| Popis: |
Quality control of protein folding is crucial to the maintainance of cellular homeostasis. Impairment of these systems and the related degradative pathways inovoed in the clearance of misfolded proteins can result in severe and varied pathologies. Peptide N-glycanase (EC 3.5.1.52) is an endoglycosidase which cleaves N-linked glycans from incorrectly folded glycoproteins exported from the endoplasmic reticulum. The hydrolysis of the β-aspartylglycosylamine bond of asparagine-linked glycopeptides occurs prior to processing by the 26S proteasome and is important for the degradation of misfolded glycoproteins during ER-associated degradation. Mutations in this enzyme are responsible for the rare congenital disorder, N-GLY1, a congenital multi-system disorder which results in a build-up of protein aggregates in the cell cytosol. \ud \ud Using a pharmacological inhibitor of peptide N-glycanase, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-fmk), we have found that inhibition of N-glycanase using 50 µM Z-VAD-fmk resulted in an increase in Thioflavin T fluorescence intensity after 48 hours which decreases to near basal levels after 72 hours. Thioflavin T is a fluorescent dye that exhibits enhanced fluorescence upon binding to β-sheets, characteristic of aggregated structures and fibril formation. \ud \ud Changes in characteristic ER stress markers were also observed; variation in the expression of BiP (GRP78), which is increased during the unfolded protein response, was observed to correlate with changes in Thioflavin T fluorescence. \ud \ud Using a tandem GFP-RFP-LC3 reporter in HEK cells we found an increase in autophagy after 72 hours of N-glycanase inhibition. The increase in autophagy corresponded to the decrease in Thioflavin T fluorescence and variation in BiP levels, suggesting that protein aggregates were removed by the induction of autophagy when deglycosylation is impaired, In an autophagy deficient cell line, ATG13-/- MEFs, we found inhibition of peptide N-glycanase by 50 µM Z-VAD-fmk lead to a 45 % decrease in cell viability within 24 hours with no loss of viability seen in the corresponding wild type MEFs or HEK cells. These results show that autophagy is essential for removal of protein aggregates resulting from the inhibition of peptide N-glycanase.\ud \ud Further work will focus on investigation of the autophagic machinery linked to clearance of protein aggregates caused by peptide N-glycanase inhibition. |
