No abstract available

[en] Thermophiles and extreme thermophiles are potential source of thermostable proteases for economical application. This study deals with cloning and over-expression of a carboxypeptidase (CBP) from the extreme thermophile archaeon Pyrococcus furiosus in E. coli. Using the forward and the reverse primers designed according to the putative CBP gene sequence analysed from the published genome sequence of P. furiosus, 1.5 kb fragment of CBP gene was PCR amplified. After TA-cloning in pTZ57R/T vector, the gene was ligated into pET-22b(+) and the recombinant plasmid thus obtained was used to transform E. coli BL21 (DE3)RIPL. On induction with IPTG for 6-8 hours CBP was expressed up to 30% of the total cell proteins. The enzyme, however, was expressed in an insoluble form which was refolded to an active state by treatment with urea. (author)

[en] Surface modified nanoparticles composed of poly(isobutylcyanoacrylate) (PIBCA) cores surrounded by a chitosan and thiolated chitosan gel layer were prepared and characterized in previous works. The presence of such biopolymers on the nanoparticle surface conferred those nanosystems interesting characteristics that might partially overcome the gastrointestinal enzymatic barrier, improving the oral administration of pharmacologically active peptides. In the present work, the antiprotease behaviour of this family of core-shell nanoparticles was in vitro tested against two model metallopeptidases present in the gastrointestinal tract (GIT): Carboxypeptidase A -CP A- (luminal protease) and Leucine Aminopeptidase M -LAP M- (membrane protease). As previous step, the zinc-binding capacity of these nanoparticles was evaluated. Interestingly, an improvement of both the zinc-binding capacity and the antiprotease effect of chitosan was observed when the biopolymers (chitosan and thiolated chitosan) were used as coating component of the core-shell nanoparticles, in comparison with their behaviour in solution, thanks to the different biopolymer chains rearrangement. The presence of amino, hydroxyl and thiol groups on the nanoparticle surface promoted zinc binding and hence the inhibition of the metallopeptidases analysed. On the contrary, the occurrence of a cross-linked structure in the gel layer surrounding the PIBCA cores of thiolated formulations, due to the formation of interchain and intrachain disulphide bonds, partially limited the inhibition of the proteases. The low accessibility of cations to the active groups of the cross-linked polymeric shell was postulated as a possible explanation of this behaviour. Results obtained in this work make this family of surface-modified nanocarriers promising candidates for the successfull administration of pharmacologically active peptides and proteins by the oral route.

[en] Highlights: • GCPII interacts specifically with HDAC1 which is present in the non-nuclear fraction. • HDAC1 regulates the stability of GCPII protein. • Mutation at lysine sites in human GCPII (K479R and K491R) reduces the ubiquitination. • Mutation at 479 lysine residue of human GCPII blocks HDAC1-mediated decrease in the level of GCPII protein. Our previous study showed that the level of glutamate carboxypeptidase II (GCPII) protein is regulated by valproic acid, a histone deacetylase (HDAC) inhibitor, through acetylation of lysine residue in the GCPII protein in human astrocytes, U-87MG. The present study further investigated which HDAC subtype is involved in the acetylation of GCPII. The results revealed that GCPII interacted with HDAC1 but not with HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6. Overexpression of catalytic domain (1–56 aa)-deleted HDAC1, which poorly binds to GCPII, enhanced lysine acetylation in GCPII and increased the level of GCPII protein when compared with that of the wild-type HDAC1. Further experiments showed that HDAC1 regulated the stability of GCPII protein. These data suggest that acetylation of GCPII is facilitated by HDAC1, and the acetylated GCPII is more stable than the non-acetylated GCPII. Additional experiments using siRNA HDAC1 and by HDAC1 overexpression confirmed the role of HDAC1 in regulating the stability of GCPII protein. Further, database search of acetylation and ubiquitination sites showed four candidate lysine sites in human GCPII protein that can be both acetylated and ubiquitinylated (K207, K479, K491, and K699). Mutation (lysine residues to arginine (R)) analysis showed that in the presence of cycloheximide K479R- and K491R-hGCPII mutants were less ubiquitinylated and degraded, and decrease in the level of GCPII protein by HDAC1 was significantly blocked by K479R mutants. These data suggest that K479 is a possible site of acetylation or ubiquitination. Furthermore, the results also demonstrate that the stability of GCPII protein is regulated by HDAC1 through acetylation at the lysine 479 residue.

No abstract available

[en] Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM₂AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

No abstract available

[en] Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase Y (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY*, and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY*-GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells.

[en] Thymopoietinsub(32-36) (TP5) is a synthetic pentapeptide that has the biological activity of its parent molecule, the 49 amino acid thymic hormone thymopoietin. Tritiated thymopoietinsub(32-36) (³H-TP5) was prepared by reductive tritiation of dibromotryrosyl-TP5. The stability of ³H-TP5 in human plasma was studied by analyzing samples by thin-layer chromatography at different time points and quantitating the radioactivity associated with TP5 (Arg-Lys-Asp-Val-Tyr) and its tyrosyl-containing breakdown products (Lys-Asp-Val-Tyr, Asp-Val-Tyr, Val-Tyr, Tyr). In plasma (but not in saline) the pentapeptide was rapidly degraded (apparent tsub(1/2)is similar to 30 seconds) with the corresponding appearance of radioactivity associated with the other tyrosyl-containing reference compounds. These data imply that the pentapeptide, which is active in vivo, may rapidly trigger changes in responsive cells; sustained circulating levels may not be required for activity. (author)

No abstract available