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1.
Cell ; 174(4): 897-907.e14, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30078705

RESUMO

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.


Assuntos
Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Células HCT116 , Humanos , Fosforilação , Domínios de Homologia à Plecstrina , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-akt/química , Serina/química , Transdução de Sinais , Treonina/química
2.
Mol Cell ; 82(8): 1411-1413, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35452613

RESUMO

Henning et al. (2022) report development of a novel class of agents, bivalent deubiquitinase (DUB)-targeting chimeras (DUBTACs), that can selectively stabilize protein targets. These findings encourage further pursuit of targeted protein stabilization as a new paradigm in chemical biology and drug discovery.


Assuntos
Enzimas Desubiquitinantes , Descoberta de Drogas , Enzimas Desubiquitinantes/metabolismo
3.
Mol Cell ; 82(3): 570-584.e8, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34951965

RESUMO

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.


Assuntos
Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Desdobramento de Proteína , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Ubiquitinadas/genética , Ubiquitinação , Proteína com Valosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Nature ; 621(7978): 404-414, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37648862

RESUMO

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.


Assuntos
Epitopos , Edição de Genes , Imunoterapia , Leucemia Mieloide Aguda , Animais , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD34/metabolismo , Transplante de Medula Óssea , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Hematopoese , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos/imunologia , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos Quiméricos/imunologia , Recidiva , Linfócitos T/imunologia , Condicionamento Pré-Transplante , Evasão Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Genes Dev ; 33(9-10): 578-589, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30846429

RESUMO

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA Polimerase II/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
PLoS Pathog ; 19(11): e1011781, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37976321

RESUMO

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV.


Assuntos
Citomegalovirus , Herpesviridae , Humanos , Núcleo Celular/metabolismo , Herpesviridae/metabolismo , Replicação Viral , Simplexvirus , Acrilamidas/metabolismo , Antivirais/farmacologia , Antivirais/metabolismo
7.
Nature ; 575(7783): 545-550, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31581174

RESUMO

RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.


Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/química , Microscopia Crioeletrônica , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sítios de Ligação , Transformação Celular Neoplásica/genética , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo
8.
Mol Cell ; 67(4): 550-565.e5, 2017 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-28803780

RESUMO

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Metilação de DNA , Replicação do DNA , DNA/biossíntese , Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/genética , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Células-Tronco Embrionárias/enzimologia , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Lisina , Metilação , Camundongos , Modelos Moleculares , Mimetismo Molecular , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transfecção , Domínio Tudor , Ubiquitina-Proteína Ligases
9.
Genes Dev ; 31(21): 2162-2174, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29203645

RESUMO

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.


Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transcrição Gênica/genética , Acetilação , Histonas/metabolismo , Mutação/genética , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores de Transcrição/metabolismo
10.
Mol Cell ; 61(2): 297-304, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26799764

RESUMO

Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."


Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Proliferação de Células , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição Gênica
11.
Nat Chem Biol ; 17(9): 954-963, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33972797

RESUMO

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
12.
Anal Chem ; 94(27): 9508-9513, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35729701

RESUMO

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.


Assuntos
Eletroforese em Microchip , Ubiquitina , Enzimas Desubiquitinantes/metabolismo , Eletroforese Capilar , Humanos , Proteoma , Ubiquitina/metabolismo
13.
Nat Chem Biol ; 16(3): 318-326, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32042200

RESUMO

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Microbioma Gastrointestinal/fisiologia , Amidoidrolases/fisiologia , Animais , Bactérias/enzimologia , Ácidos e Sais Biliares/metabolismo , Desenho de Fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bibliotecas de Moléculas Pequenas
14.
Nat Chem Biol ; 16(9): 979-987, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32483379

RESUMO

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Animais , Antineoplásicos/química , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cristalografia por Raios X , Cisteína/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
15.
Mol Cell ; 54(1): 107-118, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24657165

RESUMO

Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for proliferating cell nuclear antigen (PCNA) monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, we show that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Sobrevivência Celular , Galinhas , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células HeLa , Humanos , Inibidores da Síntese de Ácido Nucleico , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Interferência de RNA , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína 28 com Motivo Tripartido , Ubiquitina-Proteína Ligases , Ubiquitinação , Raios Ultravioleta
16.
Mol Cell ; 54(3): 512-25, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24703952

RESUMO

Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion are unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1-T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting nonhomologous end-joining. We also reveal that protein phosphatase complex PP4C/R3ß dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.


Assuntos
Quebras de DNA de Cadeia Dupla , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Fase G1 , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
17.
Chem Soc Rev ; 50(15): 8361-8381, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34143170

RESUMO

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies.


Assuntos
Descoberta de Drogas/métodos , Espectrometria de Massas , Proteômica , Desenho de Fármacos , Humanos , Proteoma/efeitos dos fármacos
18.
Genes Dev ; 28(17): 1957-75, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25184681

RESUMO

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Transcrição Gênica/genética , Proteína BRCA1/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Raios Ultravioleta
19.
Anal Chem ; 93(41): 13791-13799, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34606255

RESUMO

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.


Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Humanos , Espectrometria de Massas , Peptídeos , Proteínas
20.
Genes Dev ; 27(6): 615-26, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23512658

RESUMO

Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein associated with neurodegenerative disorders. Here, we identify PQBP1 as an alternative messenger RNA (mRNA) splicing (AS) effector capable of influencing splicing of multiple mRNA targets. PQBP1 is associated with many splicing factors, including the key U2 small nuclear ribonucleoprotein (snRNP) component SF3B1 (subunit 1 of the splicing factor 3B [SF3B] protein complex). Loss of functional PQBP1 reduced SF3B1 substrate mRNA association and led to significant changes in AS patterns. Depletion of PQBP1 in primary mouse neurons reduced dendritic outgrowth and altered AS of mRNAs enriched for functions in neuron projection development. Disease-linked PQBP1 mutants were deficient in splicing factor associations and could not complement neurite outgrowth defects. Our results indicate that PQBP1 can affect the AS of multiple mRNAs and indicate specific affected targets whose splice site determination may contribute to the disease phenotype in PQBP1-linked neurological disorders.


Assuntos
Processamento Alternativo/genética , Proteínas de Transporte/metabolismo , Deficiência Intelectual/genética , Neuritos/fisiologia , Proteínas Nucleares/metabolismo , Animais , Apoptose/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Splicing de RNA
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