Biochemistry (Moscow) (v.82, #2)

Regulatory peptides in plants by B. F. Vanyushin; V. V. Ashapkin; N. I. Aleksandrushkina (89-94).
Many different peptides regulating cell differentiation, growth, and development are found in plants. Peptides participate in regulation of plant ontogenesis starting from pollination, pollen tube growth, and the very early stages of embryogenesis, including formation of embryo and endosperm. They direct differentiation of meristematic stem cells, formation of tissues and individual organs, take part in regulation of aging, fruit maturation, and abscission of plant parts associated with apoptosis. Biological activity of peptides is observed at very low concentrations, and it has mainly signal nature and hormonal character. “Mature” peptides appear mainly due to processing of protein precursors with (or without) additional enzymatic modifications. Plant peptides differ in origin, structure, and functional properties. Their specific action is due to binding with respective receptors and interactions with various proteins and other factors. Peptides can also regulate physiological functions by direct peptide–protein interactions. Peptide action is coordinated with the action of known phytohormones (auxins, cytokinins, and others); thus, peptides control phytohormonal signal pathways.
Keywords: regulatory peptides; cell differentiation; plant growth and development; phytohormones

Solid/gas biocatalysis by L. M. Kulishova; D. O. Zharkov (95-105).
Solid/gas biocatalysis is a nontraditional reaction system that employs the ability of some enzymes, being in the solid state, to catalyze reactions of substrates in the gas phase. Manipulation of the reaction parameters (temperature and pressure) in the solid/gas system allows precise control over the thermodynamic activity of water and substrate and creation of a controlled microenvironment for the enzyme, making it an appropriate model for enzymology studies. Owing to such advantages as high stability of dry enzymes and cofactors and easy fractionation of gas mixtures, solid/gas biotechnology has already found several industrial applications. Here we review key thermodynamic factors affecting the properties of enzymes, including their activity and stability, in a solid/gas system. Examples of promising enzymes and microorganisms for development and improvement of solid/gas biocatalytic technologies in organic synthesis, biosensors, and green chemistry are discussed.
Keywords: solid/gas biocatalysis; protein hydration; lyophilized enzymes; immobilized enzymes; lipases; esterases; alcohol dehydrogenases; dehalogenases

α-Crystallins are small heat shock proteins: Functional and structural properties by T. S. Tikhomirova; O. M. Selivanova; O. V. Galzitskaya (106-121).
During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αAand αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein–protein interactions.
Keywords: aggregation; α-crystallin; apoptosis; cataract; oligomer; chaperone

Biological basis for amyloidogenesis in Alzheimer’S disease by T. V. Andreeva; W. J. Lukiw; E. I. Rogaev (122-139).
Certain cellular proteins normally soluble in the living organism under certain conditions form aggregates with a specific cross-β sheet structure called amyloid. These intraor extracellular insoluble aggregates (fibers or plaques) are hallmarks of many neurodegenerative pathologies including Alzheimer’s disease (AD), Huntington’s disease, Parkinson’s disease, prion disease, and other progressive neurological diseases that develop in the aging human central nervous system. Amyloid diseases (amyloidoses) are widespread in the elderly human population, a rapidly expanding demographic in many global populations. Increasing age is the most significant risk factor for neurodegenerative diseases associated with amyloid plaques. To date, nearly three dozen different misfolded proteins targeting brain and other organs have been identified in amyloid diseases and AD, the most prevalent neurodegenerative amyloid disease affecting over 15 million people worldwide. Here we (i) highlight the latest data on mechanisms of amyloid formation and further discuss a hypothesis on the amyloid cascade as a primary mechanism of AD pathogenesis and (ii) review the evolutionary aspects of amyloidosis, which allow new insight on human-specific mechanisms of dementia development.
Keywords: amyloidogenesis; amyloidosis; beta amyloid cleavage enzyme (BACE); β-amyloid precursor protein (APP); Alzheimer’s disease (AD); presenilins

Selection of progesterone derivatives specific to membrane progesterone receptors by A. V. Polikarpova; A. A. Maslakova; I. S. Levina; L. E. Kulikova; Y. V. Kuznetsov; A. A. Guseva; T. A. Shchelkunova; I. V. Zavarzin; O. V. Smirnova (140-148).
The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.
Keywords: membrane progesterone receptors; progesterone analogs; human pancreatic adenocarcinoma BxPC3 cell line; synthesis

Sodium orthovanadate (SOV) is a general inhibitor of tyrosine phosphatases, a large family of enzymes that catalyze the removal of phosphate groups from tyrosine residues. SOV is commonly used in the laboratory to preserve the protein tyrosyl phosphorylation state of proteins under study. It has shown promising antineoplastic activity in some human cancer cell lines; this effect has not been fully investigated in head and neck squamous cell carcinoma. In this study, the effect of SOV on cell growth, proliferation, viability, and apoptosis was assessed in Cal27 cells, an oral squamous cell carcinoma (OSCC) cell line. SOV exhibited dose-dependent inhibition of cell growth and decrease in cell viability and colony formation. The IC50 values for treatment lasting 72 h and 7 days were 25 and 10 μM, respectively. The cytotoxic effect of the drug was associated with poly(ADP-ribose)polymerase cleavage detected by immunoblot. Flow cytometry of Cal27 cells stained with annexin V-FITC and propidium iodide showed a dose-dependent increase in apoptosis that reached approximately 40% at 25 μM SOV. These findings demonstrate that SOV has in vitro antiproliferative and proapoptotic effect on OSCC cells.
Keywords: sodium orthovanadate; oral cavity cancer; squamous cell carcinoma

Preformed amyloid fibrils can act as seeds for accelerating protein fibrillation. In the present study, we examined the effects of preformed seeds on lysozyme amyloid fibrillation in the presence of two distinct inhibitors–epigallocatechin (EGC) and polyethylene glycol 2000 (PEG). The results demonstrated that the effects of fibrillar seeds on the acceleration of lysozyme fibrillation depended on the aggregation pathway directed by an inhibitor. EGC inhibited lysozyme fibrillation and modified the peptide chains with quinone moieties in a concentration-dependent manner. The resulting aggregates showed amorphous off-pathway morphology. Preformed fibril seeds did not promote lysozyme fibrillation in the presence of EGC. PEG also inhibited lysozyme fibrillation, and the resulting aggregates showed on-pathway protofibrillar morphology. In contrast, the addition of fibril seeds into the mixture of lysozyme and PEG significantly stimulated fibril growth. Assays of cell viability showed that both EGC and PEG inhibited the formation of cytotoxic species. In accordance with thioflavine T data, the seeds failed to alter the cell-damaging potency of the EGC-directed off-pathway aggregates, but increased the cytotoxicity of the PEG-directed on-pathway fibrils. We suggest that the pattern of interaction between lysozyme and an inhibitor determines the pathway of aggregation and therefore the effects of seeding on amyloid formation. EGC covalently modified lysozyme chains with quinones, directing the aggregation to proceed through an off-pathway, whereas PEG affected the protein in a noncovalent manner, and fibril growth could be stimulated under seeding through an on-pathway.
Keywords: lysozyme; amyloid fibrillation; seeding; epigallocatechin; polyethylene glycol; cytotoxicity

Chronic alcohol intoxication is not accompanied by an increase in calpain proteolytic activity in cardiac muscle of rats by Yu. V. Gritsyna; N. N. Salmov; A. G. Bobylev; I. S. Fadeeva; N. I. Fesenko; D. G. Sadikova; N. I. Kukushkin; Z. A. Podlubnaya; I. M. Vikhlyantsev (168-175).
Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of μ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the “heart weight/body weight” parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of μ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of μ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in μ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.
Keywords: cardiac muscle; μ-calpain; calpastatin; titin; chronically alcoholized rats

Characterization of two recombinant 3-hexulose-6-phosphate synthases from the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z by O. N. Rozova; S. Y. But; V. N. Khmelenina; A. S. Reshetnikov; I. I. Mustakhimov; Y. A. Trotsenko (176-185).
Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain proteinHPS–PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS–PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose 5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the k cat/K m ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS–PHI. AMP and ADP were powerful inhibitors of both HPS and HPS–PHI activities. The two-domain HPS–PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.
Keywords: methylotrophic bacteria; Methylomicrobium alcaliphilum 20Z; 3-hexulose-6-phosphate synthase; 6-phospho-3 hexulose isomerase; ribulose monophosphate cycle

High level soluble expression and ATPase characterization of human heat shock protein GRP78 by Shuang Wu; Hongpeng Zhang; Miao Luo; Ke Chen; Wei Yang; Lei Bai; Ailong Huang; Deqiang Wang (186-191).
Human GRP78 has been shown to promote cancer progression and is regarded as a novel target for anticancer drugs. However, generation of recombinant full-length GRP78 remains challenging. This report demonstrates that E. coli autoinduction is an excellent method for the preparation of active recombinant GRP78 protein. The final yield was approximately 50 mg/liter of autoinduction culture. Gel-filtration experiments confirmed that the chaperone is a monomer. The purified human GRP78 catalyzed the conversion of ATP to ADP without requiring metal ions as cofactors. Three mutants, T38A, T229A, and S300A, exhibited much lower activity than wild-type GRP78, indicating that the active sites of the ATPase are located at the negatively charged cavity. Three mutants in the negatively charged cavity region dramatically reduced GRP78 activity, further confirming the region as the site of ATPase activity.
Keywords: GRP78; ATPase; autoinduction; monomer; active site

Substrate-specific reduction of tetrazolium salts by isolated mitochondria, tissues, and leukocytes by N. I. Fedotcheva; E. G. Litvinova; M. V. Zakharchenko; N. V. Khunderyakova; R. S. Fadeev; V. V. Teplova; T. A. Fedotcheva; N. V. Beloborodova; M. N. Kondrashova (192-204).
Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate–NBT was reduced more readily during succinate oxidation, while MTT–during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors–malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.
Keywords: nitroblue tetrazolium; methyl thiazolyl tetrazolium; mitochondria; leukocytes; stress; progesterone

Cytokine profile associated with selective removal of natural anti-αGal antibodies in a sepsis model in Gal-KO mice by Magdiel Pérez Cruz; Daniel Bello Gil; Cristina Costa; Rafael Mañez (205-212).
Selective depletion of natural anti-Galα1-3Galβ1-4GlcNAc (so-called anti-αGal) antibodies is achieved in α1,3-galactosyltransferase knockout (Gal-KO) mice by administration of the soluble glycoconjugate of αGal GAS914. This molecule removed up to 90% of natural circulating anti-αGal antibodies without causing unspecific production of cytokines in wild-type (CBA) and Gal-KO mice. However, the removal of anti-αGal antibodies in Gal-KO mice with GAS914 in the context of sepsis after cecal ligation and puncture (CLP) was associated with a significant increase in the production of leptin, CXLC1, CXLC13, and TIMP-1 cytokines compared to vehicle (PBS)-treated controls. Despite the current lack of understanding of the underlying mechanism, our data suggest a putative role of natural anti-αGal antibodies in the regulation of some cytokines during sepsis.
Keywords: natural antibodies; anti-αGal; glycoconjugates; GAS914; cytokines; inflammation; sepsis

We applied dynamic light scattering (DLS) to compare aggregation properties of two isoforms of myosin subfragment 1 (S1) containing different “essential” (or “alkali”) light chains, A1 or A2, which differ by the presence of an N-terminal extension in A1. Upon mild heating (up to 40°C), which was not accompanied by thermal denaturation of the protein, we observed a significant growth in the hydrodynamic radius of the particles for S1(A1), from ~18 to ~600-700 nm, whereas the radius of S1(A2) remained unchanged and equal to ~18 nm. Similar difference between S1(A1) and S1(A2) was observed in the presence of ADP. In contrast, no differences were observed by DLS between these two S1 isoforms in their complexes S1-ADP-BeFx and S1-ADP-AlF 4 which mimic the S1 ATPase intermediate states S1*-ATP and S1**-ADP-Pi. We propose that during the ATPase cycle the A1 N-terminal extension can interact with the motor domain of the same S1 molecule, and this can explain why S1(A1) and S1(A2) in S1-ADP-BeFx and S1-ADP-AlF 4 complexes do not differ in their aggregation properties. In the absence of nucleotides (or in the presence of ADP), the A1 N-terminal extension can interact with actin, thus forming an additional actin-binding site on the myosin head. However, in the absence of actin, this extension seems to be unable to undergo intramolecular interaction, but it probably can interact with the motor domain of another S1 molecule. These intermolecular interactions of the A1 N-terminus can explain unusual aggregation properties of S1(A1).
Keywords: myosin subfragment 1; essential light chains; thermally induced aggregation; dynamic light scattering

Strain-specific single-nucleotide polymorphisms in hypertensive ISIAH rats by N. I. Ershov; A. L. Markel; O. E. Redina (224-235).
Single-nucleotide polymorphisms (SNPs) in the coding and regulatory regions of genes can affect transcription rate and translation efficiency, modify protein function, and, in some cases, cause the development of diseases. In the current study, the RNA-Seq approach has been used to discover strain-specific SNPs in ISIAH (inherited stress-induced arterial hypertension) rats, which are known as a model of stress-induced arterial hypertension. The comparison of the ISIAH SNPs with genome sequencing data available for another 42 rat strains and substrains, 11 of them known as hypertensive, showed a considerable genetic distance between the genotypes of ISIAH and all other rat strains and substrains. The study revealed 1849 novel SNPs specific for ISIAH rats and 158 SNPs present only in the genotypes of hypertensive rats. Amino acid substitutions with possible deleterious effect on protein function were detected. Several of them were found in the genes associated with hypertension. These SNPs may be considered as novel molecular targets for further studies aimed at assessing their potential in the therapy of stress-induced hypertension.
Keywords: hypertension; SNPs; RNA-Seq; ISIAH rat strain