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HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

There is some evidence supporting an association between Cullin-5 (CUL5) and cancer, but no research using pan-cancer analysis has been conducted previously. We therefore investigated the oncogenic role of CUL5 in 33 tumors from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Many cancers reduce CUL5 levels, and the prognosis of certain cancers is vitally linked with CUL5 expression. CUL5 expression is associated with CD8 + T-cell infiltration levels in uveal melanomas and head and neck squamous cell carcinomas, and we observed a positive relationship between CUL5 and Tcm (T central memory) cells, and a negative relationship between T helper (Th) cells and pDC (plasmacytoid DC). CUL5 had negative associations with NK cells, NK CD56bright cells, NK CD56dim cells, Tregs, cytotoxic cells, and Th17 cells. Functions relating to protein processing and ubiquitin were included in the CUL5 functional mechanisms. The top 100 genes that are most strongly related to CUL5 were identified, and enrichment analysis indicated that the biological process with the closest relationship was neddylation, related pathways included the TGF-beta signaling pathway and intracellular receptor signaling pathway. CUL5 is related to biological cell behaviors such as chromosome segregation and positive regulation of chromosome organization. As the first study to perform a pan-cancer analysis of CUL5, the present findings will improve the understanding of the oncogenic role of CUL5 in different tumors., Graphical abstract

The BH3-only pro-apoptotic protein BIK is regulated by the ubiquitin–proteasome system. Chen et al. identify ASB11 as an E3 ligase for BIK ubiquitination and degradation and uncover different mechanisms that regulate ASB11 activity, and thus cell survival versus death, during ER stress and DNA damage responses. Blocking this degradative pathway may increase the efficacy of active BIK gene therapy as an anti-cancer therapy., The BH3-only pro-apoptotic protein BIK is regulated by the ubiquitin–proteasome system. However, the mechanism of this regulation and its physiological functions remain elusive. Here, we identify Cul5-ASB11 as the E3 ligase targeting BIK for ubiquitination and degradation. ER stress leads to the activation of ASB11 by XBP1s during the adaptive phase of the unfolded protein response, which stimulates BIK ubiquitination, interaction with p97/VCP, and proteolysis. This mechanism of BIK degradation contributes to ER stress adaptation by promoting cell survival. Conversely, genotoxic agents down-regulate this IRE1α–XBP1s–ASB11 axis and stabilize BIK, which contributes in part to the apoptotic response to DNA damage. We show that blockade of this BIK degradation pathway by an IRE1α inhibitor can stabilize a BIK active mutant and increase its anti-tumor activity. Our study reveals that different cellular stresses regulate BIK ubiquitination by ASB11 in opposing directions, which determines whether or not cells survive, and that blocking BIK degradation has the potential to be used as an anti-cancer strategy.

HSP90 inhibitors have the potential to treat many types of cancer due to the dependence of tumor cells on HSP90 for cell growth and proliferation. The Cullin-5 (Cul5) E3 ubiquitin ligase is required for HSP90 inhibitors to induce client protein degradation and subsequent cell death. Cul5 is expressed at low levels in breast cancer cells compared to patient matched controls. This observed low Cul5 expression may play a role in the reported decreased efficacy of 17-AAG and related HSP90 inhibitors as a monotherapy. We have developed a method for delivery of 17-AAG plus Cul5 DNA to cells via gold nanoparticles (AuNPs). Delivery of AuNPs containing Cul5 DNA increases the sensitivity of Cul5 deficient AU565 cells to 17-AAG. Characterization of AuNPs by UV-vis spectrum, TEM, gel electrophoresis assay and (1)H NMR indicate attachment of both 17-AAG and DNA payload as well as AuNP stability. Studies in Cul5 deficient AU565 cells reveal that delivery of Cul5 and 17-AAG together increase cytotoxicity. Our results provide evidence that delivery of DNA with drug may serve as a method to sensitize drug resistant tumor cells.

Background The E3 Cullin 5-RING ubiquitin ligase (CRL5) is a multiprotein complex that has recently been highlighted as a major regulator of central nervous system development. Cullin 5 (Cul5) and the RING finger protein Rbx2 are two CRL5 core components required for CRL5 function in the brain, but their full expression patterns and developmental functions have not been described in detail. Results Using a gene-trap mouse model for Cul5 and a knock-in-knockout mouse model for Rbx2, we show that lack of Cul5, but not Rbx2, disrupts blastocyst formation. However, Rbx2 is required for embryo survival at later embryonic stages. We also show that cul5 is expressed in the embryo proper as early as E7.5 and its expression is mostly restricted to the central nervous system and limbs at later time points. Finally, we show that rbx2 and cul5 are co-expressed in most areas of the brain during development and in the adult. Conclusions Our results show that Cul5, but not Rbx2, is required during early embryogenesis and suggests that Cul5 has Rbx2-independent functions in early development. In the brain, Cul5 and Rbx2 are expressed in a similar fashion, allowing the nucleation of an active CRL5 complex. Developmental Dynamics 247:1227-1236, 2018. © 2018 Wiley Periodicals, Inc.

An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting., Structures and biochemistry reveal how covalent linkage of a ubiquitin-like protein elicits protein–protein interactions indirectly. NEDD8 allosterically activates CUL5-RBX2 cullin-RING E3 binding to the ARIH2 RBR-type E3 for joint ubiquitylation.

IntroductionThis study was undertaken to examine the anticancer effects of the natural product rhein on human leukaemia cells.Material and methodsCell viability was determined by MTT assay. Immunofluorescence staining, DAPI, AO/EB, and annexin V/PI staining were used for the detection of apoptosis. qRT-PCR analysis was used to examine protein expression. Western blot analysis was used to determine protein expression.ResultsThe results revealed that rhein caused a significant (p < 0.05) and dose-dependent decrease in the viability of AML-193 leukaemia cells. It was also revealed that rhein caused morphological changes such as cell rounding in rhein-treated AML-193 cells. The DAPI and AO/EB staining showed that rhein caused remarkable changes in the nuclear morphology of the AML-193 cells, characteristic of apoptosis. The percentage of apoptosis was found to be 3.12%, 14.80%, 30.31%, and 60.85% at the concentrations of 0, 3.5, 7, and 14 µM of rhein, respectively. The expression of Bax, cleaved caspase-3, 9, and cleaved PARP increased concentration dependently, while the expression of Bcl-2 decreased. The effects of rhein were also examined on 11 different miRs, and it was found that rhein specifically and significantly (p < 0.05) inhibited the expression of miR-27. Additionally, inhibition of miR-27 resulted in the decrease of AML-193 viability and upregulation of Cullin 5 (CUL5).ConclusionsTaken together, rhein triggers apoptosis in leukaemia cells and modulates the miR-27/CUL5 axis. As such, rhein may prove to be beneficial in the treatment of leukaemia.

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen–deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker., Multi-subunit Cullin (CUL)-RING ligases (CRL) form the largest family of E3 ligases and are composed of a substrate receptor, a CUL, and a RING-box (RBX) protein. Here, the authors use cryo-EM and HDX-MS to characterise the ASB9 CUL-RING E3 ligase and present the structure of ASB9-ELOB/C bound to the substrate creatine kinase and the full-length CUL5 structure in complex with RBX2, and they propose a revised allosteric mechanism for CUL-E3 ligase function.

Viral infectivity factor (Vif) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of APOBEC3 proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and CBF-β, forming a four-protein complex called VCBC. The structure of VCBC in complex with the Cullin5 (Cul5) protein has been solved by X-ray crystallography, and recently, using molecular dynamic (MD) simulations, the dynamics of VCBC and VCBC-Cul5 complexes were characterized. Here, we applied time-lapse high-speed atomic force microscopy (HS-AFM) to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of the VCBC complex, which we identified as triangle, dumbbell, and globular structures. In addition, we characterized the dynamics of each of these structures. While our data show a very dynamic behavior for all these structures, we found the triangle and dumbbell structures to be the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and support further research into the improvement of HIV treatment, as Vif is essential for virus survival in the cell.

Cullin-RING ligase 5 (CRL5) is a multi-protein complex and consists of a scaffold protien cullin 5, a RING protein RBX2 (also known as ROC2 or SAG), adaptor proteins Elongin B/C, and a substrate receptor protein SOCS. Through targeting a variety of substrates for proteasomal degradation or modulating various protein-protein interactions, CRL5 is involved in regulation of many biological processes, such as cytokine signal transduction, inflammation, viral infection, and oncogenesis. As many substrates of CRL5 are well-known oncoproteins or tumor suppressors, abnormal regulation of CRL5 is commonly found in human cancers. In this review, we first briefly introduce each of CRL5 components, and then discuss the biological processes regulated by four members of SOCS-box-containing substrate receptor family through substrate degradation. We next describe how CRL5 is hijacked by a variety of viral proteins to degrade host anti-viral proteins, which facilitates virus infection. We further discuss the regulation of CUL5 and its various roles in human cancers, acting as either a tumor suppressor or an oncoprotein in a context-dependent manner. Finally, we propose novel insights for future perspectives on the validation of cullin5 and other CRL5 components as potential targets, and possible targeting strategies to discover CRL5 inhibitors for anti-cancer and anti-virus therapies.

Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-α only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-α signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-α-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-α-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIBΔVif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-α-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-α pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIBΔVif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be essential for Vif-mediated proteasomal degradation of STAT1 and STAT3. These results reveal a target for HIV-1-Vif and demonstrate how HIV-1 impairs the anti-viral activity of Type 1 IFNs, possibly explaining why both endogenous and therapeutic IFN-α fail to activate more effective control over HIV infection., Highlights • HIV-Vif protein targets Type 1 IFN JAK/STAT pathway proteins (STAT1 and STAT3) for proteasomal degradation. • HIV-Vif uses the host ECS E3 ligase complex to degrade STAT1 and STAT3. • HIV-Vif protein blocks IFN-α-induced STAT phosphorylation and anti-viral ISG induction. Even though Type I IFNs are strongly expressed upon viral infection, IFN-α fails to cure HIV. Our study reveals that the HIV-Vif protein mediates inhibition of IFN-α's anti-viral responses through degradation of its JAK/STAT signalling cascade. These results outline the mechanism by which HIV blocks this essential anti-viral pathway and may explain why this powerful cytokine cannot eliminate the virus. These discoveries make a paradigm shift in our understanding of HIV-mediated immune evasion and may be fundamental in our global quest to develop new, immune restoring, therapy against HIV.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Upon investigation, it was discovered that 4 out of 8 images contain fabrication. Fig. 6D, E, 8E and 7F have used the same microscopic image for different experimental settings. The Figure represented in 8E is the correct image placement. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.

The APOBEC3 (A3) family of cellular cytidine deaminases comprises seven members (A, B, C, D, F, G, and H) that potently inhibit retroviral replication. Human immunodeficiency virus type 1 (HIV-1) Vif is a small pleiotropic protein that specifically inactivates these enzymes, targeting them for ubiquitin-mediated proteasomal degradation. A3 Vif-interaction sites are presumed to fall into three distinct types: A3C/D/F, A3G, and A3H. To date, two types of A3G and A3C/D/F sites have been well characterized, whereas the A3H Vif-binding site remains poorly defined. Here, we explore the residues critical for the A3H-type Vif interaction. To avoid technical difficulties in performing experiments with human A3H haplotype II (hapII), which is relatively resistant to HIV-1 Vif, we employed its ortholog chimpanzee A3H (cA3H), which displays high Vif sensitivity, for a comparison of sensitivity with that of A3H hapII. The Vif susceptibility of A3H hapII-cA3H chimeras and their substitution mutants revealed a single residue at position 97 as a major determinant for the difference in their Vif sensitivities. We further surveyed critical residues by structure-guided mutagenesis using an A3H structural model and thus identified eight additional residues important for Vif sensitivity, which mapped to the α3 and α4 helices of A3H. Interestingly, this area is located on a surface adjacent to the A3G and A3C/D/F interfaces and is composed of negatively charged and hydrophobic patches. These findings suggest that HIV-1 Vif has evolved to utilize three dispersed surfaces for recognizing three types of interfaces on A3 proteins under certain structural constraints.

Conjugation of Nedd8 (neddylation) to Cullins (Cul) in Cul-RING E3 ligases (CRLs) stimulates ubiquitination and polyubiquitination of protein substrates. CRL is made up of two Cul-flanked arms: one consists of the substrate-binding and adaptor proteins and the other consists of E2 and Ring-box protein (Rbx). Polyubiquitin chain length and topology determine the substrate fate. Here, we ask how polyubiquitin chains are accommodated in the limited space available between the two arms and what determines the polyubiquitin linkage topology. We focus on Cul5 and Rbx1 in three states: before Cul5 neddylation (closed state), after neddylation (open state), and after deneddylation, exploiting molecular dynamics simulations and the Gaussian Network Model. We observe that regulation of substrate ubiquitination and polyubiquitination takes place through Rbx1 rotations, which are controlled by Nedd8–Rbx1 allosteric communication. Allosteric propagation proceeds from Nedd8 via Cul5 dynamic hinges and hydrogen bonds between the C-terminal domain of Cul5 (Cul5CTD) and Rbx1 (Cul5CTD residues R538/R569 and Rbx1 residue E67, or Cul5CTD E474/E478/N491 and Rbx1 K105). Importantly, at each ubiquitination step (homogeneous or heterogeneous, linear or branched), the polyubiquitin linkages fit into the distances between the two arms, and these match the inherent CRL conformational tendencies. Hinge sites may constitute drug targets.

Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFβ) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFβ. Only mutants that retained the ability to bind CBFβ exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFβ and wild-type Vif. Furthermore, overexpression of CBFβ counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFβ as the key player involved in D/N interference by Vif. IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFβ), which is expressed in cells in limiting quantities. Overexpression of CBFβ neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFβ, phenocopied the D/N Vif phenotype by sequestering endogenous CBFβ. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.

Summary The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFs complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the μs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL are frequently observed in many cancers. Inhibitors targeting MCL1 are in clinical development, however numerous cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens interrogating two drugs that directly (MCL1i) or indirectly (CDK9i) target MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components was responsible for re-sensitization to CDK9 inhibitor, but not MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to multiple types of anticancer agents suggests the potential to improve combination treatments., eLife digest Organisms keep their tissues healthy by instructing damaged or unwanted cells to kill themselves via a controlled process known as apoptosis. Cancer cells, however, are able to evade death by increasing the level of proteins that block apoptosis, such as MCL1. Researchers have recently developed new drugs that can inhibit the action of the MCL1 protein. But a number of cancers have become resistant to these inhibitors. So, one important question is whether other proteins in cancer cells could be drugged, together with MCL1, to overcome or even avoid this resistance. Now, Kabir et al. have addressed this question by searching the genome of human lung cancer cells, which were resistant to treatment, for targets that could improve the performance of two MCL1 inhibitors. This involved reducing the level of every protein in these cells one by one using a genetic technique known as CRISPR-Cas9, and looking for cells that lost their resistance to the MCL1 inhibitor. From these genetic screens, Kabir et al. identified three proteins that are part of complex called CRL5. Inactivating this protein complex caused cancer cells to become more sensitive to the MCL1 inhibitor. Further biochemical experiments showed that CRL5 may contribute to drug resistance by reducing the levels of two proteins that promote apoptosis. These findings suggest that inhibiting CRL5 in combination with MCL1 could combat drug resistance. Although there are currently no drugs against CRL5, future experiments determining how CRL5 and MCL1 are linked could identify new drug targets and improve existing cancer treatments.

Clear cell renal cell carcinoma (ccRCC) is intimately associated with defects in ubiquitin-mediated protein degradation. Herein, we report that deficiency in the E3 ligase subunit cullin 5 (CUL5) promotes chromosomal instability and is an independent negative prognostic factor in ccRCC. CUL5 was initially identified in an RNA interference screen as a novel regulator of centrosome duplication control. We found that depletion of CUL5 rapidly promotes centriole overduplication and mitotic errors. Downregulation of CUL5 also caused an increase of DNA damage that was found to involve impaired DNA double-strand break repair. Using immunohistochemistry, CUL5 protein expression was found to be below detection level in the majority of RCCs. A re-analysis of the TCGA ccRCC cohort showed that a reduced CUL5 gene expression or CUL5 deletion were associated with a significantly worse overall patient survival. In conclusion, our results indicate that CUL5 functions as a novel tumor suppressor with prognostic relevance in ccRCC and is critically involved in the maintenance of genome stability.

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.

Endoplasmic reticulum protein 29 (ERp29), an endoplasmic reticulum (ER) protein, participates in ER stress (ERS), but little is known about the association of ERp29 with ERS in the metastasis and prognosis of cancerous diseases. The present study revealed that ERp29 was important to ERS and interfered with the malignant behaviors of colorectal cancer (CRC). Experiments in in vitro and in animal models revealed that ERS inhibited the cell growth and suppressed the metastatic capacity of CRC cells, but ERp29 counteracted these effects. Furthermore, it was demonstrated that ERp29 recovered the migration and metastatic behaviors of CRC cells suppressed by ERS, mediated only when it combined with cullin5 (CUL5). ERp29 also relied on CUL5 to promote epithelial-mesenchymal transition. From the immunohistochemical examination of CRC tissues, the high expression of ERp29 was revealed to predict the poor prognosis of 457 CRC cases. The retrospective analysis of the clinicopathological data of patients with CRC was consistent with the results of the in vitro and in vivo experiments. Thus, ERp29 protected CRC cells from ERS-mediated reduction of malignancy to promote metastasis and may be a potential target of medical intervention for CRC therapy.

HIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 antiviral proteins. Vif folds when it binds the Cullin-RING E3 ligase CRL5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use Molecular Dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and APOBEC3 bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, while VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation optimized spectroscopy (methyl-TROSY) NMR data, which indicates that the entire VCBC complex without CUL5 is dynamic on the μs-ms timescale. Vif binds APOBEC3 to recruit it to the complex, and methyl-TROSY NMR shows that the VCBC complex is more conformationally restricted when bound to APOBEC3F, consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjuction with the global dynamics of the complex. Like other ubiquitin substrate receptors, VCBC can exist alone or in complex with CUL5 in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Gastric cancer is one of the prevalent types of cancers and despite improvements in its treatment, the overall survival is still far from descent. The dearth of efficient biomarkers, chemotherapeutic agents and therapeutic targets form a major hurdle in the treatment of the gastric cancer. Accumulating evidences suggest that MicroRNAs (miRs) may prove important therapeutic targets/agents for the management of cancers including gastric cancer. Herein, we examined the expression of miR-19a by qRT-PCR in gastric cancer and attempted to explore its potential role. It was found that the expression of miR-19a is significantly (p 0.05) enhanced in the gastric cancer tissues as well as the gastric cancer cell lines. Inhibition of miR-19a in gastric cancer cells suppressed the proliferation migration and invasion of the gastric cancer cells. Bioinformatic analysis revealed CUL5 to be the potential target of miR-19a. Contrary, to the expression of miR-19a, the expression of CUL5 was significantly (p 0.05) downregulated in all the gastric cancer tissues and cell lines. However, inhibition of miR-19a in SNU-16 gastric cancer cells could cause upsurge of CUL5 expression. Overexpression of CUL-5 was found to exhibit similar effects on the proliferation, migration and invasion of the SNU-16 gastric cancer cells as that of miR-19a suppression. Additionally, overexpression of CUL5 could at least partially abolish the effects of miR-19a suppression on the proliferation, migration and invasion of SNU-16 gastric cancer cells. Finally, overexpression of miR-19a caused inhibition of the xenografted tumors in vivo indicating the potential of miR-19a as therapeutic target for gastric cancer.

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin-RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi-visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates., Graphical Abstract, Highlights • ARIH2 is required for ASB9CRL to polyubiquitylate 4/18 lysines on one creatine kinase subunit. • HDX-MS reveals long-range allosteric opening of a cleft in CUL5 where the ARIH2 RBRL binds. • HDX-MS reveals that neddylation of CUL5 alters the RBX2 conformation away from binding the E2∼NEDD8. • HDX-MS reveals opening of the ARIH2 active site upon binding CUL5, thus releasing its autoinhibition., In Brief Hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals how a CUL5 E3 ligase is activated for polyubiquitylation of its substrate.

E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif.IMPORTANCETwo early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of theHuman mastadenovirus A,F, andGspecies. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

Altered expression of cullin-5 (CUL5), a member of the cullin-RING E3 ubiquitin ligase family, has been implicated in a number of types of cancers including breast, cervical and hepatocellular cancers. In the present study, we found that CUL5 expression was significantly decreased in both endometrioid and serous endometrial adenocarcinomas with the more aggressive serous type displaying a higher reduction (-4.3-fold) than the less aggressive endometrioid type (-2.9-fold). Overexpression of CUL5 mRNA and protein in Ishikawa H endometrial cancer cells resulted in decreased cell proliferation and in a reduction in CUL5-RING E3 ligase downstream clients JAK2 and FAS-L. Finally, we demonstrated for the first time that CUL5 is a direct target of miR-182 that we previously showed to be significantly overexpressed in endometrial adenocarcinomas and we provided evidence that increased miR-182 expression is, at least in part, a result of demethylation of its upstream promoter. These data suggest a cascade in which miR-182 expression is epigenetically increased leading to decreased CUL5 expression and increased cellular proliferation. The final step in the cascade may be operating through a decrease in ubiquitination of pro-growth CUL5 ubiquitin ligase clients. This cascade offers a series of potential interventional steps involving epigenetic modification, miRNA and/or gene targeting and ubiquitination.