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Biomedical subjects

I V Dudich

Publications and source records attributed to I V Dudich.

At least 19 recordsLinked to original sources
PubMed1999

Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

Amino Acid Sequence↗
PubMed1997

Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.

Alkylation↗
PubMed1997

Alpha-fetoprotein as a TNF resistance factor for the human hepatocarcinoma cell line HepG2.

Human hepatocarcinoma HepG2 cells are known to be insensitive to tumor necrosis factor (TNF) cytotoxicity. In this report, preliminary washing of HepG2 cells with serum-free medium to remove endogenous and exogenous alpha-fetoprotein (AFP) from the cultivation medium transfers cells from the TNF-resistant to the TNF-sensitive state without addition of any transcriptional inhibitors. HepG2 cells sensitized to by washing again became TNF-resistant after their treatment with exogenous AFP. Protective AFP activity against TNF-induced cytotoxicity directly depends on the AFP/TNF concentration ratio, demonstrating biphasic AFP activity. Our data show that 0.2 mg/ml of AFP acts synergistically to enhance cytotoxicity of suboptimal TNF doses. In contrast, the same AFP dose significantly attenuates the cytotoxicity of high TNF doses. It is concluded that AFP can function as a protective factor against TNF cytotoxicity in human hepatoma cells. These observations suggest that AFP secretion by certain tumor cells allows a highly flexible regulation of TNF cytotoxicity, dependent on the amount of endogenous AFP.

Carcinoma, Hepatocellular↗
PubMed1997

Induction of apoptosis in human hepatoma cells by alpha-fetoprotein.

We have investigated the effects of purified human alpha-fetoprotein (AFP) on the growth of the human hepatocarcinoma-cells HepG2 in culture. Cancer-derived AFP (cAFP), isolated from the culture medium of AFP-secreting HepG2 cells and embryonal AFP (eAFP), isolated from human cord serum, were used for these studies. Both AFP pre parations studied were shown to induce strong dose-dependent inhibition of HepG2 cell proliferation and complete growth arrest at high protein concentrations (more than 0.1 mg/ml). To test whether AFP may trigger an endogenous suicide program in hepatoma cells, we examined whether DNA fragmentation preceded cell death. After exposure of the cells of the high AFP dose (1.0 mg/ml), DNA fragmentation was detected as early as 2 h after treatment, and 70% of cells were apoptotic by 24 h. DNA fragmentation was shown to precede other signs of cell death for several hours. Typical morphological changes of apoptosis were observed after 4 h of exposure of cells to high AFP doses. Low concentrations of cAFP and eAFP (less than 0.1 mg/ml) failed to induce growth inhibition of HepG2 cells, rather showing a weak stimulative effect, demonstrating a biphasic AFP activity. Cell pretreatment with the transcriptional inhibitor actinomycin D had no measurable influence on AFP cytotoxicity. These findings demonstrate that protein synthesis is not required for this mechanism of cell death. The charcoal-treated ligand-free eAFP (eAFPp) had a dose-dependent growth-inhibitory activity, similar to intact protein, but slightly less intensive. The similar growth-inhibitory activities of cAFP, eAFP and eAFPp, which have a significant difference in bound-ligand content, provide evidence that the main role in cell growth regulation may be attributed to the protein moiety of the entire AFP molecule, but not to its ligands. These biologically active AFP ligands could, however, modulate AFP-growth-regulating activity. Growth factor deprivation distinctly enhanced the cytostatic activity of high AFP concentrations and also increased the mitogenic activity of low AFP levels, showing the interdependence of the growth-regulative activity of AFP and growth factors. The findings of this study demonstrated that AFP is directly introduced into the intracellular pathways of cell growth regulation and programmed cell death.

Apoptosis↗
PubMed1995

Dimer structure as a minimum cooperative subunit of small heat-shock proteins.

Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.

Animals↗
PubMed1995

Drastic influence of a single heme axial ligand replacement on the thermostability of cytochrome c3.

The thermostability of wild type Desulfovibrio vulgaris Hildenborough tetraheme cytochrome c3 and its H22M, H25M, H35M and H70M mutants was studied by circular dichroism technique in the far UV and Soret regions. It was shown that wild type cytochrome is extremely thermostable and retains structural and functional properties up to 110 degrees C. Mutations do not change overall secondary structure and local structure of the hemes vicinity. All mutants are much more unstable to heat denaturation than the wild type cytochrome. Point mutation (His/Met replacement) results in extraordinary 30-45 degrees C decrease in the protein thermostability depending on the mutation. We may conclude therefore that the heme region is important not only for the functional properties of the cytochrome but also for the overall protein thermostability.

Circular Dichroism↗
PubMed1995

The costimulatory and differentiating activity of soluble class I MHC antigens for an autologous thymocyte population.

Combined cultivation of macrophages with syngeneic thymocytes resulted in accumulation of soluble H-2Kk antigens in culture medium. Incubation of intact autologous thymocytes with these soluble class I MHC molecules was shown to induce functional maturation of thymocytes assayed in local graft-vs-host reaction. Similar thymocyte costimulating activity was detected for the papain-solubilized purified H-2Kk antigens. Soluble class I antigens were shown to costimulate IL2 production by thymocytes in response to submitogenic doses of exogenous IL2 and to increase PHA-induced thymocyte proliferation. Soluble class I molecules were shown to increase the level of expression of function-associated membrane antigens, H-2Kk, CD8 and CD4, and to trigger thymocyte differentiation. The expression of I-Ak antigens remained invariable. It was also shown that soluble autologous class I molecules may function as direct amplifiers of thymocyte proliferation in autologous, but not allogeneic, mixed leukocyte reactions. It is concluded that soluble MHC class I molecules are capable of triggering functional and phenotype differentiation of syngeneic thymocytes and acting as restricted coaccessory molecules when thymocyte activation is induced by a submitogenic dose of different stimuli.

Animals↗
PubMed1983

Polarization fluorescence, spin label and ultracentrifugal studies of specific interaction of low molecular weight proteins with the Fc fragment of human immunoglobulin G.

Low mol. wt (about 2000) proteins which were found in normal human serum formed complexes with the Fc fragment of IgG. The interaction constant was not less than 10(6) l/mole. These complexes dissociated on dilution of the protein solution to below 2 microM or decreasing the pH below 6. The binding site of these proteins was located in the CH2 domains in close proximity to the carbohydrate moieties. The dissociation of IgG complexes with these proteins was accompanied by a conformational change in the Fc fragment.

Blood Proteins↗
PubMed1983

Release of low molecular weight proteins from the Fc fragment of rabbit immunoglobulin G induced by formation of insoluble complexes with antigen.

Serum rabbit IgG was found to be in complexes, with low mol. wt proteins (mol. wt about 1500) which are noncovalently associated with the Fc fragment. The formation of precipitating antigen-antibody complexes resulted in the dissociation of IgG complexes with these proteins and their release in solution. On formation of non-precipitating antigen-antibody complexes, the dissociation of low mol. wt proteins from IgG was not observed.

Animals↗
PubMed1980

Comparative study of dynamic structure of pig and chicken aspartate aminotransferases by measuring the rotational correlation time.

The rotational correlation time of two homologous cytoplasmic aspartate aminotransferase molecules isolated from pig and chicken hearts was obtained by spin-labeling technique. The maleimide and iodoacetamide spin-labels modifying external SH-groups of a protein were used. In the interpretation of ESR spectra a rotational motion of nitroxide group relative to the protein molecule was taken into account. To determine the macromolecule rotational correlation time two methods of the immobilization of a protein molecule were used: 1) by means of increasing protein solution viscosity and 2) by fixation of the protein molecule on adsorbent. From comparison of experimental and theoretical values of rotational correlation time it was conclude that the both enzymes exhibits an intramolecular flexibility.

Animals↗
PubMed1980

[Conformational changes of spin-labeled native and modified phosphorylase B].

The phosphorylase B labelled with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-iodacetamide (phosphorylase I) and with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-ethylmaleinimide (phosphorylase II) was studied. It was shown that label I is characterized by a greater mobility with respect to the protein as compared to label II. In spin-labelled preparations of phosphorylase B the 1,5--2,0 SH-groups of the enzyme monomer having no effect on the enzyme activity were modified. The effects of AMP, glucose-1-phosphate and glucose-6-phosphate on the EPR spectrum of phosphorylase I were studied. The greatest changes in the spectrum (especially in the high field line) were found to occur in the presence of glucose-6-phosphate. These changes are due to the increase in the degree of anisotropic spin rotation. The experimental and theoretical spectra allowing to determine the correlation time for the protein moiety (tau b = 160 ns) were shown to be similar. The local conformation changes were found to occur in the vicinity of one of the two label-bound SH-groups of phosphorylase I. The EPR spectra demonstrate the S-shaped dependence of mobility of phosphorylase I label on concentration of glucose-6-phosphate (0,1--10 mM). In the presence of AMP no S-shaped dependence is observed. Reduced NaBH4 phosphorylase I does not reveal the S-shaped dependence of the label mobility on concentration of glucose-6-phosphate. The degree of the label immobilization in the apo-phosphorylase I--pyridoxal-5-chloromethylphosphonate complex in the presence of glucose-6-phosphate and AMP is the same as in cholophosphorylase I; however, in contrast to the choloenzyme it does not depend on glucose-6-phosphate (0,1--10,0 mM). The changes in the mobility of the spin label of apophosphorylase I and its complex with the AMP analog--adenosine-5'-chloromethylphosphonate--during the choloenzyme reconstruction by pyridoxalphosphate are indicative of participation of AMP and the phosphate group of AMP in the formation of the enzyme active center.

Adenosine Monophosphate↗