Biomedical subjects

Sergey Sikora

Publications and source records attributed to Sergey Sikora.

6 recordsLinked to original sources

PubMed2006

Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase.

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.

Amino Acid Sequence↗

PubMed2005

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency.

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.

Animals↗

PubMed2005

Convergent evolution as a mechanism for pathogenic adaptation.

The survival of human pathogens depends on their ability to modulate defence pathways in human host cells. This was thought to be attained mainly by pathogen specific "virulence factors". However, pathogens are increasingly being discovered that use distant homologs of the human regulatory proteins as virulence factors. We analyzed several cases of this approach, with a particular focus on virulence proteases. The analysis reveals clear cases of bacterial proteases mimicking the specificity of their human counterparts, such as strong similarities in their active and/or binding sites. With more sensitive tools for distant homology recognition, we could expect to discover many more such cases.

Adaptation, Physiological↗

PubMed2004

Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis.

MT1-MMP, a prototypic member of a membrane-type metalloproteinase subfamily, is an invasion promoting protease and an activator of MMP-2. In addition, MT1-MMP proteolysis regulates the functionality of cell-surface adhesion/signaling receptors including tissue transglutaminase (tTG). tTG is known to serve as an adhesion coreceptor for beta1/beta3 integrins and as an enzyme that catalyzes the cross-linking of proteins and the conjugation of polyamines to proteins. Here, we report that MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated tTG, thereby further promoting the effect initiated by the activator of MMP-2. tTG, in return, preferentially associates with the activation intermediate of MMP-2. This event decreases the rate of MMP-2 maturation and protects tTG against proteolysis by MMP-2. Our cell culture, in vitro experiments, and in silico modeling indicate that the catalytic domain of MMP-2 directly associates with the core enzymatic domain II of tTG (the K(d) = 380 nM). The follow-up cleavage of the domain II eliminates both the receptor and the enzymatic activity of tTG. Our data illuminate the coordinated interplay involving the MT1-MMP/MMP-2 protease tandem in the regulation of the cell receptors and explain the underlying biochemical mechanisms of the extensive tTG proteolysis that exists at the normal tissue/tumor boundary. Our findings also suggest that neoplasms, which express functionally active MT1-MMP and, therefore, activate soluble MMP-2, can contribute to the degradation of tTG expressed in neighboring host cells. The loss of adhesive and enzymatic activities of tTG at the interface between tumor and normal tissue will decrease cell-matrix interactions and inhibit matrix cross-linking, causing multiple pathological alterations in host cell adhesion and locomotion.

Catalytic Domain↗

PubMed2004

Non-proteolytic, receptor/ligand interactions associate cellular membrane type-1 matrix metalloproteinase with the complement component C1q.

Membrane type-1 matrix metalloproteinase (MT1-MMP), a prototypic member of the membrane-tethered MMP family, is an essential component of a cellular proteolysis apparatus. Recognition of protein cleavage targets followed by proteolysis is a main function of MT1-MMP. For the first time, however, we present evidence that MT1-MMP and other structurally related membrane MMPs bind C1q, the recognition unit of the first component of complement C1 that initiates activation of the classical pathway of complement. These interactions involve the catalytic domain of MT1-MMP and the C1q globular domain. In silico modeling followed by mutagenesis and the in vitro and cell-based binding studies showed that the His(171)-Glu-Lys-Gln-Ala-Asp(176) and Val(223)-Arg-Asn(224) peptide sequences of MT1-MMP are directly involved in the binding with C1q. These sequence regions are spatially distant from the active site of the protease. As a result, the catalytically active and the catalytically latent forms of cellular MT1-MMP are both efficient in binding with C1q. In agreement, despite the MT1-MMP/C1q interactions, C1q is totally resistant to MT1-MMP proteolysis. The discovery of the unconventional, receptor/ligand-like interactions of MT1-MMP with C1q, an essential component of immunity, is a significant step toward a more complete understanding of the role of this membrane-tethered protease in cancer.

Amino Acid Sequence↗

PubMed2004

Combination of multiple alignment analysis and surface mapping paves a way for a detailed pathway reconstruction--the case of VHL (von Hippel-Lindau) protein and angiogenesis regulatory pathway.

Using the tumor suppressor VHL protein as an example, we show that detailed analysis of conservation versus variation pattern in the multiple alignment can be coupled with the genomic pathway/complex conservation analysis to provide a more complete picture of the entire interaction/regulatory network. Results from the present study have allowed us to hypothesize that two additional proteins are involved in the VHL-mediated regulation of angiogenesis. Detailed modeling also has led to a prediction of the possible interaction mode between the known and the proposed parts of the VHL complex. To aid in an analysis of the VHL protein regulation of HIF-1 alpha degradation, an important and only partially understood process that directly influences angiogenesis, we performed a comprehensive search for the orthologs of the VHL as well as for VHL-interacting proteins in all the available eukaryotic genomes. Analysis of a multiple alignment of thus identified VHL orthologs reveals an unusually high degree of conservation of the surface amino acid residues that almost exactly correspond to positions mutated in the VHL disease-associated tumors. In addition, these positions form well-defined clusters in three-dimensional space, and presence or absence of individual clusters correlates with the presence or absence of pathway elements in different genomes. We have also shown that relation trees derived from the multiple sequence alignment, functional surface-mapping, and HIF-1 alpha degradation pathway structure are in complete agreement, linking the functional and structural evolution of the VHL protein and VHL-dependent HIF-1 alpha degradation complex.

ATP-Dependent Proteases↗