Å resolution and generated a model of SAMP2. These were then compared with other Ubl molecules from eukaryotes as well as prokaryotes. The structure of SAMP1 shows a β-grasp fold of Ub, suggesting that the archaeal Ubl molecule is more closely related to eukaryotic Ub and Ubls than to its prokaryotic counterpart. The current structure identifies the location of critical elements such a single lysine residue (Lys4), C-terminal di-glycine motif, hydrophobic patches near leucine 60, and uniquely inserted α-helical segments (α1 and α3) in SAMP1. Based on the structure of SAMP1, several Ub-like features of SAMPs such as poly-SAMPylation and non-covalent interactions have been proposed, which should provide the basis for further investigations concerning the molecular function of archaeal Ubls and the large super-family of β-grasp fold proteins in the archaeal kingdom.     

Ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: Emerging patterns and molecular principles

Attachment of ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers is a reversible post-translational modification that regulates the fate and function of proteins. In particular, proteolytic enzymes with Ub/Ubl processing activity appear to be more widespread than originally anticipated. It is therefore not surprising that bacterial and viral pathogens have exploited many ways to interfere with Ub/Ubl conjugation, but also de-conjugation. On one hand, pathogens were shown to manipulate host encoded enzymes. On the other hand, pathogen derived sequences of proteases specific for Ub/Ubls are emerging as a common feature shared by many viruses, bacteria and protozoa, and we are at an early stage of understanding how these proteases contribute to the pathogenesis of infection. Whereas some of these proteases share a common origin with mammalian cell encoded hydrolases with specific properties towards Ub/Ubls, most of them have ancient intrinsic functions, such as processing pathogen protein components, and may have acquired the specificity for Ub/Ubls by interacting with mammalian hosts and their immune system throughout evolution. Since many of these proteases are clearly distinct from their mammalian counterparts, they represent attractive targets for drug design against infectious diseases.     

Leishmania donovani‐specific Ub‐related modifier‐1: an early endosome‐associated ubiquitin‐like conjugation in Leishmania donovani

Protein modification by ubiquitin (Ub) and Ub‐like molecules (Ubls) is a diverse biological process that regulates the activity of the target proteins. Systematic studies of Ubls in trypanosomatids like Leishmania, the causative organism of potentially fatal visceral leishmaniasis, would yield a better understanding of the disease pathogenesis and identify novel therapeutic targets. The present study is the first to characterize Leishmania donovani‐specific Ub‐related modifier‐1 (LdUrm1) and the associated conjugation pathway. Based on homology modeling, LdUrm1 was found to possess a β‐grasp fold and a C‐terminal di‐glycine motif unique to Ub/Ubls, essential for its conjugation to the target proteins. We identified LdUba4 as the E1 enzyme for LdUrm1 and demonstrated its energy‐dependent enzymatic activity. LdUrm1 was immunolocalized anteriorly near the flagellar reservoir, while LdUba4 was cytoplasmic, both in promastigotes and axenic amastigotes. Expression of nonconjugatable LdUrm1 in L. donovani resulted in depleted parasite growth suggesting its role in the pathogenesis. By mass spectrometry, we identified Rab5, a known mediator of early endosome regulated hemoglobin endocytosis in Leishmania, as a target of LdUrm1. Our data suggest that LdUrm1 conjugation pathway may have a role in early endosome‐mediated heme uptake in Leishmania that may be explored as a drug target.     

Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-amino group of the target lysine residue on the protein substrate. The SUMO-1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp(100) and Lys(101) of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.     

Interplay between poxviruses and the cellular ubiquitin/ubiquitin-like pathways

Post-translational polypeptide tagging by conjugation with ubiquitin and ubiquitin-like (Ub/Ubl) molecules is a potent way to alter protein functions and/or sort specific protein targets to the proteasome for degradation. Many poxviruses interfere with the host Ub/Ubl system by encoding viral proteins that can usurp this pathway. Some of these include viral proteins of the membrane-associated RING-CH (MARCH) domain, p28/Really Interesting New Gene (RING) finger, ankyrin-repeat/F-box and Broad-complex, Tramtrack and Bric-a-Brac (BTB)/Kelch subgroups of the E3 Ub ligase superfamily. Here we describe and discuss the various strategies used by poxviruses to target and subvert the host cell Ub/Ubl systems.     

Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes

Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes.     

Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes

Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes.     

Lipidation of Atg8: how is substrate specificity determined without a canonical E3 enzyme?

Atg8 and its mammalian homolog LC3, ubiquitin-like proteins (Ubls) required for autophagosome formation, are remarkably unique in that their conjugation target is the lipid phosphatidylethanolamine (PE). Although PE was identified as the sole lipid conjugated with Atg8/LC3 in vivo, phosphatidylserine (PS) can be also a good substrate for its conjugation reaction in vitro. This posed a simple, intriguing question: What confers substrate specificity to lipidation of Atg8/LC3 in vivo? Our recent in vitro studies propose that intracellular milieus such as cytosolic pH and acidic phospholipids in membranes significantly contribute to selective production of the Atg8-PE conjugate.     

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.Key words: autophagy, collaboration, gel electrophoresis, membrane, primer     

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.     

Dual roles of Atg8-PE deconjugation by Atg4 in autophagy

Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8-PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8-PE and its deconjugation.