[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

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.

[Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity].

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.

alpha-fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase-3-like proteases.

alpha-Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1-10 microM AFP produces significant suppression of tumor cell growth by inducing dose-dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase-3-like activity resulting in cleavage of poly(ADP-ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP-mediated cell death. Using fluorogenic caspase substrates, we found that caspase-3-like proteases were activated as early as 4 h after treatment of Raji cells with 15 microM AFP, whereas caspase-1, caspase-8, and caspase-9-like activity was not detected during the time interval 0.5-17 h. AFP treatment of Raji cells increased Bcl-2 protein, showing that AFP-induced apoptosis is not explained by downregulation of the Bcl-2 gene. This also suggests that AFP operates downstream of the Bcl-2-sensitive step. AFP notably decreased basal levels of soluble and membrane-bound Fas ligand. Incubation of AFP-sensitive tumor cells (HepG2, Raji) with neutralizing anti-Fas, anti-tumor necrosis factor receptor (TNFR)1 or anti-TNFR2 mAb did not prevent AFP-induced apoptosis, demonstrating its independence of Fas-dependent and TNFR-dependent signaling. In addition, it was found that cells resistant to TNF-induced (Raji) or Fas-induced (MCF-7) apoptosis are, nevertheless, sensitive to AFP-mediated cell death. In contrast, cells sensitive to Fas-mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP-mediated cell death involves activation of the effector caspase-3-like proteases, but is independent of upstream activation of the initiator caspase-1, caspase-8, and caspase-9-like proteases.

Isolation and structural and functional characterization of two stable peptic fragments of human alpha-fetoprotein.

Short-time limited peptic hydrolysis of ligand-free human alpha-fetoprotein (AFP) gave two main fragments with molecular masses of 38 and 32 kDa, which had been produced by splitting of the molecule at the position Leu(312)-Asn(313). A more prolonged proteolysis led to the further degradation of these fragments and appearance of highly proteolytically resistant 23-kDa (P23) and 26-kDa (P26) fragments, corresponding to N- and C-terminal parts of the AFP molecule, respectively. Comparative study of intact free of ligands AFP and isolated stable P23 and P26 fragments by circular dichroism, differential scanning calorimetry, and immunoprecipitation techniques demonstrated that these fragments conserved native secondary, tertiary; and antigenic structure, characteristic of the intact molecule. It was concluded that, free of ligands, the AFP molecule could be considered as a three-domain molecule, in which two compact rigid domains (N-terminal domain I and C-terminal domain III) are connected by relatively labile domain II. The structure of domain II could be approximated by a "molten globule" state, characterized by the absence of rigid tertiary structure but having a pronounced secondary structure. Tumor-suppressive activity via induction of apoptosis was recently shown for AFP [Dudich, E. I., et al. (1998) Tumor Biol. 19, 261-272]. We studied here the ability of isolated proteolytic AFP fragments to induce apoptosis in the AFP-sensitive Raji cell line, to determine possible localization of the active site responsible for apoptosis signaling. Unlike intact AFP, neither isolated fragments nor their equimolar mixture was able to induce apoptosis in a human lymphoma Raji cell line. However, it was demonstrated that both fragments P23 or P26 and their equimolar mixture P23 + P26 operated synergistically with intact AFP in suppression of Raji cell proliferation. These data suggested that two structurally determined requirements are necessary for AFP-mediated triggering of apoptosis: (i) dimerization of AFP to form the heterodimeric complex of C- and N-terminal domains and (ii) participation of the central part of AFP molecule (domain II).

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.

The effect of the intersubunit disulfide bond on the structural and functional properties of the small heat shock protein Hsp25.

The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) from. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature- or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 from oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.

Growth-regulative activity of human alpha-fetoprotein for different types of tumor and normal cells.

The dose-dependent alpha-fetoprotein (AFP) reactivity of different types of tumor cells and normal embryonal fibroblasts, which are capable of taking up AFP, was investigated. High doses (more than 100 micrograms/ml) of purified human AFP were shown to induce strongly dose-dependent growth inhibition of human hepatoma HepG2 cells, human lymphoblastoma MT4 cells, lymphoma Jurkat cells and murine fibroblastoma L929 cells. Human mammary carcinoma MCF-7 cells also revealed a growth inhibitory response to AFP, although to a lesser extent. Equivalent doses of human serum albumin (HSA) demonstrated no effect on these cells. On the contrary, normal embryonal fibroblasts of different organ origin showed dose-dependent stimulation (50-90%) of proliferation in response to AFP. A similar stimulative effect was obtained when embryonal fibroblasts were treated with the same doses of HSA. The myeloblastoma cell line U-937 and the normal epidermal fibroblast cell line M19 were shown to be resistant to the AFP action over a wide range of protein concentrations. It was demonstrated that growth factor deprivation (i.e. low serum concentration) could stimulate U-937 cell proliferation in response to high doses of AFP. It was also shown that intensive washing of U-937 and MCF-7 cells with fresh medium to remove secreted cytokines and growth factors distinctly increased cell sensitivity to high-dose-AFP-induced growth-inhibitory activity. Low AFP concentrations (less than 100 micrograms/ml) failed to induce growth inhibition in all studied cells and rather showed a slight stimulative effect. These findings demonstrate that physiological levels of AFP can exhibit a dose-dependent growth-regulatory activity toward sensitive tumor or developing cells. Our data demonstrated that AFP could reveal either stimulative or inhibitory growth activity, depending on the relative concentration of AFP and on exogenous or endogenous cytokines and growth factors in the cell culture medium. A growth-stimulative activity in normal embryonal fibroblasts and certain tumor cell lines exhibited by low AFP concentrations is supposed to result from the synergistic effects of AFP and various other secreted growth factors.

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.

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.

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.

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.

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.

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.

[Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry]. / Issledovanie strukturnykh svoistv rekombinantnogo gamma-interferona metodom krugovogo dikhroizma i skaniruiushchei differentsial'noi mikrokalkorimetrii.

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.

[Study of the structural properties of recombinant leukocyte interferon A by means of fluorescence polarization, circular dichroism, and differential microcalorimetry]. / Issledovanie strukturnykh svoistv rekombinantnogo leikotsitarnogo interferon A metodami poliarizatsii fluorestsentsii, krugovogo dikhroizma i differentsial'noi mikrokalorimetryii.

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.

[Measurements of the time of rotational correlation of bovine serum albumin molecule using iodine and mercury-containing spin-labels]. / Izmerenie vremeni vrashchatel'noi korreliatsii molekuly bych'ego syvorotochnogo al'bumina s pomoshch'iu iod- i rtut'soderzhashchikh spin-metok.

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.

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.