RESUMO
Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen-deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state.
RESUMO
Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70 , Biossíntese de Proteínas , Dobramento de Proteína , Ribossomos , Ribossomos/metabolismo , Ribossomos/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Ligação Proteica , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Modelos Moleculares , Conformação Proteica , Peptidilprolil IsomeraseRESUMO
Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Calicreínas/metabolismo , Invasividade Neoplásica , Neoplasias PancreáticasRESUMO
Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.
Assuntos
Proteoma , Proteômica , Camundongos , Animais , Proteômica/métodos , Glicoproteínas/metabolismo , Açúcares , NucleotídeosRESUMO
Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Neoplasias , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Estudos Clínicos como Assunto , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Imunidade , SARS-CoV-2RESUMO
The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.
Assuntos
Cristalografia por Raios XRESUMO
Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
Assuntos
Inibidores Enzimáticos , Esterases , Encéfalo/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esterases/metabolismo , Via de Sinalização WntAssuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Neoplasias Hematológicas/imunologia , Imunização Secundária , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/complicações , COVID-19/epidemiologia , Neoplasias Hematológicas/complicações , Humanos , Imunogenicidade da Vacina , Neoplasias/complicações , Neoplasias/imunologia , Avaliação de SintomasRESUMO
Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.
Assuntos
Galactosamina/análogos & derivados , Galactosamina/metabolismo , Galactosiltransferases/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Alcinos/química , Sequência de Aminoácidos , Animais , Azidas/química , Linhagem Celular Tumoral , Química Click , Corantes Fluorescentes/química , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Engenharia Metabólica/métodos , Camundongos , Sondas Moleculares/química , Oligossacarídeos/biossíntese , Polissacarídeos/biossíntese , Açúcares de Uridina Difosfato/biossíntese , Açúcares de Uridina Difosfato/metabolismoRESUMO
Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-ß signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-ß signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-ß responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-ß-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.
Assuntos
Aracnodactilia/genética , Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Síndrome de Marfan/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Fator de Crescimento Transformador beta/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Caderinas/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-ret/química , Proteínas de Peixe-Zebra/químicaRESUMO
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Assuntos
Acetilgalactosamina/metabolismo , Glicoproteínas/metabolismo , Acetilgalactosamina/química , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade por Substrato , Uridina Difosfato N-Acetilgalactosamina/químicaRESUMO
Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2-HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2-HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.
Assuntos
Inibidores de Proteínas Quinases , Receptor ErbB-3 , Regulação Alostérica , Animais , Células CHO , Cricetulus , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estudo de Prova de Conceito , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/química , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMO
The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a 'RIPR' motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6-aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι-FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.
Assuntos
Células Epiteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína Quinase C/metabolismo , Junções Íntimas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Células CACO-2 , Polaridade Celular , Fatores de Troca do Nucleotídeo Guanina/genética , Células HCT116 , Humanos , FosforilaçãoRESUMO
IFN-stimulated gene (ISG) 15 is a ubiquitin-like protein induced after type I IFN stimulation. There is a dearth of in vivo models to study free unconjugated ISG15 function. We found that free ISG15 enhances the production of IFN-γ and IL-1ß during murine infection with Toxoplasma gondii In our model, ISG15 is induced in a type I IFN-dependent fashion and released into the serum. Increased ISG15 levels are dependent on an actively invading and replicating parasite. Two cysteine residues in the hinge domain are necessary determinants for ISG15 to induce increased cytokine levels during infection. Increased ISG15 is concurrent with an influx of IL-1ß-producing CD8α+ dendritic cells to the site of infection. In this article, we present Toxoplasma infection as a novel in vivo murine model to study the immunomodulatory properties of free ISG15 and uniquely link it to IL-1ß production by CD8α+ dendritic cells driven by two cysteines in the hinge region of the protein.
Assuntos
Citocinas/metabolismo , Células Dendríticas/imunologia , Interleucina-1beta/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/imunologia , Animais , Antígenos CD8/metabolismo , Movimento Celular , Células Cultivadas , Cisteína/genética , Citocinas/genética , Modelos Animais de Doenças , Imunomodulação , Interferon Tipo I/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Proteica , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.
Assuntos
Antígenos CD/biossíntese , Caderinas/biossíntese , Endotélio Vascular/metabolismo , Epigênese Genética/fisiologia , Animais , Antígenos CD/genética , Caderinas/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endotélio Vascular/ultraestrutura , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica/fisiologiaRESUMO
The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Anti-Inflamatórios/síntese química , Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Biblioteca de Peptídeos , Peptídeos/síntese química , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.