RESUMO
Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiaeRESUMO
The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aß40. Strikingly, we identified the positively charged ribosome-binding domain in the N terminus of the ßNAC subunit (N-ßNAC) as a major chaperone entity of NAC. N-ßNAC by itself suppressed aggregation of PolyQ-expanded proteins in vitro, and the positive charge of this domain was critical for this activity. Moreover, we found that NAC also exerts a ribosome-independent chaperone function in vivo. Consistently, we found that a substantial fraction of NAC is non-ribosomal bound in higher eukaryotes. In sum, NAC is a potent suppressor of aggregation and proteotoxicity of mutant PolyQ-expanded proteins associated with human diseases like Huntington's disease and spinocerebellar ataxias.
Assuntos
Peptídeos beta-Amiloides/genética , Chaperonas Moleculares/genética , Agregação Patológica de Proteínas/genética , Peptídeos beta-Amiloides/química , Sítios de Ligação/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Luciferases/química , Luciferases/genética , Chaperonas Moleculares/química , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , Dobramento de Proteína , Ribossomos/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
BACKGROUND: Intravenous iron is widely prescribed in patients with chronic kidney disease (CKD) and can cause oxidative stress. The relationship of oxidative stress and renal injury in patients with CKD is unknown. Whether renal injury can occur at a time point when transferrin is incompletely saturated is also unclear. METHODS: We conducted a randomized, open-label, parallel group trial to compare the oxidative stress induced by intravenous administration of 100 mg iron sucrose over 5 minutes and its protection with N-acetylcysteine (NAC) in 20 subjects with stage 3 or 4 CKD. Transferrin saturation was measured with urea polyacrylamide gel electrophoresis, oxidative stress by malondialdehyde (MDA) measurement by high-performance liquid chromatography, and renal injury by enzymuria and proteinuria. Reduced and oxidized glutathione and free radical scavengers as well as urinary monocyte chemoattractant protein-1 were also measured. RESULTS: Parenteral iron increased plasma concentration and urinary excretion rate of MDA, a biomarker of lipid peroxidation, within 15 to 30 minutes of iron sucrose administration. This was accompanied by enzymuria and increase in proteinuria. In contrast, saturation of transferrin was not maximally seen until 3 hours after the end of infusion. Oxidative stress, enzymuria and proteinuria were transient and were completely resolved in 24 hours. NAC reduced acute generation of systemic oxidative stress but failed to abrogate proteinuria or enzymuria. CONCLUSION: Intravenous iron produces oxidative stress that is associated with transient proteinuria and tubular damage. The rapid production of oxidative stress even when transferrin is not completely saturation suggests free iron independent mechanism(s) to be operative in producing oxidative stress and transient renal injury. Long-term implications of these findings need further study.