Conjugation Cascade Research Articles

The Intrinsically Disordered N Terminus in Atg12 from Yeast Is Necessary for the Functional Structure of the Protein.

The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function. Here, we show that the IDPR in unconjugated Atg12 is positioned in proximity to the UBL domain, a configuration that is important for the functional structure of the protein. A major deletion in the IDPR disrupts intactness of the UBL domain at the unconjugated C terminus, and a mutation in the predicted α0 helix in the IDPR prevents Atg12 from binding to Atg7 and Atg10, which ultimately affects the protein function in the ubiquitin-like conjugation cascade. These findings provide evidence that the IDPR is an indispensable part of the Atg12 protein from yeast.

SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

ATG5 provides host protection acting as a switch in the atg8ylation cascade between autophagy and secretion.

ATG5 is a part of the E3 ligase directing lipidation of ATG8 proteins, a process central to membrane atg8ylation and canonical autophagy. Loss of Atg5 in myeloid cells causes early mortality in murine models of tuberculosis. This invivo phenotype is specific to ATG5. Here, we show using human cell lines that absence of ATG5, but not of other ATGs directing canonical autophagy, promotes lysosomal exocytosis and secretion of extracellular vesicles and, in murine Atg5fl/fl LysM-Cre neutrophils, their excessive degranulation. This is due to lysosomal disrepair in ATG5 knockout cells and the sequestration by an alternative conjugation complex, ATG12-ATG3, of ESCRT protein ALIX, which acts in membrane repair and exosome secretion. These findings reveal a previously undescribed function of ATG5 in its host-protective role in murine experimental models of tuberculosis and emphasize the significance of the branching aspects of the atg8ylation conjugation cascade beyond the canonical autophagy.

Insights in Post-Translational Modifications: Ubiquitin and SUMO.

Both ubiquitination and SUMOylation are dynamic post-translational modifications that regulate thousands of target proteins to control virtually every cellular process. Unfortunately, the detailed mechanisms of how all these cellular processes are regulated by both modifications remain unclear. Target proteins can be modified by one or several moieties, giving rise to polymers of different morphology. The conjugation cascades of both modifications comprise a few activating and conjugating enzymes but close to thousands of ligating enzymes (E3s) in the case of ubiquitination. As a result, these E3s give substrate specificity and can form polymers on a target protein. Polymers can be quickly modified forming branches or cleaving chains leading the target protein to its cellular fate. The recent development of mass spectrometry(MS) -based approaches has increased the understanding of ubiquitination and SUMOylation by finding essential modified targets in particular signaling pathways. Here, we perform a concise overview comprising from the basic mechanisms of both ubiquitination and SUMOylation to recent MS-based approaches aimed to find specific targets for particular E3 enzymes.

E1 Enzymes as Therapeutic Targets in Cancer.

Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.

Abstract 4435: Molecular mechanism of a novel covalent allosteric inhibitor of SUMO E1 activating enzyme

Abstract Post-translational modification of proteins by ubiquitin and ubiquitin-like proteins (collectively termed Ubls) proceeds through the sequential interactions and activities of parallel cascades of enzymes that are structurally and mechanistically related. E1 enzymes initiate Ub/Ubl conjugation cascades by catalyzing the ATP-dependent activation and transfer of their cognate Ub/Ubl to E2-conjugating enzymes, which then function with an array of E3 ligases to catalyze the formation of an isopeptide bond linking the Ub/Ubl to target proteins. Aberrations in Ub/Ubl modifications are associated with the pathogenesis of a wide range of life-threatening diseases, which has resulted in several Ubl E1 enzymes emerging as attractive targets for the development of small molecule therapeutics. We present the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a small molecule representing a new class of Ubl E1 enzyme inhibitor. The structure reveals that the covalent allosteric inhibitor targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that inhibitor binding is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. Altogether our studies provide the molecular mechanisms of inhibition SUMO E1 specificity and establishes a framework for the development of molecules targeting other Ubl E1s at a newly discovered cryptic allosteric site. Supported by NIH R01GM115568, F30CA216921, T32CA193201, and a Hollings Cancer Center Postdoctoral Fellowship. Citation Format: Zongyang Lyu, Lingmin Yuan, Katelyn M. Williams, James H. Atkison, Shaun K. Olsen. Molecular mechanism of a novel covalent allosteric inhibitor of SUMO E1 activating enzyme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4435.

Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in the first step of Ub/Ubl conjugation cascades and represent potential targets for therapeutic intervention in cancer and other life-threatening diseases. Here, we report the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a recently discovered and highly specific covalent allosteric inhibitor (COH000). The structure reveals that COH000 targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that COH000 binding to SUMO E1 is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. These structural changes include disassembly of the active site and a 180° rotation of the catalytic cysteine-containing SCCH domain, relative to conformational snapshots of SUMO E1 poised to catalyze adenylation. Altogether, our study provides a molecular basis for the inhibitory mechanism of COH000 and its SUMO E1 specificity, and also establishes a framework for potential development of molecules targeting E1 enzymes for other Ubls at a cryptic allosteric site.

Site-specific characterization of endogenous SUMOylation across species and organs

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogenous SUMO2/3 sites in human cells during heat stress and proteasomal inhibition, and quantitatively map 1963 SUMO sites across eight mouse tissues. Characterization of the SUMO equilibrium highlights striking differences in SUMO metabolism between cultured cancer cells and normal tissues. Targeting preferences of SUMO2/3 vary across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic investigation details the SUMOylation architecture across species and organs and provides a resource of endogenous SUMOylation sites on factors important in organ-specific functions.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Evolution of the ubiquitin-activating enzyme Uba1 (E1)

Ubiquitin tags diseased proteins and initiates an enzyme conjugation cascade, which has three stages. The first-stage enzyme Uba1 (E1) has evolved only modestly from slime mold to humans, and is >14 times larger than Ub. Here we use critical point thermodynamic scaling theory to connect Uba1 (E1) evolution from yeast and slime mold to fruit flies and humans to subtle changes in its amino acid sequences.

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism.

Ubiquitin-like proteins (Ubl’s) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.

S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S.pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.

The Anti-Tumor Effect of the Ubiquitin-Activating Enzyme (UAE) Inhibitor TAK-243 on Pre-Clinical Models of Multiple Myeloma

Background:The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action.Methods:We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques.Results:MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected.Conclusions:TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. DisclosuresBerger:Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

Production of Human ATG Proteins for Lipidation Assays.

Humans express several orthologs of yeast Atg8, in the LC3 and GABARAP families, which play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of LC3 and GABARAP regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. LC3 and GABARAP are families of related ubiquitin-like proteins (UBLs) (referred to here collectively as LC3/GABARAP), and their lipidation involves a divergent UBL conjugation cascade including ATG7, ATG3, and ATG12~ATG5-ATG16L1 acting as E1, E2, and E3 enzymes, respectively. ATG7 initiates LC3/GABARAP conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of ATG3. Ultimately, the ATG12~ATG5-ATG16L1 complex catalyzes LC3/GABARAP ligation to a primary amino group on PE or other acceptor lipids. This chapter describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12~ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation.

Crystallographic Characterization of ATG Proteins and Their Interacting Partners.

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.

Twists and turns in Ubiquitin Conjugation Cascades

Post‐translational modification by ubiquitin and ubiquitin‐like proteins (collectively referred to as UBLs) is a predominant eukaryotic regulatory mechanism. The vast reach of this form of regulation extends to virtually all eukaryotic processes. UBL modifications play critical roles in controlling the cell cycle, transcription, DNA repair, stress responses, signaling, immunity, plant growth, embryogenesis, circadian rhythms, and a plethora of other pathways. UBLs dynamically modulate target protein properties including half‐life, subcellular localization, enzymatic activity, conformation, and intermolecular interactions. Moreover, the enzymatic process of UBL ligation to and removal from proteins is itself dynamic, involving cascades of E1, E2, and E3 enzymes antagonized by deubiquitinating enzymes. The UBL C‐terminus is first activated, then transferred between multiple enzyme active sites, and ultimately ligated via an E3 enzyme to a target. With roughly 300 members in humans, the largest E3 family consists of the multisubunit Cullin‐RING ligases (CRLs), which regulate a staggering number of biochemical pathways and biological processes. We have found that Cullin‐RING E3 activities are modulated through protein‐protein interactions influencing enzyme conformation. In my presentation, I will discuss dynamic mechanisms underlying regulation of and by this large family of E3 ligases

Identification and characterization of the linear region of ATG3 that interacts with ATG7 in higher eukaryotes

Transfer of GABARAP thioester from the E1 ATG7 to the E2 ATG3 requires the interaction between the N-terminal domain of ATG7 and the flexible region (FR) of ATG3. This interaction has been visualized in the yeast Atg7-Atg3 complex crystal structure, but remains to be defined in higher eukaryotes. Here, our NMR data precisely define the region of the FR of human ATG3 that interacts with ATG7 (RIA7) and demonstrate RIA7 partially overlaps with the E3-interacting region, explaining how the E1-E2 and E2-E3 interactions are mutually exclusive. Mutational analyses identify critical residues of the RIA7 for the E1 interaction and GABARAP transfer, advancing our understanding of a molecular mechanism of the autophagic conjugation cascade in higher eukaryotes.

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay

Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.

Twists and Turns in Ubiquitin Conjugation Cascades

Post‐translational modification by ubiquitin‐like proteins (UBLs) is a predominant eukaryotic regulatory mechanism. The vast reach of this form of regulation extends to virtually all eukaryotic processes that involve proteins. UBL modifications play critical roles in controlling the cell cycle, transcription, DNA repair, stress responses, signaling, immunity, plant growth, embryogenesis, circadian rhythms, and a plethora of other pathways. UBLs dynamically modulate target protein properties including enzymatic activity, conformation, half‐life, subcellular localization, and intermolecular interactions. Moreover, the enzymatic process of UBL ligation to proteins is itself dynamic, involving cascades of E1, E2, and E3 enzymes. The UBL C‐terminus is first activated, then transferred between multiple enzyme active sites, and ultimately ligated via an E3 enzyme to a target. With roughly 300 members, the largest E3 family consists of Cullin‐RING ligases (CRLs), which regulate a staggering number of biochemical pathways and biological processes. In my presentation, I will discuss recent results from the lab addressing structural mechanisms underlying regulation of and by this large family of dynamic ubiquitin E3 ligases.