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Current treatments for advanced prostate cancer (PCa) primarily target androgen receptor (AR)-pathways. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR signaling inhibitors (ARSI) remains a significant clinical challenge. This study introduces BSJ-5-63, a novel triple degrader targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, with potential to transform CRPC therapy. BSJ-5-63 effectively downregulates homologous recombination repair (HRR) genes, including BRCA1 and BRCA2, through CDK12 degradation, and attenuates AR signaling through CDK7 and CDK9 degradation, further enhancing its therapeutic impact. Importantly, BSJ-5-63 induces a "BRCAness" state that persists for a significant duration, enabling sequential combination therapy with PARP inhibitors (PARPis) while potentially minimizing drug-related toxicity and resistance. In both in vitro and in vivo studies, BSJ-5-63 exhibited potent antiproliferative effects in both AR-positive and AR-negative CRPC models. This study presents a promising multi-pronged approach for CRPC treatment, addressing both DNA repair mechanisms and AR signaling, with the potential to benefit a wide range of patients regardless of their BRCA1/2 mutational status., Significance: This study introduces BSJ-5-63, a triple degrader designed to target CDK12, CDK7, and CDK9, making a significant advancement in CRPC therapy. The distinctive mechanism of BSJ-5-63 involves downregulating HRR genes and inhibiting AR signaling, thereby inducing a BRCAness state. This enhances sensitivity to PARP inhibition, effectively addressing ARSI resistance and improving the overall efficacy of treatment. The development of BSJ-5-63 represents a promising therapeutic approach, with the potential to benefit a broad spectrum of CRPC patients.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca 2+ ] I flux in neuronal PC12 cells and this TRPV2-specific [Ca 2+ ] I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation., Competing Interests: Declaration of interests A.C.K. is a co-founder and consultant for biotechnology companies Tectonic Therapeutic Inc. and Seismic Therapeutic Inc., as well as for the Institute for Protein Innovation, a non-profit research institute. S.C.B. is on the scientific advisory board for and receives funding from Erasca, Inc. for an unrelated project; is an advisor to MPM Capital; and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, Droia Ventures, and Ayala Pharmaceuticals for unrelated projects., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroinflammation is a pathological feature of many neurodegenerative diseases, including Alzheimer's disease (AD) 1,2 and amyotrophic lateral sclerosis (ALS) 3 , raising the possibility of common therapeutic targets. We previously established that cytoplasmic double-stranded RNA (cdsRNA) is spatially coincident with cytoplasmic pTDP-43 inclusions in neurons of patients with C9ORF72-mediated ALS 4 . CdsRNA triggers a type-I interferon (IFN-I)-based innate immune response in human neural cells, resulting in their death 4 . Here, we report that cdsRNA is also spatially coincident with pTDP-43 cytoplasmic inclusions in brain cells of patients with AD pathology and that type-I interferon response genes are significantly upregulated in brain regions affected by AD. We updated our machine-learning pipeline DRIAD-SP (Drug Repurposing In Alzheimer's Disease with Systems Pharmacology) to incorporate cryptic exon (CE) detection as a proxy of pTDP-43 inclusions and demonstrated that the FDA-approved JAK inhibitors baricitinib and ruxolitinib that block interferon signaling show a protective signal only in cortical brain regions expressing multiple CEs. Furthermore, the JAK family member TYK2 was a top hit in a CRISPR screen of cdsRNA-mediated death in differentiated human neural cells. The selective TYK2 inhibitor deucravacitinib, an FDA-approved drug for psoriasis, rescued toxicity elicited by cdsRNA. Finally, we identified CCL2, CXCL10, and IL-6 as candidate predictive biomarkers for cdsRNA-related neurodegenerative diseases. Together, we find parallel neuroinflammatory mechanisms between TDP-43 associated-AD and ALS and nominate TYK2 as a possible disease-modifying target of these incurable neurodegenerative diseases., Competing Interests: COMPETING INTERESTS M.W.A. is a consultant for TLL, LLC, Transposon Therapeutics, and has received in kind support from Eli Lilly that is not related to this work. A.S. is an employee at Flagship Labs 84, Inc., a subsidiary of Flagship Pioneering. F.P. is an employee of Merck Research Laboratories.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

We performed quantitative proteomics on 60 human-derived breast cancer cell line models to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics., (© 2023. Springer Nature Limited.)

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity.

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility., Competing Interests: Competing interests Columbia University, Harvard University and NY Presbyterian Hospital have filed a patent (WO/2021/003389), which is published and pending review, reporting a FRET-based method for screening small molecules that increase contractility for the treatment of heart failure. Inventors on this patent application are S.O.M., H.M.C., M.K., S.I.Z., A.N.K., M.B.J. and G.L. The FRET-based assay was utilized in this manuscript for assessing the effects of calyculin and whether 3DA-β2B and 2DA-β2B Ca2+ channel subunits bind to Rad.

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis., (© 2022. The Author(s).)

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate 1-3 . Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex 4-6 . Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells., (© 2022. The Author(s).)

The cell stress-responsive transcription factor p53 influences the expression of its target genes and subsequent cellular responses based in part on its dynamics (changes in level over time). The mechanisms decoding p53 dynamics into subsequent target mRNA and protein dynamics remain unclear. We systematically quantified p53 target mRNA and protein expression over time under two p53 dynamical regimes, oscillatory and rising, using RNA-sequencing and TMT mass spectrometry. Oscillatory dynamics allowed for a greater variety of dynamical patterns for both mRNAs and proteins. Mathematical modeling of empirical data revealed three distinct mechanisms that decode p53 dynamics. Specific combinations of these mechanisms at the transcriptional and post-transcriptional levels enabled exclusive induction of proteins under particular dynamics. In addition, rising induction of p53 led to higher induction of proteins regardless of their functional class, including proteins promoting arrest of proliferation, the primary cellular outcome under rising p53. Our results highlight the diverse mechanisms cells employ to distinguish complex transcription factor dynamics to regulate gene expression., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Rapidly changing and transient protein-protein interactions regulate dynamic cellular processes in the cardiovascular system. Traditional methods, including affinity purification and mass spectrometry, have revealed many macromolecular complexes in cardiomyocytes and the vasculature. Yet these methods often fail to identify in vivo or transient protein-protein interactions. To capture these interactions in living cells and animals with subsequent mass spectrometry identification, enzyme-catalyzed proximity labeling techniques have been developed in the past decade. Although the application of this methodology to cardiovascular research is still in its infancy, the field is developing rapidly, and the promise is substantial. In this review, we outline important concepts and discuss how proximity proteomics has been applied to study physiological and pathophysiological processes relevant to the cardiovascular system.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs., (© 2021 The Authors.)

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions., (© 2021 May et al.)

Despite objective responses to PARP inhibition and improvements in progression-free survival compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in BRCA-associated TNBC. Through multi-omics profiling we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming driven by the sterol regulatory element-binding protein 1 (SREBP-1) pathway. Combined PARP inhibitor therapy with CSF-1R blocking antibodies significantly enhanced innate and adaptive anti-tumor immunity and extends survival in BRCA-deficient tumors in vivo and is mediated by CD8 + T-cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate combined PARP inhibition and macrophage targeting therapy induces a durable reprogramming of the tumor microenvironment, thus constituting a promising therapeutic strategy for TNBC., Competing Interests: Competing Interests Statement J.L.G. is a consultant for Glaxo-Smith Kline (GSK), Codagenix, Verseau, Kymera and Array BioPharma and receives sponsored research support from GSK, Array BioPharma and Eli Lilly. G.I.S. has served on advisory boards for Pfizer, Eli Lilly, G1 Therapeutics, Roche, Merck KGaA/EMD-Serono, Sierra Oncology, Bicycle Therapeutics, Fusion Pharmaceuticals, Cybrexa Therapeutics, Astex, Almac, Ipsen, Bayer, Angiex, Daiichi Sankyo, Seattle Genetics, Boehringer Ingelheim, ImmunoMet, Asana, Artios, Atrin, Concarlo Holdings, Syros and Zentalis, and has received sponsored research support from Merck, Eli Lilly, Merck/EMD Serono and Sierra Oncology. Clinical trial support from Pfizer and Array Biopharma has been provided to Dana-Farber Cancer Institute for the conduct of investigator-initiated studies led by G.I.S. He holds a patent entitled, “Dosage regimen for sapacitabine and seliciclib,” also issued to Cyclacel Pharmaceuticals, and a pending patent, entitled, “Compositions and Methods for Predicting Response and Resistance to CDK4/6 Inhibition,” together with Liam Cornell. E.A.M is on the SAB for Astra-Zeneca/Medimmune, Celgene, Genentech, Genomic Health, Merck, Peregrine Pharmaceuticals, SELLAS Lifescience, and Tapimmune and has clinical trial support to her former institution (MDACC) from Astra-Zeneca/Medimmune, EMD-Serono, Galena Biopharma and Genentech as well as Genentech support to a SU2C grant, as well as sponsored Research Support to the laboratory from GSK and Eli Lilly. S.R. receives research funding from Merck, Bristol-Myers Squibb, Gilead and Affimed, and on the scientific advisory board for Immunitas. S.S is a consultant for Rarecyte, Inc. N.T. receives research support from Astra-Zeneca. PKS serves on the SAB or BOD of Glencoe Software, Applied Biomath and RareCyte Inc. and has equity in these companies; he is a member of the NanoString SAB and is also co-founder of Glencoe Software, which contributes to and supports the open-source OME/OMERO image informatics software used in this paper. D.D. consults for Novartis and is on the advisory board for Oncology Analytics, Inc. S.J. receives consulting fees from Venn Therapeutics.

Background: The developing field of osteoimmunology supports importance of an interferon (IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus (ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts., Hypothesis: Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN., Methods: Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells., Results: Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome, exemplified by HELZ2 protein which is upregulated by IFN but non responsive to ZIKV. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KI20A and UBP18), and ZIKV induces potential entry factor PLVAP., Conclusions: Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance., Competing Interests: GS, AD, MK, SG, DM No competing interests declared, (© 2020, Shipkovenska et al.)

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo 1 . To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln z24 ) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.

Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Hippo pathway regulates organ size, regeneration, and cell growth by controlling the stability of the transcription factor, YAP (Yorkie in Drosophila ). When there is tissue damage, YAP is activated allowing the restoration of homeostatic tissue size. The exact signals by which YAP is activated are still not fully understood, but its activation is known to affect both cell size and cell number. Here we used cultured cells to examine the coordinated regulation of cell size and number under the control of YAP. Our experiments in isogenic HEK293 cells reveal that YAP can affect cell size and number by independent circuits. Some of these effects are cell autonomous, such as proliferation, while others are mediated by secreted signals. In particular CYR61, a known secreted YAP target, is a non-cell autonomous mediator of cell survival, while another unidentified secreted factor controls cell size., Competing Interests: DM, MK, XL, JG, LP, MK No competing interests declared, (© 2020, Mugahid et al.)

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults 1 . No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities 2,3 . How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å 2 of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers-which are largely disordered in their free state-demonstrates an example of a 'folding-upon-partner-binding' mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs.

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 voltage-gated calcium channels 1-4 . However, this augmentation persists in transgenic murine hearts expressing mutant Ca V 1.2 α 1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α 1C or β 2B subunits conjugated to ascorbate peroxidase 5 in mouse hearts, and use multiplexed quantitative proteomics 6,7 to track hundreds of proteins in the proximity of Ca V 1.2. We observe that the calcium-channel inhibitor Rad 8,9 , a monomeric G protein, is enriched in the Ca V 1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of Ca V 1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca V 1.3 and Ca V 2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

A fundamental challenge in chemical biology and medicine is to understand and expand the fraction of the human proteome that can be targeted by small molecules. We recently described a strategy that integrates fragment-based ligand discovery with chemical proteomics to furnish global portraits of reversible small-molecule/protein interactions in human cells. Excavating clear structure-activity relationships from these 'ligandability' maps, however, was confounded by the distinct physicochemical properties and corresponding overall protein-binding potential of individual fragments. Here, we describe a compelling solution to this problem by introducing a next-generation set of fully functionalized fragments differing only in absolute stereochemistry. Using these enantiomeric probe pairs, or 'enantioprobes', we identify numerous stereoselective protein-fragment interactions in cells and show that these interactions occur at functional sites on proteins from diverse classes. Our findings thus indicate that incorporating chirality into fully functionalized fragment libraries provides a robust and streamlined method to discover ligandable proteins in cells.

Cyclin-dependent kinase 14 (CDK14) and other TAIRE family kinases (CDKs 15-18) are proteins that lack functional annotation but are frequent off-targets of clinical kinase inhibitors. In this study we develop and characterize FMF-04-159-2, a tool compound that specifically targets CDK14 covalently and possesses a TAIRE kinase-biased selectivity profile. This tool compound and its reversible analog were used to characterize the cellular consequences of covalent CDK14 inhibition, including an unbiased investigation using phospho-proteomics. To reduce confounding off-target activity, washout conditions were used to deconvolute CDK14-specific effects. This investigation suggested that CDK14 plays a supporting role in cell-cycle regulation, particularly mitotic progression, and identified putative CDK14 substrates. Together, these results represent an important step forward in understanding the cellular consequences of inhibiting CDK14 kinase activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peroxidase-catalyzed proximity labeling is a powerful technique for defining the molecular environment of proteins in vivo. Expressing a protein of interest fused to a modified plant peroxidase (APEX2) allows labeling of nearby polypeptides. Addition of hydrogen peroxide (H 2 O 2 ) and biotin-tyramide (biotin-phenol) generates short-lived radicals around the peroxidase. Labeling is thus restricted to proteins in close proximity, providing a snapshot of the local environment around the APEX2 fusion protein. Combined with an initial perturbation, progressive changes in interaction partners can be tracked, e.g., after drug treatment. Multiplexed quantitative mass spectrometry permits the parallel analysis of several experimental replicates or of up to 11 time points. Here we describe the denaturing purification of biotin-labeled proteins with magnetic streptavidin beads, and subsequent sample preparation for multiplexed quantitative mass spectrometry. Proximity-labeled proteins are enriched under strong denaturing conditions. Tryptic on-bead digest of purified proteins is combined with tandem mass tag peptide labeling (TMT), alkaline reversed-phase peptide fractionation, and SPS MS 3 mass spectrometry. This analysis pipeline enables studies of complex protein environment changes in perturbed biological systems, as well as comparative studies of functional protein proximity in different cell lines. Through multiplexing, hundreds of proteins can be quantified in each experimental condition in parallel.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression 1-4 . The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3 5-11 . This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me 12-15 . However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me-which would lead to aberrant gene silencing and epigenetic instability-is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue 16 suggest that the mechanism described here is broadly conserved.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the β2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of β2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Homologous recombination (HR) is crucial for genetic exchange and accurate repair of DNA double-strand breaks and is pivotal for genome integrity. HR uses homologous sequences for repair, but how homology search, the exploration of the genome for homologous DNA sequences, is conducted in the nucleus remains poorly understood. Here, we use time-resolved chromatin immunoprecipitations of repair proteins to monitor homology search in vivo. We found that homology search proceeds by a probing mechanism, which commences around the break and samples preferentially on the broken chromosome. However, elements thought to instruct chromosome loops mediate homology search shortcuts, and centromeres, which cluster within the nucleus, may facilitate homology search on other chromosomes. Our study thus reveals crucial parameters for homology search in vivo and emphasizes the importance of linear distance, chromosome architecture, and proximity for recombination efficiency., (Copyright © 2013 Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) are acutely hazardous for cells, as they can cause genome instability. DSB repair involves the sequential recruitment of repair factors to the DSBs, followed by Rad51-mediated homology probing, DNA synthesis, and ligation. However, little is known about how cells react if no homology is found and DSBs persist. Here, by monitoring a single persistent DNA break, we show that, following DNA resection and RPA recruitment, Rad51 spreads chromosome-wide bidirectionally from the DSB but selectively only on the broken chromosome. Remarkably, the persistent DSB is later fixed to the nuclear periphery in a process that requires Rad51, the histone variant H2A.Z, its SUMO modification, and the DNA-damage checkpoint. Indeed, H2A.Z is deposited close to the break early but transiently and directs DNA resection, single DSB-induced checkpoint activation, and DSB anchoring. Thus, a persistent DSB induces a multifaceted response, which is linked to a specific chromatin mark.

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle.