Eugene V. Gasanov

Propeptides as modulators of functional activity of proteases

Abstract Most proteases are synthesized in the cell as precursor-containing propeptides. These structural elements can determine the folding of the cognate protein, function as an inhibitor/activator peptide, mediate enzyme sorting, and mediate the protease interaction with other molecules and supramolecular structures. The data presented in this review demonstrate modulatory activity of propeptides irrespective of the specific mechanism of action. Changes in propeptide structure, sometimes minor, can crucially alter protein function in the living organism. Modulatory activity coupled with high variation allows us to consider propeptides as specific evolutionary modules that can transform biological properties of proteases without significant changes in the highly conserved catalytic domains. As the considered properties of propeptides are not unique to proteases, propeptide-mediated evolution seems to be a universal biological mechanism.

Structural Organization of Precursors of Thermolysin-like Proteinases

The primary structures of the full-length precursors of thermolysin-like proteinases (TLPs) were systemically analyzed. Structural comparison of the precursor amino-terminal regions (ATRs) removed during maturation allowed us to divide the family into two groups: peptidases with short (about 50 amino acids) and long (about 200 amino acids) ATRs. The accumulation of mutations in the ATRs of both types proved to correlate with that in the catalytic domains. No classical signal peptides were identified in the short ATRs, but they contained a conserved PPL-motif near the initiation methionine. The functional role of the short ATRs and PPL-motif is currently unclear. The C-terminal regions (CTRs) of TLP precursors, which are often removed during maturation, too, are found in about a half of precursors with long ATRs, but occur more rarely in precursors with short ATRs. CTRs in TLP precursors contain previously identified conserved domains typical for many other proteins and likely underlie the interaction with high molecular weight substrates.

Alterations in Gene Expression of Proprotein Convertases in Human Lung Cancer Have a Limited Number of Scenarios

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p<0.00005) and decreased mRNA levels of PCSK2 (p<0.007), PCSK5 (p<0.0002), PCSK7 (p<0.002), PCSK9 (p<0.00008), and MBTPS1 (p<0.00004) as well as a tendency to increase in the level of PCSK1 mRNA. Four distinct groups of samples have been identified by cluster analysis of the expression patterns of PC genes in tumor vs. normal tissue. Three of these groups covering 80% of samples feature a strong elevation in the expression of a single gene in cancer: FURIN, PCSK1, or PCSK6. Thus, the changes in the expression of PC genes have a limited number of scenarios, which may reflect different pathways of tumor development and cryptic features of tumors. This finding allows to consider the mRNAs of PC genes as potentially important tumor markers.

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

Neurotrophin Propeptides: Biological Functions and Molecular Mechanisms.

Neurotrophins constitute a family of growth factors that play a key role in the regulation of the development and function of the central and peripheral nervous systems. A common feature of all the neurotrophins is their synthesis in cells as long precursors (pre-pro-neurotrophins) that contain an N-terminal signal peptide, a following propeptide and the mature neurotrophin. Although the signal peptide functions have been well studied, the role of neurotrophin propeptides is not so clear. Here, we briefly summarize the biochemistry of neurotrophin propeptides, including their role as folding-assistants for the mature factor and their role in processing and in secretion of neurotrophins. In the main part of the review we summarize our current state of knowledge of the biological activity of neurotrophin propeptides, their possible mechanisms of action, and their potential influence on the activity of the mature neurotrophins.

Hetero- and auto-activation of recombinant glutamyl endopeptidase from Bacillus intermedius

Glutamyl endopeptidase from Bacillus intermedius (BIGEP) is a secretory serine proteinase specifically hydrolyzing peptide bonds involving alpha-carboxyl groups of glutamic and aspartic acids. In this work, different BIGEP forms (full-length precursor, precursor without signal peptide and mature part) were expressed in Escherichia coli and the process of enzyme maturation was studied in vitro. BIGEP precursor renaturation leads to autocatalytic hydrolysis of the propeptide at Glu(-16). At the same time, the enzyme activation requires the complete removal of the prosequence by other proteinases. The mature part of BIGEP cannot be activated, which indicates that the propeptide is required for the active protein formation. The data obtained allowed us to apply directed mutagenesis of the processing site to obtain a BIGEP form that matured autocatalytically. This approach makes it possible to produce the enzyme without extrinsic proteinases, which is a prerequisite for using it in limited hydrolysis of proteins and peptides.

BDNF-TrkB axis regulates migration of the lateral line primordium and modulates the maintenance of mechanoreceptor progenitors.

BDNF and its specialized receptor TrkB are expressed in the developing lateral line system of zebrafish, but their role in this organ is unknown. To tackle this problem in vivo, we used transgenic animals expressing fluorescent markers in different cell types of the lateral line and combined a BDNF gain-of-function approach by BDNF mRNA overexpression and by soaking embryos in a solution of BDNF, with a loss-of-function approach by injecting the antisence ntrk2b-morpholino and treating embryos with the specific Trk inhibitor K252a. Subsequent analysis demonstrated that the BDNF-TrkB axis regulates migration of the lateral line primordium. In particular, BDNF-TrkB influences the expression level of components of chemokine signaling including Cxcr4b, and the generation of progenitors of mechanoreceptors, at the level of expression of Atoh1a-Atp2b1a.

Oligomeric organization of recombinant human neurotrophins expressed in Escherichia coli cells

Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3λ) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure.

Effect of the modification of the processing site of Bacillus intermedius glutamyl endopeptidase on the production of active enzyme by Bacillus subtilis cells

A site-directed modification at position P1 of the processing site of Bacillus intermedius glutamyl endopeptidase was carried out. Variants of the protease gene were obtained that correspond to the protein with an Ala, Asn, Ser, or Glu residue at this position substituted for the Lys residue. The residue in the P1 position of the processing site was shown to affect substantially the production of the active enzyme; however, none of the mutations leads to the complete termination of active protein production by cells.

Precursors and Propeptides of Neurotrophic Factors as Modulators of the Biological Activity of Mature Forms

Problems of the maturation of neurotrophic factors, the role of their precursors (proneurotrophins), and the contribution of elements that are removed in the course of maturation (propeptides) to the biological functions of these growth factors, are reviewed.