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1.
Mol Cell ; 81(12): 2549-2565.e8, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33957083

RESUMO

Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.


Assuntos
Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas rab de Ligação ao GTP/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723042

RESUMO

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.


Assuntos
Sequência Conservada , Modelos Moleculares , Conformação Proteica , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Aminoácidos , Autofagia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Evolução Molecular , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas R-SNARE/genética , Relação Estrutura-Atividade
3.
Nat Methods ; 17(11): 1093-1096, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33020655

RESUMO

Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently than existing platforms.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fatores de Transcrição Kruppel-Like/genética , Domínio Catalítico , Genes Reporter , Células HEK293 , Humanos , Células K562 , Regiões Promotoras Genéticas , RNA Guia/genética , Proteínas Repressoras/genética , Transcrição Genética
4.
Science ; 370(6514): 351-356, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060361

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.


Assuntos
Aminoácidos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas R-SNARE/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Pinocitose , Proteólise
5.
Nat Chem Biol ; 16(11): 1170-1178, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32778845

RESUMO

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Talidomida/análogos & derivados , Ubiquitina/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimera/metabolismo , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Terapia de Alvo Molecular , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteólise , Transdução de Sinais , Relação Estrutura-Atividade , Talidomida/química
6.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
7.
Nat Commun ; 11(1): 3498, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641823

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Cell ; 182(2): 345-356.e16, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32589945

RESUMO

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium sordellii/metabolismo , Semaforinas/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Linhagem Celular , Microscopia Crioeletrônica , Edema/patologia , Edema/prevenção & controle , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Semaforinas/química , Semaforinas/genética
9.
Nat Commun ; 10(1): 5317, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757954

RESUMO

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.


Assuntos
RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Levivirus , Luciferases/metabolismo , Camundongos , Oligopeptídeos/metabolismo
10.
Biochem Cell Biol ; 97(1): 46-57, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29693415

RESUMO

Mutations in protein-coding regions underlie almost all Mendelian disorders, drive tumorigenesis, and contribute to susceptibility to common diseases. Despite the great diversity of diseases that are caused by coding mutations, the cellular processes that affect, and are affected by, pathogenic variants at the molecular level are fundamentally conserved. Experimental and computational approaches have revealed that a substantial fraction of disease mutations are not simple loss-of-function alleles. Rather, these pathogenic variants disrupt protein function in more subtle ways by tuning protein folding pathways, altering subcellular trafficking, interrupting signaling cascades, and rewiring highly connected interaction networks. Focusing mainly on Mendelian disorders, this review discusses the common mechanisms by which deleterious mutations disrupt protein function and how these disruptions can be exploited in the development of novel therapies.


Assuntos
Mutação , Pneumonia Aspirativa/fisiopatologia , Dobramento de Proteína , Multimerização Proteica , Proteínas/metabolismo , Humanos , Proteínas/química , Proteínas/genética
11.
Nat Genet ; 51(1): 196, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429576

RESUMO

In the version of this article originally published, the main-text sentence "In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor" misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.

12.
Cell Stress Chaperones ; 24(1): 7-15, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478692

RESUMO

Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.


Assuntos
Evolução Molecular , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Animais , Doença , Proteínas de Choque Térmico HSP70/classificação , Humanos , Agregados Proteicos , Domínios Proteicos , Redobramento de Proteína
13.
Nat Genet ; 50(12): 1650-1657, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30374066

RESUMO

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.


Assuntos
Mutação em Linhagem Germinativa , Receptor Celular 2 do Vírus da Hepatite A/genética , Linfo-Histiocitose Hemofagocítica/genética , Linfoma de Células T/genética , Paniculite/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Diagnóstico Diferencial , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Linfo-Histiocitose Hemofagocítica/classificação , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfoma de Células T/classificação , Linfoma de Células T/diagnóstico , Masculino , Pessoa de Meia-Idade , Paniculite/classificação , Paniculite/diagnóstico , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
14.
Mol Syst Biol ; 14(7): e8485, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-30021847
15.
Methods Mol Biol ; 1709: 47-58, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29177650

RESUMO

Chaperones associate with hundreds or thousands of diverse client proteins and regulate their function. Chaperone/client interactions are generally very transient and involve a highly orchestrated assembly and disassembly of regulatory co-factors. This poses specific challenges for identifying and characterizing these interactions in a scalable and sensitive manner. LUMIER assay, which takes advantage of the high sensitivity and linear range of luminescence-based detection, has proven to be an ideal assay to quantitatively profile chaperone/client interactions in a high-throughput manner. This article provides step-by-step instructions for quantitatively profiling these interactions with LUMIER.


Assuntos
Chaperonas Moleculares/metabolismo , Mapeamento de Interação de Proteínas/métodos , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Chaperonas Moleculares/química
16.
Mol Syst Biol ; 12(12): 893, 2016 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-27986836

RESUMO

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/patogenicidade , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Legionella pneumophila/metabolismo , Modelos Biológicos , Mapas de Interação de Proteínas , Biologia de Sistemas/métodos
17.
Mol Syst Biol ; 12(4): 865, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27107014

RESUMO

In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical-versus-functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an "inter-interactome" approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter-interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra-species networks. Although substantially reduced relative to intra-species networks, the levels of functional overlap in the yeast-human inter-interactome network uncover significant remnants of co-functionality widely preserved in the two proteomes beyond human-yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co-functionality. Such non-functional interactions, however, represent a reservoir from which nascent functional interactions may arise.


Assuntos
Proteínas Fúngicas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Evolução Molecular , Humanos
18.
Cell ; 161(3): 647-660, 2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25910212

RESUMO

How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.


Assuntos
Doença/genética , Mutação de Sentido Incorreto , Mapas de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Fases de Leitura Aberta , Dobramento de Proteína , Estabilidade Proteica
19.
Cell ; 158(2): 434-448, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25036637

RESUMO

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct ß-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mapas de Interação de Proteínas , Humanos , Dobramento de Proteína , Proteínas de Ligação a Tacrolimo/metabolismo
20.
PLoS Comput Biol ; 10(6): e1003693, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24967846

RESUMO

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-xL and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models.


Assuntos
Genômica/métodos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Reprodutibilidade dos Testes , Alinhamento de Sequência
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