14 research outputs found
Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4
Abstract Purpose The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes. Methods O-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA). Results O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules. Conclusion These results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy.</p
ModProt: a database for integrating laboratory and literature data about protein post-translational modifications
Proteomic analysis in usual and nonspecific interstitial pneumonia
Differentiating nonspecific interstitial
pneumonia (NSIP) from usual interstitial pneumonia
(UIP) is important for the determination of both
treatment and prognosis. Using two-dimensional
fluorescence difference gel electrophoresis (2D-DIGE),
we examined 8 UIPs, 8 NSIPs, and 30 normal lung
tissues. Comparisons with control in 2D-DIGE showed
that (a) in UIP, nine protein spots were significantly
upregulated and seven were significantly downregulated,
(b) in NSIP, four protein spots were significantly
upregulated and nine were significantly downregulated.
The detected proteins were analyzed by MALDI-TOF
mass spectrometry, allowing qualitative differences in
vimentin subtypes to be characterized. One vimentin
subtype was upregulated in UIP, while another one was
downregulated in NSIP (vs. control). These different
characteristics were partially supported by the results of
Western blot analysis. Our immunohistochemistry
revealed vimentin expression within fibroblasts (a) in
fibroblastic foci in UIP and (b) in fibrotic alveolar walls
in NSIP. Differences in vimentin subtypes may provide
useful biomarkers for separating NSIP from UIP,
alongside differences in histological characteristics
Proteomic analysis of the lung in rats with hypobaric hypoxia-induced pulmonary hypertension
Experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the lung. Proteomics - which may be the most powerful way to uncover unknown remodeling proteins involved in enhancing cardiovascular performance - was used to study 150 male Wistar rats housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level. After 14 days’ exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In lung tissue, about 140 matching protein spots were found among 8 groups (divided according to their hypobaric period) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH4.5-pH6.5, 30 kDa-100 kDa). In hypobaric rats, three spots were increased two-fold or more (vs. control rats) in two-dimensional differential in-gel electrophoresis (2D-DIGE). The increased proteins were identified, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), as one isoform of heat shock protein 70 (HSP70) and two isoforms of protein disulfide isomerase associated 3. This result was confirmed by Western blotting analysis of 2D-PAGE. Conceivably, HSP70 and PDIA3 may play roles in modulating the lung structural remodeling that occurs due to pulmonary hypertension in hypobaric hypoxia