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ABSTRACT: PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCβ with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCβ in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCβ inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCβ might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCβΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCβ acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.
Mitochondrion 06/2011; 12(1):138-43. · 3.62 Impact Factor
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ABSTRACT: Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.
Biochimica et Biophysica Acta 02/2011; 1814(5):610-21. · 4.66 Impact Factor
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ABSTRACT: The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.
Archives of Biochemistry and Biophysics 421(2):260-266. · 2.93 Impact Factor
