A high-throughput MALDI-TOF MS biochemical screen for small molecule inhibitors of the antigen aminopeptidase ERAP1.

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.

Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages.

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.

A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling.

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d0-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets.

SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Ki = 14 nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.

Overview of peptide and protein analysis by mass spectrometry.

Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells.

BACKGROUND: Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. METHODS: High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. RESULTS: 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells. CONCLUSIONS: Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation.

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.

GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition.

PURPOSE: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. EXPERIMENTAL DESIGN: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. RESULTS: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27(Kip1/CDKN1B), and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. CONCLUSIONS: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors.

Overview of peptide and protein analysis by mass spectrometry.

Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide.

Antitumor activity of an allosteric inhibitor of centromere-associated protein-E.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

Novel mechanism of lapatinib resistance in HER2-positive breast tumor cells: activation of AXL.

HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)-positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic.

The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP.

Aurora kinases are a family of serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. AurB (Aurora B kinase) has shown a clear link to cancer and is being pursued as an attractive cancer target. Multiple small molecules targeting AurB have entered the clinic for the treatment of cancer. A protein cofactor, INCENP (inner centromere protein), regulates the cellular localization and activation of AurB. In the present study, we examined the effect of INCENP on the activation kinetics of AurB and also elucidated the kinetic mechanism of AurB-catalysed substrate phosphorylation. We have concluded that: (i) substoichoimetric concentrations of INCENP are sufficient for AurB autophosphorylation at the activation loop residue Thr(232), and hence INCENP plays a catalytic role in AurB autophosphorylation; (ii) AurB/INCENP-catalysed phosphorylation of a peptide substrate proceeds through a rapid equilibrium random Bi Bi kinetic mechanism; and (iii) INCENP has relatively minor effects on the specific activity of AurB using a peptide substrate when compared with its role in AurB autoactivation. These results indicate that the effects of INCENP, and probably accessory proteins in general, may differ when enzymes are acting on different downstream targets.

TULA proteins bind to ABCE-1, a host factor of HIV-1 assembly, and inhibit HIV-1 biogenesis in a UBA-dependent fashion.

TULA, a recently identified UBA- and SH3-containing protein, has previously been shown to regulate cell signaling through protein tyrosine kinases. In order to search for novel functions of TULA, we identified, using mass spectrometry, proteins associated with TULA. ABCE-1 also known as RLI and HP68, a host factor of HIV-1 assembly, was found among TULA-associated proteins in these experiments. Considering an important role of ABCE-1 in HIV-1 assembly, we were compelled to analyze the effect of TULA on HIV-1 biogenesis. Our study provides evidence that TULA proteins substantially inhibit production of both sub-genomic and full-length HIV-1 viral particles and that the effect of TULA is dependent on UBA domain-mediated interactions. The primary role of ABCE-1 in the effect of TULA appears to be the recruitment of TULA to the sites of HIV-1 assembly where TULA interferes with the late steps of the HIV-1 life cycle, most likely by disrupting essential ubiquitylation-dependent events that remain to be identified.