RESUMO
O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.
Assuntos
Aptâmeros de Nucleotídeos , beta Catenina/metabolismo , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismoRESUMO
A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
Assuntos
Cromatina , Proteoma , Acilação , Mapeamento Cromossômico , Histonas , Sobrevivência CelularRESUMO
Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.
Assuntos
Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas do Complexo SMN/química , Proteínas do Complexo SMN/metabolismo , Animais , Arginina/metabolismo , Núcleo Celular/metabolismo , Corpos Enovelados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Metilação , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.
Assuntos
Arrestina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Arrestina/química , Simulação por Computador , Células HEK293 , Humanos , Fosfatos/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Análise EspectralRESUMO
Selective autophagy of organelles is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few autophagosomal receptors and remodelers. By using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence human ER-phagy factors. Two pathways were unexpectedly required for ER-phagy. First, reduced mitochondrial metabolism represses ER-phagy, which is opposite of general autophagy and is independent of AMPK. Second, ER-localized UFMylation is required for ER-phagy to repress the unfolded protein response via IRE1α. The UFL1 ligase is brought to the ER surface by DDRGK1 to UFMylate RPN1 and RPL26 and preferentially targets ER sheets for degradation, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network.
Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Autofagia/genética , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Estudo de Associação Genômica Ampla/métodos , Células HCT116 , Células HEK293 , Células HeLa , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Ribossômicas/metabolismo , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteogenômica , Neoplasias Encefálicas/imunologia , Criança , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Glioma/genética , Glioma/patologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Mutação/genética , Gradação de Tumores , Recidiva Local de Neoplasia/patologia , Fosfoproteínas/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.
Assuntos
Lisina , Fosfoproteínas , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , Fosforilação , Lisina/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Nucleolina , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Animais , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Polifosfatos/metabolismo , Polifosfatos/química , Concentração OsmolarRESUMO
Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.
Assuntos
Amidas , Lisina , Ácidos Fosfóricos , Polifosfatos , Lisina/metabolismo , Lisina/química , Polifosfatos/química , Polifosfatos/metabolismo , Fosforilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Proteínas/genéticaRESUMO
Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.
Assuntos
Linfócitos T CD8-Positivos , Serotonina , Linfócitos T CD8-Positivos/metabolismo , Serotonina/metabolismo , Serotonina/farmacologia , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
Assuntos
Proteostase , Fatores de Transcrição , Proteostase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta ao Choque Térmico/genética , Chaperonas Moleculares/genética , Cromatina/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismoRESUMO
CRISPR screens have empowered the high-throughput dissection of gene functions; however, more explicit genetic elements, such as codons of amino acids, require thorough interrogation. Here, we establish a CRISPR strategy for unbiasedly probing functional amino acid residues at the genome scale. By coupling adenine base editors and barcoded sgRNAs, we target 215,689 out of 611,267 (35%) lysine codons, involving 85% of the total protein-coding genes. We identify 1,572 lysine codons whose mutations perturb human cell fitness, with many of them implicated in cancer. These codons are then mirrored to gene knockout screen data to provide functional insights into the role of lysine residues in cellular fitness. Mining these data, we uncover a CUL3-centric regulatory network in which lysine residues of CUL3 CRL complex proteins control cell fitness by specifying protein-protein interactions. Our study offers a general strategy for interrogating genetic elements and provides functional insights into the human proteome.
Assuntos
Lisina , Proteoma , Humanos , Proteoma/genética , Lisina/genética , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , CódonRESUMO
Programmed cell death and caspase proteins play a pivotal role in host innate immune response combating pathogen infections. Blocking cell death is employed by many bacterial pathogens as a universal virulence strategy. CopC family type III effectors, including CopC from an environmental pathogen Chromobacterium violaceum, utilize calmodulin (CaM) as a co-factor to inactivate caspases by arginine ADPR deacylization. However, the molecular basis of the catalytic and substrate/co-factor binding mechanism is unknown. Here, we determine successive cryo-EM structures of CaM-CopC-caspase-3 ternary complex in pre-reaction, transition, and post-reaction states, which elucidate a multistep enzymatic mechanism of CopC-catalyzed ADPR deacylization. Moreover, we capture a snapshot of the detachment of modified caspase-3 from CopC. These structural insights are validated by mutagenesis analyses of CopC-mediated ADPR deacylization in vitro and animal infection in vivo. Our study offers a structural framework for understanding the molecular basis of arginine ADPR deacylization catalyzed by the CopC family.
Assuntos
Calmodulina , Caspases , Animais , Calmodulina/genética , Calmodulina/metabolismo , Caspases/metabolismo , Caspase 3/metabolismo , Arginina , Catálise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
ADP-ribosylation (ADPRylation) is a post-translational modification of proteins catalyzed by ADP-ribosyl transferase (ART) enzymes, including nuclear PARPs (e.g., PARP1 and PARP2). Historically, studies of ADPRylation and PARPs have focused on DNA damage responses in cancers, but more recent studies elucidate diverse roles in a broader array of biological processes. Here, we summarize the expanding array of molecular mechanisms underlying the biological functions of nuclear PARPs with a focus on PARP1, the founding member of the family. This includes roles in DNA repair, chromatin regulation, gene expression, ribosome biogenesis, and RNA biology. We also present new concepts in PARP1-dependent regulation, including PAR-dependent post-translational modifications, "ADPR spray," and PAR-mediated biomolecular condensate formation. Moreover, we review advances in the therapeutic mechanisms of PARP inhibitors (PARPi) as well as the progress on the mechanisms of PARPi resistance. Collectively, the recent progress in the field has yielded new insights into the expanding universe of PARP1-mediated molecular and therapeutic mechanisms in a variety of biological processes.
Assuntos
ADP-Ribosilação , Reparo do DNA , Cromatina/genética , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Processamento de Proteína Pós-Traducional , RNA/metabolismoRESUMO
Redox reactions are intrinsically linked to energy metabolism. Therefore, redox processes are indispensable for organismal physiology and life itself. The term reactive oxygen species (ROS) describes a set of distinct molecular oxygen derivatives produced during normal aerobic metabolism. Multiple ROS-generating and ROS-eliminating systems actively maintain the intracellular redox state, which serves to mediate redox signaling and regulate cellular functions. ROS, in particular hydrogen peroxide (H2O2), are able to reversibly oxidize critical, redox-sensitive cysteine residues on target proteins. These oxidative post-translational modifications (PTMs) can control the biological activity of numerous enzymes and transcription factors (TFs), as well as their cellular localization or interactions with binding partners. In this review, we describe the diverse roles of redox regulation in the context of physiological cellular metabolism and provide insights into the pathophysiology of diseases when redox homeostasis is dysregulated.
Assuntos
Metabolismo Energético/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Cisteína/metabolismo , Homeostase , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Estresse Oxidativo , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Bacteriophages have been a treasure trove for the discovery of fundamental biological principles and the expansion of our enzymatic toolkit since the dawn of molecular biology. In a recent study by Wolfram-Schauerte et al. these ubiquitous bacteria-infecting viruses reveal yet another new biological concept: post-translational modification through covalent RNA-protein linkages.
Assuntos
Bacteriófagos , Bacteriófagos/genética , RNA , Processamento de Proteína Pós-TraducionalRESUMO
Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions. This review focuses on the mechanisms of lipoylation and its significant impact on cell metabolism and cuproptosis, emphasizing the key genes involved and their implications for human diseases. It offers valuable insights into targeting dysregulated cellular metabolism for therapeutic purposes.
Assuntos
Cobre , Lipoilação , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Cobre/metabolismo , Animais , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Metabolismo EnergéticoRESUMO
The interactions of molecular chaperones with clients can be regulated by chaperone post-translational modification (PTMs) collectively known as the 'chaperone code'. What is less understood is how PTMs on client proteins may impact chaperone-client interactions. In this forum, we discuss the possibility of a 'client code'.
Assuntos
Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Humanos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Choque Térmico HSP70/metabolismo , Ligação ProteicaRESUMO
Due to their capacity to mediate repetitive protein interactions, intrinsically disordered regions (IDRs) are crucial for the formation of various types of protein-RNA complexes. The functions of IDRs are strongly modulated by post-translational modifications (PTMs). Phosphorylation is the most common and well-studied modification of IDRs, which can alter homomeric or heteromeric interactions of proteins and impact their ability to phase separate. Moreover, phosphorylation can influence the RNA-binding properties of proteins, and recent studies demonstrated its selective impact on the global profiles of protein-RNA binding and regulation. These findings highlight the need for further integrative approaches to understand how signalling remodels protein-RNA networks in cells.
Assuntos
Proteínas Intrinsicamente Desordenadas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA , RNA , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , RNA/genética , Processamento de Proteína Pós-Traducional/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
In response to infection, plants can induce the production of reactive oxygen species (ROS) to restrict pathogen invasion. In turn, adapted pathogens have evolved a counteracting mechanism of enzymatic ROS detoxification, but how it is activated remains elusive. Here, we show that in the tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici (Fol) this process is initiated by deacetylation of the FolSrpk1 kinase. Upon ROS exposure, Fol decreases FolSrpk1 acetylation on the K304 residue by altering the expression of the acetylation-controlling enzymes. Deacetylated FolSrpk1 disassociates from the cytoplasmic FolAha1 protein, thus enabling its nuclear translocation. Increased accumulation of FolSrpk1 in the nucleus allows for hyperphosphorylation of its phosphorylation target FolSr1 that subsequently enhances transcription of different types of antioxidant enzymes. Secretion of these enzymes removes plant-produced H2 O2 , and enables successful Fol invasion. Deacetylation of FolSrpk1 homologs has a similar function in Botrytis cinerea and likely other fungal pathogens. These findings reveal a conserved mechanism for initiation of ROS detoxification upon plant fungal infection.
Assuntos
Antioxidantes , Fusarium , Espécies Reativas de Oxigênio/metabolismo , Doenças das Plantas/microbiologiaRESUMO
Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.