Mitochondria play a major role in cellular health, yet their contribution to chronic diseases has been underestimated. Mitochondria are essential for all tissues and are the major source of ATP in high-energy-demand organs such as brain and heart, which consequently are vulnerable to mitochondrial dysfunction. Failure to repair or remove damaged mitochondria contributes to aging and chronic diseases. Cells have evolved quality control mechanisms, including mitophagy to eliminate damaged mitochondria and mitobiogenesis to replenish them. The ubiquitin-proteasome system (UPS) is responsible for removing misfolded proteins, a process that is highly ATP dependent and therefore reliant on mitochondrial function. In turn, damaged mitochondria are eliminated through coordinated actions of the UPS and lysosomal degradation through mitophagy. Many neurodegenerative diseases are characterized by the presence of disease-specific protein aggregates, such as α-synuclein aggregates in Parkinson's disease and tau neurofibrillary tangles in Alzheimer's disease. These aggregates impair mitochondrial function, while dysfunctional mitochondria generate reactive oxygen species that further exacerbate proteotoxic stress, creating a pathogenic cycle. This highlights the functional interplay between mitochondria and the UPS. Recent studies have uncovered phosphorylation of ubiquitin at serine 65 by the mitochondrial kinase PINK1 as a key signal of mitochondrial dysfunction. Phospho-Ser65-ubiquitin (pUb) has emerged as an indicator of mitochondrial health and a potential biomarker for aging and neurodegenerative disease. However, due largely to a lack of tools, little is known about the role of pUb in cellular physiology. Here, we review the current landscape of pUb biology, the phospho-ubiquitome, and its role as biomarker for mitochondrial health and neurodegeneration.

Polyubiquitination of proteins serves distinct functions that are governed by the nature of polyubiquitin chains built on target proteins. Among the eight distinct type of ubiquitin chains, lysine 48 (K48)-linked chains are specifically associated with proteasomal degradation, while lysine 63 (K63)-linked chains are primarily involved in regulating signal transduction and protein trafficking. The ubiquitin-proteasome system (UPS) has recently been exploited in drug discovery and introduced PROTACs (Proteolysis Targeting Chimeras), or molecular glues (MGs), to hijack ubiquitin E3 ligases, to facilitate the targeted degradation of specific proteins. However, assessment of PROTAC or MG mediated endogenous target protein ubiquitination in a linkage-specific manner in high throughput format remains a challenge. In this study, we applied chain-specific TUBEs (Tandem Ubiquitin Binding Entities) with nanomolar affinities for polyubiquitin chains in HTS assays to investigate the ubiquitination dynamics of RIPK2, a key regulator of inflammatory signaling. Using L18-MDP to induce K63 ubiquitination of RIPK2 and RIPK degrader-2, a RIPK2 PROTAC to induce K48 ubiquitination, we demonstrate that chain-selective TUBEs can differentiate and unravel context dependent linkage specific ubiquitination of endogenous RIPK2. Potential application of this technology to other target proteins and cellular contexts will be discussed.

BACKGROUND AND AIMS:Almost 30% of survivors of myocardial infarction (MI) develop heart failure (HF), in part due to damage caused by the accumulation of dysfunctional mitochondria. Organelle quality control through Parkin-mediated mitochondrial autophagy (mitophagy) is known to play a role in mediating protection against HF damage post-ischaemic injury and remodelling of the subsequent deteriorated myocardium. METHODS:This study has shown that a single i.p. dose (2 h post-MI) of the selective small molecule Parkin activator PR-364 reduced mortality, preserved cardiac ejection fraction, and mitigated the progression of HF. To reveal the mechanism of PR-364, a multi-omic strategy was deployed in combination with classical functional assays using in vivo MI and in vitro cardiomyocyte models. RESULTS:In vitro cell data indicated that Parkin activation by PR-364 increased mitophagy and mitochondrial biogenesis, enhanced adenosine triphosphate production via improved citric acid cycle, altered accumulation of calcium localization to the mitochondria, and initiated translational reprogramming with increased expression of mitochondrial translational proteins. In mice, PR-364 administered post-MI resulted in widespread proteome changes, indicating an up-regulation of mitochondrial metabolism and mitochondrial translation in the surviving myocardium. CONCLUSIONS:This study demonstrates the therapeutic potential of targeting Parkin-mediated mitophagy using PR-364 to protect surviving cardiac tissue post-MI from progression to HF.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with k inact /K I = 10,000 M- 1 s- 1, achieved sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

SARS-CoV-2 protease Nsp3 is a therapeutic target for developing anti-SARS-CoV-2 drugs. Nsp3 is a large multi-spanning membrane protein, and its characterization in vitro has been challenging. Here we describe an in vitro assay to characterize the biochemical activity of full-length Nsp3 isolated from cells. The assay can be used to evaluate Nsp3 inhibitors.

The ongoing pandemic of Coronavirus Disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health. Currently no approved drug or vaccine exists against SARS-CoV-2. Drug repurposing, represented as an effective drug discovery strategy from existing drugs, is a time efficient approach to find effective drugs against SARS-CoV-2 in this emergency situation. Both experimental and computational approaches are being employed in drug repurposing with computational approaches becoming increasingly popular and efficient. In this study, we present a robust experimental design combining deep learning with molecular docking experiments to identify most promising candidates from the list of FDA approved drugs that can be repurposed to treat COVID-19. We have employed a deep learning based Drug Target Interaction (DTI) model, called DeepDTA, with few improvements to predict drug-protein binding affinities, represented as KIBA scores, for 2,440 FDA approved and 8,168 investigational drugs against 24 SARS-CoV-2 viral proteins. FDA approved drugs with the highest KIBA scores were selected for molecular docking simulations. We ran docking simulations for 168 selected drugs against 285 total predicted and/or experimentally proven active sites of all 24 SARS-CoV-2 viral proteins. We used a recently published open source AutoDock based high throughput screening platform virtualflow to reduce the time required to run around 50,000 docking simulations. A list of 49 most promising FDA approved drugs with best consensus KIBA scores and AutoDock vina binding affinity values against selected SARS-CoV-2 viral proteins is generated. Most importantly, anidulafungin, velpatasvir, glecaprevir, rifabutin, procaine penicillin G, tadalafil, riboflavin 5’-monophosphate, flavin adenine dinucleotide, terlipressin, desmopressin, elbasvir, oxatomide, enasidenib, edoxaban and selinexor demonstrate highest predicted inhibitory potential against key SARS-CoV-2 viral proteins.

TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases (RTKs) that are expressed by multiple immune cells including NK cells. Although RTKs typically enhance cellular functions, TAM receptor ligation blocks NK‐cell activation. The mechanisms by which RTKs block NK‐cell signaling downstream of activating receptors are unknown. In this report, we demonstrate that TAM receptors attenuate NK cell responses via the activity of E3 ubiquitin ligase Casitas B lineage lymphoma b (Cbl‐b). Specifically, we show that Tyro3, Axl, and Mer phosphorylate Cbl‐b, and Tyro3 ligation activates Cbl‐b by phosphorylating tyrosine residues 133 and 363. Ligation of TAM receptors by their ligand Gas6 suppresses activating receptor‐stimulated NK‐cell functions such as IFN‐γ production and degranulation, in a TAM receptor kinase‐ and Cbl‐b‐dependent manner. Moreover, Gas6 ligation induces the degradation of LAT1, a transmembrane adaptor protein required for NK cell activating receptor signaling, in WT but not in Cbl‐b knock‐out NK cells. Together, these results suggest that TAM receptors may attenuate NK‐cell function by phosphorylating Cbl‐b, which in turn dampens NK‐cell activation signaling by promoting the degradation of LAT1. Our data therefore support a mechanism by which RTKs attenuate, rather than stimulate, signaling pathways via the activation of ubiquitin ligases.

Abstract Tumors employ diverse strategies to suppress and evade the immune system’s ability to recognize and destroy tumor cells. Immune-suppressive Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment correlate with poor prognosis in solid tumors. Therefore, selective depletion of tumor-associated Tregs or impairment of Treg function is considered an attractive cancer immunotherapy approach. The pro-tumorigenic deubiquitylase (DUB) USP7 is also essential for Treg functions. USP7 controls Treg function largely by regulating post-translational modification of Foxp3 and TIP60. Deletion of USP7 in Tregs results in impairment of Treg functions and autoimmunity in mice. Progenra has developed potent, selective covalent irreversible USP7 inhibitors that impair Treg functions ex vivo and in vivo. Most importantly, USP7 inhibitors exhibit robust antitumor activity against several syngeneic solid tumor models in immunocompetent mice. In addition, Progenra’s USP7 inhibitors enhance the efficacy of anti-PD1 antibody, anti-CTLA4 antibody, and cancer vaccines. These studies strongly suggest that USP7 inhibitors alone or in combination regimens can improve the efficacy and expand the scope of cancer immunotherapy. Citation Format: Suresh Kumar, Jian Wu, Feng Wang, Liqing Wang, Lee Chen, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Brigid Cunnion, Joseph Weinstock, Michael Mattern, Wayne Hancock. Covalent irreversible usp7 inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B202.

The Habitable-zone Planet Finder (HPF) is a highly stabilized fiber fed precision radial velocity (RV) spectrograph working in the Near Infrared (NIR): 810 - 1280 nm. In this paper we present an overview of the preparation of the optical fibers for HPF. The entire fiber train from the telescope focus down to the cryostat is detailed. We also discuss the fiber polishing, splicing and its integration into the instrument using a fused silica puck. HPF was designed to be able to operate in two modes, High Resolution (HR- the only mode mode currently commissioned) and High Efficiency (HE). We discuss these fiber heads and the procedure we adopted to attach the slit on to the HR fibers.

The ubiquitin-specific protease 7 (USP7) has emerged as an attractive oncology/immune-oncology target owing to its critical roles in several cancer-related signaling pathways as well as its essential role in maintaining functions of Foxp3+ T-regulatory cells (Tregs), the key players in tumor immune evasion. Progenra has developed a series of compounds that inhibit purified USP7 selectively and attenuate USP7 activity in cells and in vivo; these inhibitors exert antitumor activity directly and also facilitate immune-mediated antitumor activity by suppressing Treg functions. However, the precise mechanism of action of these compounds remains unclear. In this study, using a combination of NMR spectroscopy, mass spectrometry, and single amino-acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic pocket of USP7 and modify its active site cysteine (Cys223) by forming a covalent adduct. Consistent with the covalent binding mechanism, pharmacokinetic studies revealed long-lasting, irreversible USP7 inhibition after a short pulse treatment with inhibitor, accompanied by changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage-specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition will permit the rational design of improved inhibitors as a new class of anticancer agent. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Lee Chen, Brigid Cunnion, David Sterner, Charles Grove, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Irina Bezsonova, Wayne W. Hancock, Suresh Kumar. Characterization of selective active-site targeted covalent inhibitors of usp7 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B193.

Tumor microenvironment (TME) plays critical role in suppressing the immune system’s ability to recognize and destroy cancer cells. Although T cell checkpoint blockers such as anti-PD1 antibodies unleash anti-tumor immune response against a variety of tumors, the complex immunosuppressive tumor milieu necessitates development of additional therapeutic agents to potentiate active drugs and expand the realm of the revolutionary cancer immunotherapy. The presence of highly immunosuppressive Foxp3+ T-regulatory cells (Tregs) in the TME has been correlated with poor prognosis. Thus, depletion of Tregs or impairment of Treg function is considered an attractive therapeutic approach. USP7, a deubiquitylase (DUB) implicated as a critical node in several cancer signaling pathways has emerged as an essential factor in maintaining Treg functions. Treg specific deletion of USP7 impairs Treg function leading to lethal autoimmunity. Using the UbiProTM discovery platform, Progenra has identified selective covalent USP7 inhibitors that were subjected to lead optimization and preclinical evaluation. Potent and selective candidate USP7 inhibitors have been shown to impair Treg functions ex vivo and in vivo as well as exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. Most importantly, Progenra’s USP7 inhibitors enhances the efficacy of anti-PD1 antibody and cancer vaccines. Along with the already established direct anti-tumor activities of USP7 inhibitors, these studies provide a strong rationale for combining USP7 inhibitors to suppress Treg functions and improve the efficacy of currently approved cancer immunotherapy agents. Citation Format: Suresh Kumar, Jian Wu, Liqing Wang, Feng Wang, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Joseph Weinstock, Michael Mattern, Wayne Hancock. USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2017-1691

USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.

Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.

Abstract Ubiquitylation and deubiquitylation control the stability, localization and activity of many cellular proteins. Deubiquitylation is performed by a family of ∼80 enzymes (DUBs) and many of these DUBs have been implicated in a wide range of diseases including cancer. In particular, USP2 removes ubiquitin selectively from Cyclin D1, Cyclin A1, Aurora A, MDM2 and FAS which are all implicated in oncogenic processes. Importantly, USP2 is overexpressed in prostate and ovarian cancers and inhibition of USP2 has been shown to sensitize prostate carcinoma cells to apoptosis. Therefore USP2 is an attractive therapeutic target. Progenra's unique UbiProTM discovery platform was utilized to screen ∼200K member diversity based library of small molecules for identifying novel USP2 inhibitors. Hits from the screen were subjected to orthogonal selectivity assays to identify USP2 specific inhibitors. Here we report discovery and hit to lead optimization of novel USP2 inhibitors showing cellular efficacy and anti-cancer activities against relevant cancer cell models. The most promising compounds are being further developed as novel anti-cancer agents. Citation Format: Suresh Kumar, James P. LaRocque, Jeffrey G. Marblestone, Jian Wu, Joseph Weinstock, David E. Sterner, Michael R. Mattern. Discovery and development of novel USP2 inhibitors for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2015-5433

Despite widespread use of statins and other therapeutics, hypercholesterolemia remains a significant medical issue for a many patients unable to effectively regulate cholesterol levels. Cholesterol uptake is primarily mediated by the hepatic low density lipoprotein receptor (LDLR), which binds and internalizes plasma LDL. Elevated LDLR is associated with reduced LDL levels. Recently, the E3 ligase Idol (Inducible Degrader of LDLR) was shown to be a key regulator of LDLR levels. IDOL is a unique E3 ligase that utilizes its FERM domain to specifically target LDLR for polyubiquitylation and subsequent lysosomal degradation. Genetic ablation of IDOL raises levels of LDLR. Thus, inhibition of IDOL may be beneficial for the treatment of hypercholesterolemia. The ubiquitin‐proteasome system is a rich landscape for drug discovery. E3 ligases in particular are attractive therapeutic targets in various disorders, including metabolic disorders. Nevertheless, it has been challenging to discover and develop E3 ligase inhibitors as first in class clinical candidates. Here, we report the identification of novel IDOL inhibitors that modulate cellular cholesterol homeostasis. These compounds increased LDLR levels and increased LDL association in various cellular models. Biophysical characterization revealed direct binding of the compounds to IDOL and perturbation of IDOL:LDLR interactions. The most promising compounds were used as starting points to develop novel drug like molecules and the lead compounds are being evaluated in translational models of hypercholesterolemia. Data will be presented summarizing our progress to date targeting IDOL for the treatment of hypercholesterolemia. Work supported in part by NIH grant HL127893.

Abstract The degradation of most cellular proteins is regulated by coordinated addition and removal of ubiquitin by families of ubiquitin E3 ligases and deubiquitylating enzymes (DUBs) respectively. DUBs proteolytically cleave ubiquitin molecules from proteins resulting in modifications of protein activity, localization and function. Several DUBs are aberrantly regulated in cancer, including the best studied, USP7, selective inhibitors of which are active in cancer models. USP22, is another validated anticancer target, being one of 11 genes in the death-from-cancer gene signature, a component of the human SAGA transcriptional cofactor complex regulating myc transcription, and a regulator of the expression of p21, the histone deacetylase Sirt 1, and p53 activity. USP22 is overexpressed in oral squamous cell carcinoma, breast, non-small cell lung, colorectal, and other cancers and its expression is inversely correlated with survival. Unlike most other DUBs, USP22 exhibits robust activity only as a component of a multi-subunit complex. Initial studies reported activity solely as a member of the 2MDa SAGA complex, but more recently it has been demonstrated that USP22 exhibits similar activity in a four-protein DUB module derived from the SAGA complex. USP22 inhibitors are expected to have broad anti-cancer activities. Progenra's UbiProTM discovery platform was utilized to screen ∼200K member diversity based library of small molecules for identifying novel USP22 inhibitors. Data will be presented describing these results. Citation Format: Feng Wang, Timothy R. Stanek, Leelabati Biswas, Matthew Kodrasov, James LaRocque, Jian Wu, David Sterner, Joseph Weinstock, Michael Mattern, Steven B. McMahon, Suresh Kumar. Discovery and characterization of USP22 inhibitors as novel anti-cancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5432. doi:10.1158/1538-7445.AM2015-5432

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

Molecular oncology has the potential to revolutionize cancer treatment owing to its focus on discrete, cancer-selective targets, as evident in the recent success of kinase inhibitors and antibody-based therapies. Because of the heterogeneous nature of cancer, however, not every tumor type can be addressed with an appropriately selective therapy and some respond best to drug combinations that include classical "toxic" agents. The ubiquitin-proteasome pathway, recently harnessed for cancer treatment with the clinical use of "toxic" proteasome inhibitors bortezomib and carfilzomib, affords targets that intuitively are highly selective, exemplified by inhibitors of E3 ligases, the ubiquitin-conjugating enzymes, as well as those that are intuitively nonselective, exemplified by the proteasomal proteases. In the last two decades, anticancer drug development based on these two target classes has proceeded in parallel, with the early results suggesting that the nonselective proteasome is the better target. Lately, however, it has become clear that (1) the "nonselective" proteasome target may be addressed in selective ways and (2) a clearer understanding of the E3 ligase reaction can lead to the design or discovery of efficacious inhibitors. Evidence supporting these notions and implications for cancer treatment going forward will be discussed.

The ubiquitin pathway regulates diverse functions including protein localization and stability. The complexity of the pathway involving nearly 40 identified E2 conjugating enzymes and over 600 E3 ligases raises the issue of specificity. With the E2s and E3s fitting into a limited number of classes based on bioinformatics, structures, and proven activities, there is not a clear picture as to what would determine which E2/E3 enzyme pair would be functional. There have been many reports of limited E2/E3 activity profiling with a small number of E2s and E3s. We have expanded on this to investigate the activity of ubiquitin E2s covering the majority of the reported classes/families in concert with a number of E3s implicated in a variety of diseases. Using an ELISA-based assay we screened 10 E3 ligases against a panel of 11 E2s to determine which E2/E3 pairs exhibited E3 autoubiquitylation activity. In addition, the ubiquitin chain linkage preference by certain E2/E3 pairs was investigated. Finally, substrate ubiquitylation was assayed for the E3 ligase MuRF1 using various E2/MuRF1 pairs. These studies demonstrate the utility of identifying the correct E2/E3 pair to monitor specific substrate ubiquitylation.