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A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins.

ABSTRACT: Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.

SUBMITTER: Katiyar S

PROVIDER: S-EPMC518832 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins.

Katiyar Samiksha S Li Guangtao G Lennarz William J WJ

Proceedings of the National Academy of Sciences of the United States of America 20040909 38

Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) ...[more]

PMID: 15358861

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Project description:BackgroundProteasome functional insufficiency is implicated in a large subset of cardiovascular diseases and may play an important role in their pathogenesis. The regulation of proteasome function is poorly understood, hindering the development of effective strategies to improve proteasome function.Methods and resultsProtein kinase G (PKG) was manipulated genetically and pharmacologically in cultured cardiomyocytes. Activation of PKG increased proteasome peptidase activities, facilitated proteasome-mediated degradation of surrogate (enhanced green fluorescence protein modified by carboxyl fusion of degron CL1) and bona fide (CryAB(R120G)) misfolded proteins, and attenuated CryAB(R120G) overexpression-induced accumulation of ubiquitinated proteins and cellular injury. PKG inhibition elicited the opposite responses. Differences in the abundance of the key 26S proteasome subunits Rpt6 and β5 between the PKG-manipulated and control groups were not statistically significant, but the isoelectric points were shifted by PKG activation. In transgenic mice expressing a surrogate substrate (GFPdgn), PKG activation by sildenafil increased myocardial proteasome activities and significantly decreased myocardial GFPdgn protein levels. Sildenafil treatment significantly increased myocardial PKG activity and significantly reduced myocardial accumulation of CryAB(R120G), ubiquitin conjugates, and aberrant protein aggregates in mice with CryAB(R120G)-based desmin-related cardiomyopathy. No discernible effect on bona fide native substrates of the ubiquitin-proteasome system was observed from PKG manipulation in vitro or in vivo.ConclusionsPKG positively regulates proteasome activities and proteasome-mediated degradation of misfolded proteins, likely through posttranslational modifications to proteasome subunits. This may be a new mechanism underlying the benefit of PKG stimulation in treating cardiac diseases. Stimulation of PKG by measures such as sildenafil administration is potentially a new therapeutic strategy to treat cardiac proteinopathies.

Dataset Information

A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins.

Publications

A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins.

Similar Datasets

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