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2.
FASEB J ; 35(2): e21185, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33191543

RESUMO

Autophagy, a cellular stress response to starvation and bacterial infection, is executed by double-membrane-bound organelles called autophagosomes. Autophagosomes transfer cytosolic material to acidified lysosomes for degradation following soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-dependent fusion processes. Many of the autophagy-related disorders stem from defective end-step proteolysis inside lysosomes. The role of epithelial cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel has been argued to be critical for efficient lysosomal clearance; however, its context to autophagic clearance and the underlying mechanism is poorly defined. Here, we report that syntaxin17 (Stx17), an autophagic SNARE protein interacts with CFTR under nutritional stress and bacterial infection and incorporates it into mature autophagosomes to mediate an efficient lysosomal clearance. Lack of CFTR function and Stx17 and loss of CFTR-Stx17 interaction impairs bacterial clearance. We discover a specialized role of the Stx17-CFTR protein complex that is critical to prevent defective autophagy as has been the reported scenario in CF airway epithelial cells, infectious diseases, and lysosomal clearance disorders.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais/genética , Estresse Fisiológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lisossomos/metabolismo , Nutrientes/deficiência , Ligação Proteica , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Proteínas Qa-SNARE/genética , Transfecção
3.
Sci Rep ; 7(1): 17383, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234107

RESUMO

Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N 2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2-dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.


Assuntos
Benzo(a)pireno/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Simulação de Dinâmica Molecular , Sulfolobus solfataricus/enzimologia , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA
4.
RNA Biol ; 10(1): 133-48, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23353571

RESUMO

RNA helicases are a diverse group of RNA-dependent ATPases known to play a large number of biological roles inside the cell, such as RNA unwinding, remodeling, export and degradation. Understanding how helicases mediate changes in RNA structure is therefore of fundamental interest. The advent of single-molecule spectroscopic techniques has unveiled with unprecedented detail the interplay of RNA helicases with their substrates. In this review, we describe the characterization of helicase-RNA interactions by single-molecule approaches. State-of-the-art techniques are presented, followed by a discussion of recent advancements in this exciting field.


Assuntos
RNA Helicases/metabolismo , RNA/química , RNA/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Dobramento de RNA , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
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