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Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4‐DDB1‐RBX1 E3 ligase to change its substrate specificity and induce degradation of ‘neosubstrate’ transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2‐dependent proximity labelling (BioID2‐CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2‐CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating ‘neointeractors’ distinct from previously disclosed ‘neosubstrates’. Here we identify Non‐Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3‐ligase‐modulating drugs. [ABSTRACT FROM AUTHOR]

The article presents a study which explores that Tankyrase-mediated ADP-ribosylation, a regulator of Tumor necrosis factor (TNF)-induced death . It mentions that findings of the study suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

The article presents a study which explores about the Tankyrase-mediated ADP-ribosylation as a regulator of Tumor necrosis factor (TNF)-induced death. It mentions that TNF, a key component of the innate immune response and suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self‐associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane‐bound BAK, we employed hydrogen‐deuterium exchange mass spectrometry (HDX‐MS). The HDX‐MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX‐MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX‐MS profiling revealed that the BCL‐2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N‐terminal region preceding the BAK oligomerisation domains became disordered post‐activation and remained disordered in the activated oligomer. Removal of the disordered N‐terminus did not impair, but rather slightly potentiated, BAK‐mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX‐MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting. SYNOPSIS: While BAK is a known executioner protein in apoptotic cell death, how it changes conformation to form oligomeric mitochondrial pores to kill cells remains unclear. Hydrogen‐deuterium exchange mass spectrometry and site‐directed mutagenesis now resolve key events in BAK activation as it occurs on membranes and mitochondria. Hydrogen‐deuterium exchange mass spectrometry provides new insight into conformation changes of BAK on a mitochondria‐like membrane.The BAK BH4 domain constrains BAK activity in the absence of a death stimulus.During BAK activation and oligomerisation, amino acids preceding the BH3 domain become exposed and disordered.The disordered N‐terminus is not required for BAK to permeabilise membranes and mediate cytochrome c release and may rather inhibit it. [ABSTRACT FROM AUTHOR]

RNA-binding proteins are customarily regarded as important facilitators of gene expression. In recent years, RNA–protein interactions have also emerged as a pervasive force in the regulation of homeostasis. The compendium of proteins with provable RNA-binding function has swelled from the hundreds to the thousands astride the partnership of mass spectrometry-based proteomics and RNA sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that can extract bound protein that has been cross-linked in its native environment. These methods reveal snapshots in time displaying an extensive network of regulation and a wealth of data that can be used for both the discovery of RNA-binding function and the molecular interfaces at which these interactions occur. This review will focus on the impact of these developments on our broader perception of post-transcriptional regulation, and how the technical features of current capture methods, as applied in mammalian systems, create a challenging medium for interpretation by systems biologists and target validation by experimental researchers. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, EV biogenesis remains poorly understood. We previously defined a role for Arrdc4 (Arrestin domain containing protein 4), an adaptor for Nedd4 family ubiquitin ligases, in the biogenesis of EVs. Here we report that ubiquitination of Arrdc4 is critical for its role in EV secretion. We identified five potential ubiquitinated lysine residues in Arrdc4 using mass spectrometry. By analysing Arrdc4 lysine mutants we discovered that lysine 270 (K270) is critical for Arrdc4 function in EV biogenesis. Arrdc4K270R mutation caused a decrease in the number of EVs released by cells compared to Arrdc4WT, and a reduction in trafficking of divalent metal transporter (DMT1) into EVs. Furthermore, we also observed a decrease in DMT1 activity and an increase in its intracellular degradation in the presence of Arrdc4K270R. K270 was found to be ubiquitinated with K‐29 polyubiquitin chains by the ubiquitin ligase Nedd4‐2. Thus, our results uncover a novel role of K‐29 polyubiquitin chains in Arrdc4‐mediated EV biogenesis and protein trafficking. [ABSTRACT FROM AUTHOR]

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein. [ABSTRACT FROM AUTHOR]

Background: Low‐density neutrophils (LDN) are a distinct subset of neutrophils rarely detected in healthy people but appear in the blood of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), and are mobilised in response to granulocyte colony‐stimulating factor (G‐CSF). The aim of this study was to identify novel mechanisms responsible for the pathogenic capacity of LDN in SLE. Methods: Neutrophils were isolated from donors treated with G‐CSF, and whole‐cell proteomic analysis was performed on LDN and normal‐density neutrophils. Results: CD98 is significantly upregulated in LDN from G‐CSF donors and defines a subset of LDN within the blood of SLE patients. CD98 is a transmembrane protein that dimerises with L‐type amino acid transporters. We show that CD98 is responsible for the increased bioenergetic capacity of LDN. CD98 on LDN mediates the uptake of essential amino acids that are used by mitochondria to produce adenosine triphosphate, especially in the absence of glucose. Inhibition of CD98 reduces the metabolic flexibility of this population, which may limit their pathogenic capacity. CD98+ LDN produce more proinflammatory cytokines and chemokines than their normal density counterparts and are resistant to apoptosis, which may also contribute to tissue inflammation and end organ damage in SLE. Conclusions: CD98 provides a phenotypic marker for LDN that facilitates identification of this population without density‐gradient separation and represents a novel therapeutic target to limit its pathogenic capacity. [ABSTRACT FROM AUTHOR]

The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the "function-to-find" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immuno-precipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity as-says. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes. [ABSTRACT FROM AUTHOR]

Cells are constantly subjected to a vast range of potentially lethal insults, which may activate specific molecular pathways that have evolved to kill the cell. Cell death pathways are defined partly by their morphology, and more specifically by the molecules that regulate and enact them. As these pathways become more thoroughly characterized, interesting molecular links between them have emerged, some still controversial and others hinting at the physiological and pathophysiological roles these death pathways play. We describe specific molecular programs controlling cell death, with a focus on some of the distinct features of the pathways and the molecular links between them. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α‐arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4‐mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4–/– mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two‐cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4–/‐ epididymal epithelial cells, and further, supplementation of Arrdc4–/– sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis. [ABSTRACT FROM AUTHOR]

Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored. Unlabelled Image [ABSTRACT FROM AUTHOR]

The article discusses a study according to which cyclin-dependent kinases (CDKs)13 cooperates with CDK12 to control global RNA polymerase II processivity. It mentions that single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation tomembranes where activatedMLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobodybinding site centered on the a4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the a4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediatemembrane translocation, distinct from known phospholipid binding sites. [ABSTRACT FROM AUTHOR]

T cell receptors (TCRs) recognize antigens presented by major histocompatibility complex (MHC) and MHC class I–like molecules. We describe a diverse population of human γδ T cells isolated from peripheral blood and tissues that exhibit autoreactivity to the monomorphic MHC-related protein 1 (MR1). The crystal structure of a γδTCR–MR1–antigen complex starkly contrasts with all other TCR–MHC and TCR–MHC-I-like complex structures. Namely, the γδTCR binds underneath the MR1 antigen-binding cleft, where contacts are dominated by the MR1 α3 domain. A similar pattern of reactivity was observed for diverse MR1-restricted γδTCRs from multiple individuals. Accordingly, we simultaneously report MR1 as a ligand for human γδ T cells and redefine the parameters for TCR recognition. [ABSTRACT FROM AUTHOR]

Abstract: The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function. Characterisation of the structural and dynamic requirements for the activation of the βc receptor family and the molecular definition of downstream signalling pathways are providing new insights into cytokine receptor signalling as well as new therapeutic opportunities. [Copyright &y& Elsevier]

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically. [Display omitted] • Loss of the INTS6 subunit of Integrator confers resistance to CDK9 inhibition • INTS6 recruits PP2A to chromatin and forms a submodule of Integrator (Int-PP2A) • Int-PP2A opposes CDK9 at the phosphorylation level to fine-tune transcription • PP2A activators synergize therapeutically with CDK9 inhibitors in cancer Interplay between PP2A and CDK9 provides a control point for gene expression that can be exploited to inhibit tumorigenesis. [ABSTRACT FROM AUTHOR]

Human type 1 insulin-like growth factor receptor (IGF-1R) signals chiefly in response to the binding of insulin-like growth factor I. Relatively little is known about the role of insulin-like growth factor II signaling via IGF-1R, despite the affinity of insulin-like growth factor II for IGF-1R being within an order of magnitude of that of insulin-like growth factor I. Here, we describe the cryoelectron microscopy structure of insulin-like growth factor II bound to a leucine-zipper-stabilized IGF-1R ectodomain, determined in two conformations to a maximum average resolution of 3.2 Å. The two conformations differ in the relative separation of their respective points of membrane entry, and comparison with the structure of insulin-like growth factor I bound to IGF-1R reveals long-suspected differences in the way in which the critical C domain of the respective growth factors interact with IGF-1R. • Cryo-EM structure of IGF-II bound to leucine-zippered human IGF-1R ectodomain • IGF-II C domain interacts with receptor differently to that of IGF-I Insulin-like growth factors I and II differ in the length of their C domain, the segment that links growth factor B and A domains. Xu et al. show that the different 3D modes of C-domain engagement with type 1 insulin-like growth factor receptor align with their receptor affinity. [ABSTRACT FROM AUTHOR]

Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2V617F mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1V658F). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations. [ABSTRACT FROM AUTHOR]

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co‐ordinate the removal of damaged mitochondria. We report that following mitochondrial damage‐induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3‐only proteins and its homo‐dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX‐mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK‐dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1‐dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro‐inflammatory effect. Synopsis: The ubiquitin ligase Parkin plays a protective role in neurodegenerative disease by removing damaged mitochondria and preventing apoptosis, and by limiting the functions of pro‐apoptotic effector proteins BAK and BAX. Defective control of apoptosis may contribute to the pathogenesis of early onset Parkinson's Disease caused by Parkin mutations. Parkin activity is induced by mitochondrial damage during apoptosis.Parkin mono‐ and di‐ubiquitinates BAK at a conserved lysine in its hydrophobic groove.Ubiquitination reduces BAK oligomerisation and apoptotic activity on mitochondria.Parkin prevents BAX mitochondrial localisation and apoptotic activity independent of ubiquitination.Certain Parkinson's Disease‐associated Parkin mutants cannot ubiquitinate BAK and restrain it on mitochondria. Autophagic clearance of damaged mitochondria and suppression of apoptotic effector proteins are coordinated functions of the Parkin ubiquitin ligase that are lost upon Parkinson's Disease‐associated mutations. [ABSTRACT FROM AUTHOR]