Biomedical subjects

E V Apletalina

Publications and source records attributed to E V Apletalina.

5 recordsLinked to original sources

PubMed2000

Processing and sorting of the prohormone convertase 2 propeptide.

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.

Amino Acid Sequence↗

PubMed2000

Mutations in the catalytic domain of prohormone convertase 2 result in decreased binding to 7B2 and loss of inhibition with 7B2 C-terminal peptide.

Prohormone convertases 1 (PC1) and 2 (PC2) are members of a family of subtilisin-like proprotein convertases responsible for proteolytic maturation of a number of different prohormones and proneuropeptides. Although sharing more than 50% homology in their catalytic domains, PC1 and PC2 exhibit differences in substrate specificity and susceptibility to inhibitors. In addition to these differences, PC2, unlike PC1 and other members of the family, specifically binds the neuroendocrine protein 7B2. In order to identify determinants responsible for the specific properties of the PC2 catalytic domain, we compared its primary sequence with that of other PCs. This allowed us to distinguish a PC2-specific sequence at positions 242-248. We constructed two PC2 mutants in which residues 242 and 243 and residues 242-248 were replaced with the corresponding residues of PC1. Studies of in vivo cleavage of proenkephalin, in vivo production of alpha-MSH from proopiomelanocortin, and in vitro cleavage of a PC2-specific artificial substrate by mutant PC2s did not reveal profound alterations. On the other hand, both mutant pro-PC2s exhibited a considerably reduced ability to bind to 21-kDa 7B2. In addition, inhibition of mutant PC2-(242-248) by the potent natural inhibitor 7B2 CT peptide was almost completely abolished. Taken together, our results show that residues 242-248 do not play a significant role in defining the substrate specificity of PC2 but do contribute greatly to binding 7B2 and are critical for inhibition with the 7B2 CT peptide.

Amino Acid Sequence↗

PubMed2000

Structure-function analysis of the 7B2 CT peptide.

Prohormone convertases play important roles in the proteolytic conversion of many protein precursors. The neuroendocrine protein 7B2 and its 31-residue carboxyl-terminal (CT) peptide potently and specifically inhibit prohormone convertase 2 (PC2). We have analyzed the residues contributing to inhibition using N-terminal truncation and alanine scanning. Removal of more than 3 residues from the amino-terminal end of CT1-18 resulted in a more than 190-fold drop in inhibitory activity, showing that most of the residues between 3 and 18 are required for inhibition. In agreement, an Ala scan indicated that only 4 residues could be replaced with Ala without losing mid-nanomolar inhibitory potency; in particular, Gln7, Gln9, and Asp12 could be Ala-substituted to yield peptides with a similar inhibitory potency to the starting peptide. The all-d-retro-inverso, all-l-inverso, and all-d analogues of CT peptide were completely inactive, indicating that amino acid side chains and the CT peptide main chain interact with PC2. CT peptide inhibition could not be competitively blocked by preincubation with truncated CT peptide forms, supporting an absolute requirement for the Lys-Lys pair in initial binding of the CT peptide to the active site.

Amino Acid Sequence↗

PubMed1995

[Surface of cytochrome P-450 2B4: structure and function].

The review is devoted to the identification and structure of one of the cytochromes P450s-cytochrome P-450 2B4 derived from the rabbit liver endoplasmic reticulum. A critical review is made of the data on this enzyme membrane topology, its active site's structure and localization of its membrane and water-exposed regions. The paper is based on the data available in the literature and the authors' own findings. Various experimental and calculating methods used to identify the topography of cytochrome P450 are covered in the paper.

Amino Acid Sequence↗

PubMed1995

The comparative study of peroxidase activity and substrate binding properties of cytochrome P450 2B4 incorporated into phospholipid vesicles with the use of two different methods.

The comparative study of peroxidase activities and substrate binding properties of cytochrome p450 2B4 in reconstituted vesicles prepared with the use of two different techniques, and microsomal cytochrome P450 was carried out. The data obtained show that the two types of cytochrome P450 2B4-containing proteoliposomes do not differ substantially from each other with respect to H2O2- or cumene hydroperoxide-dependent substrate hydroxylation activities as well as substrate binding properties of hemoprotein reconstituted. However, some parameters measured with proteoliposomal cytochrome P450 are markedly different from those measured with microsomal hemoprotein, suggesting the existence of conformational differences between the molecules of these two cytochromes or the differences in the depth of their immersion into lipid bilayer.

Animals↗