Search

The bromodomain and extra-terminal (BET) family of proteins includes BRD2, BRD3, BRD4, and the testis-specific protein, BRDT, each containing two N-terminal tandem bromodomain (BRD) modules. Potent and selective inhibitors targeting the two bromodomains are required to elucidate their biological role(s), with potential clinical applications. In this study, we designed and synthesized a series of benzimidazole-6-sulfonamides starting from the azobenzene compounds MS436 (7 a) and MS611 (7 b) that exhibited preference for the first (BD1) over the second (BD2) BRD of BET family members. The most-promising compound (9 a) showed good binding potency and improved metabolic stability and selectivity towards BD1 with respect to the parent compounds., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells., Competing Interests: RF, CP, KL No competing interests declared, (© 2022, Fujisawa et al.)

DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition., (© 2021 Su et al.)

To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction., (© 2021 Doodhi et al.)

Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

Background and Aims: Little is known about genetic factors that affect development of alcohol-related cirrhosis. We performed a genome-wide association study (GWAS) of samples from the United Kingdom Biobank (UKB) to identify polymorphisms associated with risk of alcohol-related liver disease., Methods: We performed a GWAS of 35,839 participants in the UKB with high intake of alcohol against markers of hepatic fibrosis (FIB-4, APRI, and Forns index scores) and hepatocellular injury (levels of aminotransferases). Loci identified in the discovery analysis were tested for their association with alcohol-related cirrhosis in 3 separate European cohorts (phase 1 validation cohort; n=2545). Variants associated with alcohol-related cirrhosis in the validation at a false discovery rate of less than 20% were then directly genotyped in 2 additional European validation cohorts (phase 2 validation, n=2068)., Results: In the GWAS of the discovery cohort, we identified 50 independent risk loci with genome-wide significance (P < 5 × 10 -8 ). Nine of these loci were significantly associated with alcohol-related cirrhosis in the phase 1 validation cohort; 6 of these 9 loci were significantly associated with alcohol-related cirrhosis in phase 2 validation cohort, at a false discovery rate below 5%. The loci included variants in the mitochondrial amidoxime reducing component 1 gene (MARC1) and the heterogeneous nuclear ribonucleoprotein U like 1 gene (HNRNPUL1). After we adjusted for age, sex, body mass index, and type-2 diabetes in the phase 2 validation cohort, the minor A allele of MARC1:rs2642438 was associated with reduced risk of alcohol-related cirrhosis (adjusted odds ratio, 0.76; P=.0027); conversely, the minor C allele of HNRNPUL1:rs15052 was associated with an increased risk of alcohol-related cirrhosis (adjusted odds ratio, 1.30; P=.020)., Conclusions: In a GWAS of samples from the UKB, we identified and validated (in 5 European cohorts) single-nucleotide polymorphisms that affect risk of alcohol-related cirrhosis in opposite directions: the minor A allele in MARC1:rs2642438 decreases risk, whereas the minor C allele in HNRNPUL1:rs15052 increases risk., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.

Congenital dyserythropoietic anaemia (CDA) type I is a rare blood disorder characterised by moderate to severe macrocytic anaemia and hepatomegaly, with spongy heterochromatin and inter-nuclear bridges seen in bone marrow erythroblasts. The vast majority of cases of CDA type I are caused by mutations in the CDAN1 gene. The product of CDAN1 is Codanin-1, which interacts the histone chaperone ASF1 in the cytoplasm. Codanin-1 is a negative regulator of chromatin replication, sequestering ASF1 in the cytoplasm, restraining histone deposition and thereby limiting DNA replication. The remainder of CDA-I cases are caused by mutations in the C15ORF41 gene, but very little is known about the product of this gene. Here, we report that C15ORF41 forms a tight, near-stoichiometric complex with Codanin1 in human cells, interacting with the C-terminal region of Codanin-1. We present the characterisation of the C15ORF41-Codanin-1 complex in humans in cells and in vitro, and demonstrate that Codanin-1 appears to sequester C15ORF41 in the cytoplasm as previously shown for ASF1. The findings in this study have major implications for understanding the functions of C15ORF41 and Codanin-1, and the aetiology of CDA-I., (© 2020 The Author(s).)

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear., Objectives: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease., Methods: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples., Results: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein., Conclusion: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The vertebrate body forms by continuous generation of new tissue from progenitors at the posterior end of the embryo. The study of these axial progenitors has proved to be challenging in vivo largely because of the lack of unique molecular markers to identify them. Here, we elucidate the expression pattern of the transcription factor Nkx1-2 in the mouse embryo and show that it identifies axial progenitors throughout body axis elongation, including neuromesodermal progenitors and early neural and mesodermal progenitors. We create a tamoxifen-inducible Nkx1-2CreER T2 transgenic mouse and exploit the conditional nature of this line to uncover the lineage contributions of Nkx1-2 -expressing cells at specific stages. We show that early Nkx1-2 -expressing epiblast cells contribute to all three germ layers, mostly neuroectoderm and mesoderm, excluding notochord. Our data are consistent with the presence of some self-renewing axial progenitors that continue to generate neural and mesoderm tissues from the tail bud. This study identifies Nkx1-2 -expressing cells as the source of most trunk and tail tissues in the mouse and provides a useful tool to genetically label and manipulate axial progenitors in vivo ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC 50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention., (Copyright © 2018 American Society for Microbiology.)

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomized left-right body asymmetry. PCD is mostly caused by mutations affecting the core axoneme structure of motile cilia that is essential for movement. Genes that cause PCD when mutated include a group that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified two unrelated families (three affected children) with mutations in the uncharacterized C11orf70 gene (official gene name CFAP300). The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Tagged C11orf70 in Paramecium and Chlamydomonas localizes mainly in the cytoplasm with a small amount in the ciliary component. IFT139/TTC21B (IFT-A protein) and FLA10 (IFT kinesin) depletion experiments show that its transport within cilia is IFT dependent. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores., (© 2017 The Authors.)

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Pluripotent stem cells (PSCs) hold great clinical potential, as they possess the capacity to differentiate into fully specialised tissues such as pancreas, liver, neurons and cardiac muscle. However, the molecular mechanisms that coordinate pluripotent exit with lineage specification remain poorly understood. To address this question, we perform a small molecule screen to systematically identify novel regulators of the Smad2 signalling network, a key determinant of PSC fate. We reveal an essential function for BET family bromodomain proteins in Smad2 activation, distinct from the role of Brd4 in pluripotency maintenance. Mechanistically, BET proteins specifically engage Nodal gene regulatory elements (NREs) to promote Nodal signalling and Smad2 developmental responses. In pluripotent cells, Brd2-Brd4 occupy NREs, but only Brd4 is required for pluripotency gene expression. Brd4 downregulation facilitates pluripotent exit and drives enhanced Brd2 NRE occupancy, thereby unveiling a specific function for Brd2 in differentiative Nodal-Smad2 signalling. Therefore, distinct BET functionalities and Brd4-Brd2 isoform switching at NREs coordinate pluripotent exit with lineage specification., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK 169 DE 171 ) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by a covalent post-translational modification.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4 BD2 ). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4 BD2 . Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity., Competing Interests: The authors declare no conflict of interest.

Left ventricular (LV) hypertrophy (LVH) is a heritable trait that is common in type 2 diabetes and is associated with the development of heart failure. The transcriptional factor Kruppel like factor 15 (KLF15) is expressed in the heart and acts as a repressor of cardiac hypertrophy in experimental models. This study investigated if KLF15 gene variants were associated with LVH in type 2 diabetes. In stage 1 of a 2-stage approach, patients with type 2 diabetes and no known cardiac disease were prospectively recruited for a transthoracic echocardiographic assessment (Melbourne Diabetes Heart Cohort) (n=318) and genotyping of two KLF15 single nucleotide polymorphisms (SNPs) (rs9838915, rs6796325). In stage 2, the association of KLF15 SNPs with LVH was investigated in the Genetics of Diabetes Audit and Research in Tayside Scotland (Go-DARTS) type 2 diabetes cohort (n=5631). The KLF15 SNP rs9838915 A allele was associated in a dominant manner with LV mass before (P=0.003) and after (P=0.001) adjustment for age, gender, body mass index (BMI) and hypertension, and with adjusted septal (P<0.0001) and posterior (P=0.004) wall thickness. LVH was present in 35% of patients. Over a median follow up of 5.6years, there were 22 (7%) first heart failure hospitalizations. The adjusted risk of heart failure hospitalization was 5.5-fold greater in those with LVH and the rs9838915 A allele compared to those without LVH and the GG genotype (hazard ratio (HR) 5.5 (1.6-18.6), P=0.006). The association of rs9838915 A allele with LVH was replicated in the Go-DARTS cohort. We have identified the KLF15 SNP rs9838915 A allele as a marker of LVH in patients with type 2 diabetes, and replicated these findings in a large independent cohort. Studies are needed to characterize the functional importance of these results, and to determine if the SNP rs9838915 A allele is associated with LVH in other high risk patient cohorts., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The BRE (brain and reproductive expression) gene, highly expressed in nervous and reproductive system organs, plays an important role in modulating DNA damage repair under stress response and pathological conditions. Folliculogenesis, a process that ovarian follicle develops into maturation, is closely associated with the interaction between somatic granulosa cell and oocyte. However, the regulatory role of BRE in follicular development remains undetermined. In this context, we found that BRE is normally expressed in the oocytes and granulosa cells from the primordial follicle stage. There was a reduction in follicles number of BRE mutant (BRE -/- ) mice. It was attributed to increase the follicular atresia in ovaries, as a result of retarded follicular development. We established that cell proliferation was inhibited, while apoptosis was markedly increased in the granulosa cells in the absence of BRE. In addition, expressions of γ-H2AX (marker for showing DNA double-strand breaks) and DNA damage-relevant genes are both upregulated in BRE -/- mice. In sum, these results suggest that the absence of BRE, deficiency in DNA damage repair, causes increased apoptosis in granulosa cells, which in turn induces follicular atresia in BRE -/- mice.

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper β-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRβ and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at β-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Embryonic stem cells (ESCs) can self-renew or differentiate into any cell type, a phenomenon known as pluripotency. Distinct pluripotent states, termed naive and primed pluripotency, have been described. However, the mechanisms that control naive-primed pluripotent transition are poorly understood. Here, we perform a targeted screen for kinase inhibitors, which modulate the naive-primed pluripotent transition. We find that XMD compounds, which selectively inhibit Erk5 kinase and BET bromodomain family proteins, drive ESCs toward primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, we reveal distinct functions for Erk5 and Brd4 in pluripotency regulation. We show that Erk5 signaling maintains ESCs in the naive state and suppresses progression toward primed pluripotency and neuroectoderm differentiation. Additionally, we identify a specialized role for Erk5 in defining ESC lineage selection, whereby Erk5 inhibits a cardiomyocyte-specific differentiation program. Our data therefore reveal multiple critical functions for Erk5 in controlling ESC identity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways.

Several human diseases, including cancer, show altered signaling pathways resulting from changes in the activity levels of epigenetic modulators. In the past few years, small-molecule inhibitors against specific modulators, including the bromodomain and extra-terminal (BET) bromodomain family of acetylation readers, have shown early promise in the treatment of the genetically defined midline carcinoma and hematopoietic malignancies. We have recently developed a novel potent inhibitor of BET proteins, 1 (XD14[ Angew. Chem., Int. Ed. 2013, 52, 14055]), which exerts a strong inhibitory potential on the proliferation of specific leukemia cell lines. In the study presented here, we designed analogues of 1 to study the potential of substitutions on the 4-acyl pyrrole backbone to occupy additional sites within the substrate recognition site of BRD4(1). The compounds were profiled using ITC, DSF, and X-ray crystallography. We could introduce several substitutions that address previously untargeted areas of the substrate recognition site. This work may substantially contribute to the development of therapeutics with increased target specificity against BRD4-related malignancies.

We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra-terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl rings, in addition to the diazepine ternary center and the side chain methylene moiety. Substitution at the level of the side chain methylene afforded compounds targeting specifically and potently engineered BET bromodomains designed as part of a bump and hole approach. We further demonstrate that marked selectivity for the second over the first bromodomain can be achieved with an indole derivative that exploits differential interaction with an aspartate/histidine conservative substitution on the BC loop of BET bromodomains.

Promyelocytic leukemia (PML) protein forms the basis of PML nuclear bodies (PML NBs), which control many important processes. We have screened an shRNA library targeting ubiquitin pathway proteins for effects on PML NBs, and identified RNF8 and RNF168 DNA-damage response proteins as negative regulators of PML NBs. Additional studies confirmed that depletion of either RNF8 or RNF168 increased the levels of PML NBs and proteins, whereas overexpression induced loss of PML NBs. RNF168 partially localized to PML NBs through its UMI/MIU1 ubiquitin-interacting region and associated with NBs formed by any PML isoform. The association of RNF168 with PML NBs resulted in increased ubiquitylation and SUMO2 modification of PML. In addition, RNF168 was found to associate with proteins modified by SUMO2 and/or SUMO3 in a manner dependent on its ubiquitin-binding sequences, suggesting that hybrid SUMO-ubiquitin chains can be bound. In vitro assays confirmed that RNF168, preferentially, binds hybrid SUMO2-K63 ubiquitin chains compared with K63-ubiquitin chains or individual SUMO2. Our study identified previously unrecognized roles for RNF8 and RNF168 in the regulation of PML, and a so far unknown preference of RNF168 for hybrid SUMO-ubiquitin chains., (© 2016. Published by The Company of Biologists Ltd.)

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.

The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.

RING E3 ligase-catalyzed formation of K63-linked ubiquitin chains by the Ube2V2-Ubc13 E2 complex is required in many important biological processes. Here we report the structure of the RING-domain dimer of rat RNF4 in complex with a human Ubc13∼Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with K63 in a position favorable for attack on the linkage between Ubc13 and the donor (second) ubiquitin held in the active 'folded back' conformation by the RING domain of RNF4. We verified the interfaces identified in the structure by in vitro ubiquitination assays of site-directed mutants. To our knowledge, this represents the first view of synthesis of K63-linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase-mediated catalysis.

During the early steps of ribosome biogenesis in mammals, the two ribosomal subunits 40S and 60S are produced via splitting of the large 90S pre-ribosomal particle (90S) into pre-40S and pre-60S pre-ribosomal particles (pre-40S and pre-60S). We previously proposed that replacement of fibrillarin by Nop52 (RRP1/NNP-1) for the binding to p32 (C1QBP) is a key event that drives this splitting process. However, how the replacement by RRP1 is coupled with the endo- and/or exo-ribonucleolytic cleavage of pre-rRNA remains unknown. In this study, we demonstrate that RRP1 deficiency suppressed site 2 cleavage on ITS1 of 47S/45S, 41S and 36S pre-rRNAs in human cells. RRP1 was also present in 90S and was localized in the dense fibrillar component of the nucleolus dependently on active RNA polymerase I transcription. In addition, double knockdown of XRN2 and RRP1 revealed that RRP1 accelerated the site 2 cleavage of 47S, 45S and 41S pre-rRNAs. These data suggest that RRP1 is involved not only in competitive binding with fibrillarin to C1QBP on 90S but also in site 2 cleavage in ITS1 of pre-rRNAs at early stages of human ribosome biogenesis; thus, it is likely that RRP1 integrates the cleavage of site 2 with the physical split of 90S into pre-40S and pre-60S., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

PLK (Polo-like kinase) inhibitors, such as BI-2536, have been reported to suppress IFNB (encoding IFNβ, interferon β) gene transcription induced by ligands that activate TLR3 (Toll-like receptor 3) and TLR4. In the present study, we found that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 (interferon-regulatory factor 3) and c-Jun with the IFNB promoter, but without affecting the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}-catalysed phosphorylation of IRF3 at Ser³⁹⁶, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2 (activating transcription factor 2). Although BI-2536 inhibits few other kinases tested, it interacts with BET (bromodomain and extra-terminal) family members and displaces them from acetylated lysine residues on histones. We found that BET inhibitors that do not inhibit PLKs phenocopied the effect of BI-2536 on IFNB gene transcription. Similarly, BET inhibitors blocked the interaction of IRF5 with the IFNB promoter and the secretion of IFNβ induced by TLR7 or TLR9 ligands in the human plasmacytoid dendritic cell line GEN2.2, but without affecting the nuclear translocation of IRF5. We found that the BET family member BRD4 (bromodomain-containing protein 4) was associated with the IFNB promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors. Our results establish that BET family members are essential for TLR-stimulated IFNB gene transcription by permitting transcription factors to interact with the IFNB promoter. They also show that the interaction of the IFNB promoter with BRD4 is regulated by TLR ligation and that BI-2536 is likely to suppress IFNB gene transcription by targeting BET family members., (© 2015 The Author(s).)

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.

p63 and p73, the two other members of the p53 family, were identified almost 15 years ago. Here, we review their potential use for diagnosis, prognosis and prediction of response to therapy in various cancers. The two genes show distinct expression patterns in both normal and cancer tissues and each gene gives rise to multiple protein isoforms with different activities, including those with tumour-suppressor or oncogenic effects. Despite such complexity, some common themes emerge; p63 is commonly overexpressed as the ΔNp63 isoform and sometimes associated with TP63 amplification, whereas p73 is often reduced (by methylation or gene loss), or there is an increase in the ratio of ΔNp73 to TAp73. These generalisations do not apply universally; TAp63 is overexpressed in haematological malignancies, TP63 mis-sense mutations have been reported in squamous cancers and TP63 translocations occur in lymphomas and some lung adenocarcinomas. There are associations with disease prognosis and response to specific therapies in individual cancer types for both p63 and p73, making their analysis of clinical relevance. We also discuss their utility for aiding in differential diagnosis, which has been demonstrated for p63, but not yet for p73. Throughout, we highlight the discrepant nature of many studies due to the variable methodologies employed, the lack of systematic evaluation of isoforms and the problems of poor antibody characterization and cross-reactions within the p63/p73 family. Finally, we emphasize the value of recently developed isoform-specific reagents that have clear advantages for the study of p63 and p73 experimentally and clinically.

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

Glucocorticoids play an important role in the treatment of inflammation and immune disorders, despite side effects, which include metabolic derangements such as central adiposity. These studies examine the role of protein phosphatase 5 (Ppp5) in glucocorticoid receptor (GR) complexes which mediate response to glucocorticoids. Mice homozygous for inactivated Ppp5 (Ppp5D274A/D274A) exhibit decreased adipose tissue surrounding the gonads and kidneys compared with wild-type mice. Adipocyte size is smaller, more preadipocytes/stromal cell are present in their gonadal fat tissue and differentiation of preadipocytes to adipocytes is retarded. Glucocorticoid levels are raised and the GR is hyperphosphorylated in adipose tissue of Ppp5D274A/D274A mice at Ser212 and Ser220 (orthologous to human Ser203 and Ser211) in the absence of glucocorticoids. Preadipocyte cultures from Ppp5D274A/D274A mice show decreased down regulation of Delta-like protein-1/preadipocyte factor-1, hyperphosphorylation of extra-cellular signal regulated kinase 2 (ERK2) and increased concentration of (sex determining region Y)-box 9 (SOX9), changes in a pathway essential for preadipocyte differentiation, which leads to decreased concentrations of the transcription factors CEBPβ and CEBPα necessary for the later stages of adipogenesis. The data indicate that Ppp5 plays a crucial role in modifying GR-mediated initiation of adipose tissue differentiation, suggesting that inhibition of Ppp5 may potentially be beneficial to prevent obesity during glucocorticoid treatment.

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs., (© 2014 Bizarro et al.)

Small molecules are useful tools for probing the biological function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The Bromo and Extra-Terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-molecule inhibitors, but these inhibitors lack selectivity for individual family members. We developed an ethyl derivative of an existing small-molecule inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiology and disease., (Copyright © 2014, American Association for the Advancement of Science.)

Chromosome replication is initiated by a universal mechanism in eukaryotic cells, involving the assembly and activation at replication origins of the CMG (Cdc45-MCM-GINS) DNA helicase, which is essential for the progression of replication forks. Disassembly of CMG is likely to be a key regulated step at the end of chromosome replication, but the mechanism was unknown until now. Here we show that the ubiquitin ligase known as SCF(Dia2) promotes ubiquitylation of CMG during the final stages of chromosome replication in Saccharomyces cerevisiae. The Cdc48/p97 segregase then associates with ubiquitylated CMG, leading rapidly to helicase disassembly. These findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step., (Copyright © 2014, American Association for the Advancement of Science.)

The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.

Background: FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons., Results: We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation., Conclusions: The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings indicate that the repertoire of VAPB interactors is more diverse than previously anticipated and link VAPB to the function of ATPase complexes such as p97/FAF1 and ASNA1/TRC.