Biomedical subjects

Andrey E Kalinin

Publications and source records attributed to Andrey E Kalinin.

4 recordsLinked to original sources

PubMed2005

Breaking the connection: caspase 6 disconnects intermediate filament-binding domain of periplakin from its actin-binding N-terminal region.

Periplakin is a member of the plakin family of cytolinkers that connect cytoskeletal networks to each other as well as to the cell junctional complexes. Here, we demonstrate a direct molecular interaction between actin and periplakin. Furthermore, the oligomerization state of periplakin was shown to determine specificity of its binding to intermediate filaments (IF) in vitro. Both the filament association and the cell membrane localization of periplakin were confirmed in the cells overexpressing human periplakin. Double labeling of the N- and C-terminally tagged periplakin revealed unexpected lack of co-localization of periplakin ends in a confluent culture, and separation of the periplakin ends was even more pronounced in apoptotic cells. Western analysis revealed that after induction of apoptosis, periplakin becomes cleaved close to its C-terminal tail. Only the distinct cleavage products, but not the full-length periplakin, were present in the cells detached from the solid support during the apoptotic process. We show that caspase 6 cleaves periplakin at an unconventional recognition site, amino acid sequence TVAD. Thus, the separation of periplakin ends disconnects the actin-binding head-rod domain from the IF-binding C-terminal domain. We show that specific cleavage products co-exist with the full-length periplakin in cells, suggesting physiological consequences due to their altered binding specificities.

Actins↗

PubMed2004

Co-assembly of envoplakin and periplakin into oligomers and Ca(2+)-dependent vesicle binding: implications for cornified cell envelope formation in stratified squamous epithelia.

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca(2+). These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca(2+)] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.

Anions↗

PubMed2002

Processing of Escherichia coli alkaline phosphatase. Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide.

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor. The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation. These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.

Alkaline Phosphatase↗

PubMed2002

Epithelial barrier function: assembly and structural features of the cornified cell envelope.

Terminally differentiating stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery termed the cornified cell envelope (CE). It is composed of numerous structural proteins that become cross-linked by several transglutaminase enzymes into an insoluble macromolecular assembly. Several proteins are involved in the initial stages of CE assembly, but only certain proteins from a choice of more than 20 different proteins are used in the final stages of CE reinforcement, apparently to meet tissue-specific requirements. In addition, a variable selection of proteins may be upregulated in response to genetic defects of one of the CE proteins or tissue injury, in an effort to maintain an effective barrier. Additionally, in the epidermis and hair fiber cuticle, a layer of lipids is covalently attached to the proteins, which provides essential water barrier properties. Here we describe our current understanding of CE structure, a possible mechanism of its assembly, and various disorders that cause a defective barrier.

Animals↗