Ubiquitin-activating Enzyme Uba1 Research Articles

P04 A vexing case of polymyalgia rheumatica

Abstract Introduction VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a recently described autoinflammatory condition characterised by somatic mutations in the UBA1 gene, resulting in significant morbidity. Disease presentation can mimic several rheumatological conditions, delaying diagnosis and appropriate management. This case report presents the clinical course of a patient with a complex history of musculoskeletal symptoms and systemic inflammation, first diagnosed with polymyalgia rheumatica (PMR), who was later identified as having VEXAS syndrome almost six years after first referral to the rheumatology clinic. It illustrates the importance of considering rare autoinflammatory conditions in patients with atypical presentations of common rheumatological disorders. Case description A 76-year-old gentleman was referred from the haematology clinic where he had undergone investigation for pancytopenia and raised inflammatory markers. Initial investigations under the haematology team revealed normal CT chest, abdomen and pelvis, unremarkable bone marrow biopsy and cytogenetics. He presented with muscle stiffness and proximal limb girdle pain. Additional symptoms included fatigue and a diffuse non-specific erythematous rash. There was no significant past medical history and he was previously very fit and active. Rheumatology investigations were normal, apart from persistently elevated inflammatory markers (ESR 86 mm/h and CRP 70 mg/L). A diagnosis of polymyalgia rheumatica was made and prednisolone 15 mg was started. Although he initially had a good response to this, he continued to experience significant inflammatory and constitutional symptoms. These included small joint polyarthritis, night sweats and weight loss. He was pancytopenic with associated reduced exercise tolerance, requiring transfusions and erythropoietin. To exclude other differentials, a repeat bone marrow biopsy was performed. This revealed dysplasia likely secondary to underlying inflammation, although no clonal abnormalities or vacuoles were detected. MRI right hand showed synovitis, and he was therefore treated for myalgic-onset rheumatoid arthritis. Prednisolone dose was increased temporarily and methotrexate was added, resulting in temporary improvement of his ESR 16 mm/h and CRP 25 mg/L. The patient continued to have ongoing constitutional symptoms and developed new ataxia. Further investigations, including MRI head, gastroscopy and Whipple’s PCR were all normal. Repeat CT chest, abdomen and pelvis and PET-CT were unremarkable. His symptoms and inflammatory markers were responsive to steroids. Given his symptoms and macrocytosis, he was tested for ubiquitin-activating enzyme UBA1 gene which was positive, confirming the diagnosis of VEXAS syndrome. Methotrexate was stopped due to worsening cytopenia, prednisolone continued and tocilizumab started. He showed significant improvement in pancytopenia and symptoms following initiation of tocilizumab. Discussion VEXAS is characterised by somatic mutations in the UBA1 gene; its pathogenesis leads to malfunctioning methionine-41, an enzyme that initiates ubiquitylation. This leads to increased activation of innate immune pathways and induces systemic inflammation, manifesting in a variable clinical presentation. This variability makes the diagnosis challenging and leads to a median diagnostic delay of 5.5 years, similar to our patient’s six-year delay. Our patient was initially managed as PMR due to his clinical presentation and lack of an obvious alternative diagnosis. This misdiagnosis is not unique. Often, patients later diagnosed with VEXAS are first managed as either inflammatory conditions, notably polychondritis, vasculitis or Sweet’s syndrome, or myelodysplastic syndrome (MDS). VEXAS is hallmarked by haematological dysfunction as much as it is by inflammatory features. In fact, our patient’s first signs included a mild cytopenia which later developed into a progressive macrocytic anaemia. This is classical of VEXAS. Other haematological manifestations include thrombotic events and MDS, with minimal response to EPO-stimulating agents as seen here. His ongoing deterioration triggered an alternative diagnosis to be sought. VEXAS is unusual as an autoinflammatory disease: its causative mechanism is a somatic mutation acquired later in life. Its diagnosis can be confirmed by inactivation of the x-linked UBA1 gene. This case highlights the need for UBA1 testing when dealing with an unexplained inflammatory phenotype, especially in men with cytopenia, multiorgan autoinflammation or MDS-like features. VEXAS presents a management challenge. The term ‘haeamatoinflammatory’ has been created to reflect its unique nature, warranting input from both specialist teams as well as the wider MDT. Key learning points • VEXAS should be considered as a differential when dealing with unexplained inflammatory symptoms, especially in men, multiorgan autoinflammation, vasculitis and/or MDS-like features. • VEXAS is often progressive and transfusion-dependence is associated with a poorer prognosis. A macrocytic anaemia is a prominent feature of the condition. • The absence or low proportion of vacuoles in bone marrow should not prevent us from testing for VEXAS if clinically indicated. • No established protocols for VEXAS yet exist. Haematological management is supportive. Anti-inflammatory treatment is based on collective observed evidence, including the use of tocilizumbab (anti-IL6) and janus kinase (JAK) inhibitors. Allogenic haemopoietic stem cell transplantation has been successful in few patients and may represent an option for patients with severe disease.

Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes.

SUMMARYProtein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear. Here, we show that the E2 Rad6 associates and modifies ribosomes during stress. We further demonstrate that Rad6 and its human homolog UBE2A are redox regulated by forming a reversible disulfide with the E1 ubiquitin-activating enzyme (Uba1). This redox regulation is part of a negative feedback regulation, which controls the levels of K63 ubiquitination under stress. Finally, we show that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress, thus providing an additional physiological role for this multifunctional enzyme.

A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade

SummaryUbiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6∼ubiquitin thioester formation, and the Rad6–Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression.

SLFN11 Inactivation Induces Proteotoxic Stress and Sensitizes Cancer Cells to Ubiquitin Activating Enzyme Inhibitor TAK-243

Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1,978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2α, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than in WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors. SIGNIFICANCE: This study uncovers that SLFN11 deficiency induces proteotoxic stress and sensitizes cancer cells to TAK-243, suggesting that profiling SLFN11 status can serve as a therapeutic biomarker for cancer therapy.

Mode of inhibitory binding of epigallocatechin gallate to the ubiquitin-activating enzyme Uba1 via accelerated molecular dynamics.

The green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) and some of its analogs potently inhibit the ubiquitin-activating enzyme Uba1. In an effort to understand the possible molecular basis of inhibitory activity of EGCG, we conducted a molecular docking and molecular dynamics simulation study. We found that EGCG and its two selected analogs, (−)-epicatechin-3-gallate (ECG) and (−)-epigallocatechin (EGC), bind favorably at two likely hot spots for small-molecule ligand binding on human Uba1. The compounds bind with energetics that mirror their experimental potency for inhibition of Uba1∼ubiquitin thioester formation. The binding of EGCG, ECG, and EGC at one of the hot spots, in particular, recapitulates the rank order of potency determined experimentally and suggests a possible mechanism for inhibition. A hinge-like conformational change of the second catalytic cysteine domain and the opposing ubiquitin-fold domain observed during accelerated molecular dynamics simulations of the EGCG-bound Uba1 complex that results in disruption of the ubiquitin-binding interfaces could explain the compounds' inhibitory activity. These results shed light on the possible molecular mechanism of EGCG and related catechins in the inhibition of Uba1.

Multilevel structure\u2013activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure–activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum-associated degradation pathway.

Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum–associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)–resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin-­conjugating enzymes Ubc6 and Ubc7, and the ubiquitin–protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.

Administration of ubiquitin-activating enzyme UBA1 inhibitor PYR-41 attenuates angiotensin II-induced cardiac remodeling in mice

Pathological cardiac hypertrophy is the main risk factor for heart diseases. The ubiquitin-proteasome system (UPS) is the major intracellular protein degradation system involved in the development of cardiac hypertrophic remodeling. Ubiquitin-activating enzyme E1, a key component of the UPS, catalyzes the first step in ubiquitin conjugation to mark cellular proteins for degradation via proteasome. However, the functional role of E1 (UBA1) in regulation of hypertrophic remodeling in angiotensin II (Ang II)-infused mice remains unknown. In this study, male wild-type mice were treated with UBA1 inhibitor PYR-41 at two doses of 5 and 10 mg and infused with Ang II (1000 ng/kg/min) for 14 days. Systolic blood pressure was detected by using tail-cuff system. Cardiac function was assessed by echocardiography. Hypertrophic remodeling was analyzed examined by histological examinations. The expressions of genes and proteins were detected by quantitative real-time PCR and immunoblotting analysis. After 14 days, Ang II infusion significantly increased UBA1 expression at both mRNA and protein levels in the hearts. Furthermore, Ang II-infused mice showed a significant increase in systolic blood pressure compensatory cardiac function, hypertrophy, interstitial fibrosis, inflammation and oxidative stress compared with saline-treated controls, whereas these effects were dose-dependently attenuated in PYR-41-treated mice. These beneficial actions were associated mainly with inhibition of PTEN degradation and multiple downstream mediators (AKT, ERK1/2, STAT3, TGF-β/Smad2/3 and NF-kB(p65)). In conclusion, these results indicate that inhibition of UBA1 suppresses Ang II-induced hypertrophic remodeling, and suggest that administration of low dose PYR-41 may be a new potential therapeutic approach for treating hypertensive heart diseases.

Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling.

The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.

Multiple chemo-genetic interactions between a toxic metabolite and the ubiquitin pathway in yeast.

AICAR is the precursor of ZMP, a metabolite with antiproliferative properties in yeast and human. We aim at understanding how AICAR (and its active form ZMP) affects essential cellular processes. In this work, we found that ZMP accumulation is synthetic lethal with a hypomorphic allele of the ubiquitin-activating enzyme Uba1. A search for gene-dosage suppressors revealed that ubiquitin overexpression was sufficient to restore growth of the uba1 mutant upon AICAR treatment, suggesting that the ubiquitin pool is critical for cells to cope with AICAR. Accordingly, two mutants with constitutive low ubiquitin, ubp6 and doa1, were highly sensitive to AICAR, a phenotype that could be suppressed by ubiquitin overexpression. We established, by genetic means, that these new AICAR-sensitive mutants act in a different pathway from the rad6/bre1 mutants which were previously reported as sensitive to AICAR (Albrecht et al., Genetics 204:1447-1460, 2016). Two ubiquitin-conjugating enzymes (Ubc4 and Cdc34) and a ubiquitin ligase (Cdc4) were found to contribute to the ability of cells to cope with ZMP. This study illustrates the complexity of chemo-genetic interactions and shows how genetic analyses allow deciphering the implicated pathways, the individual gene effects, and their combined phenotypic contribution. Based on additivity and suppression patterns, we conclude that AICAR treatment shows synthetic interactions with distinct branches of the yeast ubiquitin pathway.

Ubiquitin System

Ever since the discovery of ubiquitin in 1975[...].

Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance

The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health [1], and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin [2]. Here we screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes. We identify the endosomal sorting complex required for transport (ESCRT) components TSG101 and Vps36, as well as the novel gene Vps13D. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, and mitochondrial clearance in Drosophila. Interestingly, a similar mitochondrial phenotype is observed in VPS13D mutant human cells. The ubiquitin-associated (UBA) domain of Vps13D binds K63 ubiquitin chains, and mutants lacking the UBA domain have defects in mitochondrial size and clearance and exhibit semi-lethality, highlighting the importance of Vps13D ubiquitin binding in both mitochondrial health and development. VPS13D mutant cells possess phosphorylated DRP1 and mitochondrial fission factor (MFF) as well as DRP1 association with mitochondria, suggesting that VPS13D functions downstream of these known regulators of mitochondrial fission. In addition, the large Vps13D mitochondrial and cell size phenotypes are suppressed by decreased mitochondrial fusion gene function. Thus, these results provide a previously unknown link between ubiquitin, mitochondrial size regulation, and autophagy.

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.

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function.

ABSTRACTCell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response.

A Novel in Vitro Three Organ System Assay Identifies a Small Molecule Ubiquitin Analog with in Vivo Activity Against Myeloma

Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one “sandwich” setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our “sandwich” assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb

The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.

Timely activation of budding yeast APCCdh1 involves degradation of its inhibitor, Acm1, by an unconventional proteolytic mechanism.

Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles can be established independent of proteolysis mediated by the APC and SCF enzymes.

Characterization of ubiquitin-activating enzyme Uba1 in the nucleus by its mammalian temperature-sensitive mutant.

Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.

Structure of the ubiquitin-activating enzyme loaded with two ubiquitin molecules.

The activation of ubiquitin by the ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent modification of target proteins with ubiquitin. This activation is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to a catalytic cysteine residue of Uba1 and the subsequent adenylation of a second ubiquitin. Here, a ubiquitin E1 structure loaded with two ubiquitin molecules is presented for the first time. While one ubiquitin is bound in its adenylated form to the active adenylation domain of E1, the second ubiquitin represents the status after transfer and is covalently linked to the active-site cysteine. The covalently linked ubiquitin enables binding of the E2 enzyme without further modification of the ternary Uba1-ubiquitin2 arrangement. This doubly loaded E1 structure constitutes a missing link in the structural analysis of the ubiquitin-transfer cascade.