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1.
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
2.
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
3.
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
4.
FEBS Lett ; 594(6): 1081-1087, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31764998

RESUMO

The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop-transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23-mediated stop-transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2-deletion or -overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix-facing positive charges, is reduced. These results imply that the Mgr2-mediated gatekeeper function is important for controlling membrane sorting of marginal stop-transfer signals.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/genética , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
5.
J Biol Chem ; 292(49): 20058-20066, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29030426

RESUMO

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m-AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m-AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m-AAA protease subunit. Furthermore, m-AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m-AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m-AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates.


Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Membranas Mitocondriais/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Mitocondriais/metabolismo , Domínios Proteicos , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras
6.
Biochem Biophys Res Commun ; 450(4): 1587-92, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-25034329

RESUMO

Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6102, SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in Cin form. MRP6102 and SP-C(Leu/Val) are inserted into the membrane in Cout form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Animais , Células Cultivadas , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Glicosilação , Humanos , Microscopia de Fluorescência
7.
Biochem Biophys Res Commun ; 453(2): 268-76, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24938127

RESUMO

Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.


Assuntos
Proteínas de Membrana/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Potenciais da Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico
8.
J Biol Chem ; 289(22): 15845-55, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24753257

RESUMO

The evolutionarily conserved Sec61 translocon mediates the translocation and membrane insertion of proteins. For the integration of proteins into the membrane, the Sec61 translocon opens laterally to the lipid bilayer. Previous studies suggest that the lateral opening of the channel is mediated by the helices TM2b and TM7 of a pore-forming subunit of the Sec61 translocon. To map key residues in TM2b and TM7 in yeast Sec61 that modulate lateral gating activity, we performed alanine scanning and in vivo site-directed photocross-linking experiments. Alanine scanning identified two groups of critical residues in the lateral gate, one group that leads to defects in the translocation and membrane insertion of proteins and the other group that causes faster translocation and facilitates membrane insertion. Photocross-linking data show that the former group of residues is located at the interface of the lateral gate. Furthermore, different degrees of defects for the membrane insertion of single- and double-spanning membrane proteins were observed depending on whether the mutations were located in TM2b or TM7. These results demonstrate subtle differences in the molecular mechanism of the signal sequence binding/opening of the lateral gate and membrane insertion of a succeeding transmembrane segment in a polytopic membrane protein.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Canais de Translocação SEC , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
9.
Biochem Biophys Res Commun ; 427(4): 780-4, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-23047006

RESUMO

Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.


Assuntos
Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Sequência de Aminoácidos , Animais , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Fluorescência , Glicosilação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas , Saccharomyces cerevisiae/metabolismo
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