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1.
Prikl Biokhim Mikrobiol ; 52(2): 193-9, 2016.
Artigo em Russo | MEDLINE | ID: mdl-27266248

RESUMO

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.


Assuntos
Proteínas Mutantes/biossíntese , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/biossíntese , alfa-Fetoproteínas/biossíntese , Linhagem Celular Tumoral , Fermentação , Humanos , Proteínas Mutantes/administração & dosagem , Proteínas Mutantes/genética , Pichia/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , alfa-Fetoproteínas/administração & dosagem , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/isolamento & purificação
2.
Bioorg Khim ; 38(5): 524-34, 2012.
Artigo em Russo | MEDLINE | ID: mdl-23342486

RESUMO

Recombinant human alpha-fetoprotein (rhAFP) expressed in yeast system as a glycoprotein, was isolated and purified to 98% by multistep method. The testing of the rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration as well as vascular endothelial growth factor production, with no significant influence on cell invasion and matrix metalloproteinase-2 and -9 secretion. It has been also estimated that rhAFP is internalized in hASC via clathrin-dependent mechanism. A study in the murine experimental model of hindlimb ischemia has shown the capability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.


Assuntos
Tecido Adiposo/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regeneração/efeitos dos fármacos , alfa-Fetoproteínas/farmacologia , Tecido Adiposo/citologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Isquemia/patologia , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Regeneração/fisiologia , Células Estromais/citologia , Células Estromais/metabolismo , alfa-Fetoproteínas/química , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/isolamento & purificação
4.
J Bacteriol ; 181(8): 2422-9, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10198004

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Proteínas de Membrana/química , Chaperonas Moleculares/química , Periplasma/química , Proteínas Periplásmicas , Yersinia pestis , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Genes Bacterianos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Periplasma/genética , Periplasma/metabolismo , Conformação Proteica , Dobramento de Proteína , Deleção de Sequência , Espectrometria de Fluorescência , Fatores de Transcrição , Tripsina
5.
Biochem J ; 324 ( Pt 2): 571-8, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9182720

RESUMO

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.


Assuntos
Cistina/fisiologia , Chaperonas Moleculares/química , Yersinia pestis/química , Alquilação , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Dicroísmo Circular , Cistina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Isomerases/fisiologia , Cinética , Proteínas de Membrana/fisiologia , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Isomerases de Dissulfetos de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
6.
Tumour Biol ; 18(1): 30-40, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-8989923

RESUMO

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.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Fator de Necrose Tumoral alfa/toxicidade , alfa-Fetoproteínas/farmacologia , alfa-Fetoproteínas/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Células Tumorais Cultivadas
7.
Tumour Biol ; 18(5): 261-73, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9276026

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Hepáticas/patologia , alfa-Fetoproteínas/farmacologia , Carcinoma Hepatocelular/patologia , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Células Tumorais Cultivadas , alfa-Fetoproteínas/farmacocinética
9.
Biochim Biophys Acta ; 1253(2): 163-8, 1995 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-8519797

RESUMO

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.


Assuntos
Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas de Neoplasias/química , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Proteínas de Choque Térmico HSP27 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Temperatura , Termodinâmica
10.
Biochem Biophys Res Commun ; 211(3): 742-7, 1995 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-7598701

RESUMO

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.


Assuntos
Grupo dos Citocromos c/química , Desulfovibrio vulgaris/química , Dicroísmo Circular , Grupo dos Citocromos c/genética , Desulfovibrio vulgaris/genética , Histidina/química , Histidina/genética , Temperatura Alta , Ligantes , Metionina/química , Metionina/genética , Mutação Puntual , Desnaturação Proteica , Espectrofotometria Ultravioleta
11.
Res Immunol ; 146(1): 45-57, 1995 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7569312

RESUMO

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.


Assuntos
Antígenos H-2/fisiologia , Ativação Linfocitária , Linfócitos T/imunologia , Timo/imunologia , Animais , Diferenciação Celular/imunologia , Reação Enxerto-Hospedeiro , Antígenos H-2/isolamento & purificação , Interleucina-2/biossíntese , Isoantígenos/imunologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fito-Hemaglutininas/farmacologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia
12.
Mol Biol (Mosk) ; 26(2): 441-51, 1992.
Artigo em Russo | MEDLINE | ID: mdl-1339959

RESUMO

Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.


Assuntos
Interferon gama/química , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Cinética , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Termodinâmica
13.
Mol Biol (Mosk) ; 25(4): 1061-70, 1991.
Artigo em Russo | MEDLINE | ID: mdl-1795700

RESUMO

Human leukocyte interferon-A1 (IFN-alpha A) structure in solution was investigated by fluorescence polarization, circular dichroism and scanning microcalorimetry techniques. Using gel-filtration it was established that at neutral pH values and at concentration not exceeding 0.3 mg/ml IFN-alpha A has a dimeric configuration in solution. At pH below 5, IFN-alpha A exists as a monomer. Using circular dichroism technique the IFN-alpha A molecule was shown to preserve a native structure upon decreasing pH to 3.5. The rotational correlation time of IFN-alpha A molecule in dimeric and monomeric form was measured using fluorescence of DNS, conjugated with the protein, and fluorescence of tryptophan residues. Our data indicate that the shape of IFN-alpha A molecule may be approximated by the rigid ellipsoid of revolution with the axis ratio = 4:1. The intramolecular melting of IFN-alpha A was studied by scanning microcalorimetry and circular dichroism in the acidic pH range. Thermodynamic analysis reveals two independent cooperative transitions. These transitions can be explained by assuming that the IFN-alpha A molecule consists of two structural domains.


Assuntos
Interferon-alfa/metabolismo , Leucócitos/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Polarização de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Proteínas Recombinantes/metabolismo
15.
Mol Biol (Mosk) ; 22(4): 1045-61, 1988.
Artigo em Russo | MEDLINE | ID: mdl-2847022

RESUMO

New spin-labels based on iodine and hydrargirum containing imidazolids were approbated on the bovine serum albumin (BSA) molecule. It is shown that all hydrargirum labels are binded to the external SH-group of BSA practically immediately in comparison with earlier known spin-labels based on piperidine with maleimide and iodacetamide groups, requiring some minutes of hours, correspondingly. Rotational correlation times and the character of relative mobility of the spin-label were measured. Values of the rotational correlation times of the protein molecule obtained by hydrargirum containing labels were found in the range of correlation times typical for the BSA molecule and represent at pH 7.0 a rigid stretch ellipsoid of rotation. The result obtained revealed that the relative reorientation nature of spin-labels is essentially different and is taken into account in different values of order parametres S according to model--fast anisotropic rotator on slow isotropic rotator. ESR spectra based on theoretical calculations by means of computer are given.


Assuntos
Soroalbumina Bovina/análise , Marcadores de Spin , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Imidazóis , Iodo , Mercúrio , Compostos de Sulfidrila/análise , Viscosidade
16.
Mol Immunol ; 20(12): 1267-72, 1983 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6318093

RESUMO

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.


Assuntos
Proteínas Sanguíneas , Fragmentos Fc das Imunoglobulinas , Fragmentos de Imunoglobulinas , Proteínas Sanguíneas/imunologia , Cromatografia em Gel , Espectroscopia de Ressonância de Spin Eletrônica , Polarização de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G , Técnicas In Vitro , Substâncias Macromoleculares , Peso Molecular , Ligação Proteica , Conformação Proteica , Ultracentrifugação
17.
Mol Immunol ; 20(12): 1273-6, 1983 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6656773

RESUMO

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.


Assuntos
Complexo Antígeno-Anticorpo , Proteínas Sanguíneas , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Animais , Proteínas Sanguíneas/isolamento & purificação , Cromatografia em Gel , Imunoglobulina G , Substâncias Macromoleculares , Peso Molecular , Coelhos , Soroalbumina Bovina/imunologia
18.
Mol Biol (Mosk) ; 17(2): 279-85, 1983.
Artigo em Russo | MEDLINE | ID: mdl-6304490

RESUMO

A selective modification of external SH-groups of riboluse 1,5-diphosphate carboxylase (EC 4.1.1.39) from spinach by a spin label on the basis of maleimide was performed. From the EPR spectra of spin labels of three types of SH-groups were classified. It is shown that part of spin labels pre-reduced with dithiothreitol are reoxidized by the enzyme after the removal of dithiothreitol from the reaction mixture. The reoxidation is connected with the fact that one of the spin labels is bound to the SH-group at the enzyme active site and can act as its substrate, which results in alteration of the electron structure of the greater than N--O fragment in the spin label upon interaction of the spin-labeled enzyme with the substrate and Mg2+.


Assuntos
Carboxiliases/metabolismo , Plantas/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Ditiotreitol/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Magnésio/metabolismo , Maleimidas , Oxirredução , Ribulosefosfatos/metabolismo , Marcadores de Spin
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