Biochemistry (Moscow) (v.73, #6)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic protein with energy production as its implied occupation. It has established itself lately as a multifunctional protein. Recent studies have found GAPDH to be involved in a variety of nuclear and cytosolic pathways ranging from its role in apoptosis and regulation of gene expression to its involvement in regulation of Ca2+ influx from endoplasmic reticulum. Numerous studies also indicate that GAPDH interacts with microtubules and participates in cell membrane fusion. This review is focused on the cytosolic functions of the protein related to vesicular transport. Suggestions for future directions as well as the model of protein polymer structure and possible post-translational modifications as a basis for its multifunctional activities in the early secretory pathway are given.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; microtubules; phosphorylation; membranes; vesicles; Golgi apparatus; endoplasmic reticulum; COPI; COPII

The centrosome is a polyfunctional multiprotein cell complex by I. B. Alieva; R. E. Uzbekov (626-643).
Contemporary knowledge about centrosome proteins and their ensembles, which can be divided into several functional groups—microtubule-nucleating proteins, microtubule-anchoring proteins, centriole-duplication proteins, cell cycle control proteins, primary cilia growth regulation proteins, and proteins of regulation of cytokinesis—is reviewed. Structural—temporal classification of centrosomal proteins and the scheme of interconnection between the different centrosomal protein complexes are presented.
Keywords: proteins; centrosome; centriole; microtubules; cell cycle

Analysis of proteins interacting with TRIP8b adapter by N. V. Popova; A. N. Plotnikov; R. Kh. Ziganshin; I. E. Deyev; A. G. Petrenko (644-651).
Calcium-independent receptor of latrotoxin (CIRL) is an orphan heptahelical receptor implicated in regulation of exocytosis. To characterize molecular mechanisms of CIRL functioning, we searched for its intracellular partners using the yeast two-hybrid SR system with the cytoplasmic C-terminal fragment of CIRL as bait. One of the interacting proteins was identified as TRIP8b, a putative cytosolic adapter protein with multiple tetratricopeptide repeats. To understand functional significance of CIRL-TRIP8b interaction, we further isolated TRIP8b-interacting proteins by affinity chromatography of brain extracts on immobilized recombinant TRIP8b. Sixteen proteins were identified by mass spectrometry in the purified preparations. Clathrin and subunits of AP2 complex appeared to be the major TRIP8b-interacting proteins. Our data suggest a role of TRIP8b in receptor-mediated endocytosis.
Keywords: CIRL/latrophilin; TRIP8b; endocytosis; clathrin

Selection of random RNA fragments as method for searching for a site of regulation of translation of E. coli streptomycin mRNA by ribosomal protein S7 by A. V. Surdina; T. I. Rassokhin; A. V. Golovin; V. A. Spiridonova; B. Kraal; A. M. Kopylov (652-659).
In E. coli cells ribosomal small subunit biogenesis is regulated by RNA—protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 ± 5 and 60 ± 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.
Keywords: ribosome biogenesis; S7 protein; streptomycin operon; bacteria; translation regulation; SELEX; SERF

Hepatitis C virus helicase/NTPase: an efficient expression system and new inhibitors by A. V. Mukovnya; V. L. Tunitskaya; A. L. Khandazhinskaya; N. A. Golubeva; N. F. Zakirova; A. V. Ivanov; M. K. Kukhanova; S. N. Kochetkov (660-668).
A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2′-deoxythymidine 5′-phosphoryl-β,γ-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2′-deoxythymidine 5′-phosphorylβ,γ-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.
Keywords: hepatitis C virus; protein NS3; NTPase; helicase; NTP analogs; inorganic pyrophosphate analogs

Anti-inflammatory activity of polysaccharide from Pholiota nameko by Haiping Li; Xiaoxiang Lu; Shuhai Zhang; Meijun Lu; Hongmei Liu (669-675).
Pholiota nameko polysaccharide (PNPS-1) has been isolated and purified by enzymatic hydrolysis, hot water extraction, ethanol precipitation, and ion-exchange and gel-filtration chromatography. The anti-inflammatory activity of PNPS-1 was evaluated in rodents using xylene-induced ear edema, egg albumin-, carrageenin-, and formaldehyde-induced paw edema, cotton pellet granuloma test, adhesion of peritoneal leukocytes in vitro, and ulcerogenic activity. The results showed that PNPS-1 (5 mg/ear) inhibited topical edema in the mouse ear and at 100, 200, and 400 mg/kg (intraperitoneally) it significantly suppressed the development of egg albumin-, carrageenin-, and formaldehyde-induced paw edema in the animals. PNPS-1 (100, 200, and 400 mg/kg, per oral) significantly inhibited the growth of granuloma tissues induced by subcutaneously implanted cotton pellets in rats by 10.96, 18.07, and 43.75%, respectively. PNPS-1 also inhibited spontaneous and phorbol-12-myristate-13-acetate-activated adhesion of peritoneal leukocytes in vitro. Further, both acute as well as chronic administration of PNPS-1 (100, 200, and 400 mg/kg, per oral) did not produce any gastric lesion in rats. In conclusion, these data indicated that PNPS-1 possesses significant anti-inflammatory activity suggesting its potential as an anti-inflammatory agent for use in the treatment of various inflammatory-related diseases.
Keywords: Pholiota nameko ; polysaccharide; anti-inflammatory activity; leukocyte adhesion

Proteomic characterization of royal jelly proteins in Chinese (Apis cerana cerana) and European (Apis mellifera) honeybees by Ning Qu; Jie Jiang; Liangxian Sun; Changcheng Lai; Lifang Sun; Xueji Wu (676-680).
In this study, the proteins contained in royal jelly (RJ) derived from Chinese and European honeybees have been analyzed in detail and compared. Remarkable differences were found in the heterogeneity of major royal jelly proteins (MRJPs), MRJP2 and MRJP3, in terms of molecular weight and isoelectric points between the two species of RJ. MRJP2 and MRJP3 produced by Chinese honeybee are less polymorphic than those produced by European honeybee. This study is a contribution to the description of the royal jelly proteome.
Keywords: Apis cerana ; Apis mellifera ; royal jelly; major royal jelly proteins; two-dimensional gel electrophoresis

The sequence-reversed form of a small heat shock protein, HSP12.6 (retro-HSP12.6), has been reported to fold and assemble into structured tetramers in aqueous solution. Upon raising the protein concentration to ∼1.0–1.5 mg/ml, tetrameric retro-HSP12.6 is known to display a tendency to associate further into spherical beads of 18–20 nm in diameter containing folded protein subunits. Here we report that storage of this protein at low temperatures leads to further association of the beaded structures into linear and ring-shaped amyloid nanofibers of 18–20 nm in diameter. The electron micrographs presented in this communication provide the best visual evidence yet that amyloids can form through the association of smaller structured bead-like intermediates. The results also suggest that folded β-sheet-rich subunits can participate in amyloid formation.
Keywords: protein aggregation; amyloid-like aggregates; linear and ring-shaped amyloids; bead-amyloid transformation; amyloid filament assembly; folded protein aggregation

Nucleoside diphosphate kinase (NDPK) is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP. NDPK regulates a variety of eukaryotic cellular activities including cell proliferation, development, and differentiation. The ndpk cDNA was cloned from the hemocytes of the scallop Chlamys farreri and designated Cf-ndpk. The full-length sequence of Cf-ndpk consists of 715 bp encoding a polypeptide of 153 amino acids with a calculated molecular mass of 16927.52 daltons and pI of 7.64. The mRNA expression and distribution of Cf-ndpk in both bacterially challenged and unchallenged scallops were studied by Northern blotting and in situ hybridization. The results showed that Cf-ndpk transcripts were present in hemocytes, gill, adductor muscle, mantle, digestive gland, foot, and gonad, and the expression level increased in hemocytes after bacterial challenge. Recombinant Cf-NDPK expressed in Escherichia coli could transfer the terminal phosphate between UDP and ATP. The Cf-NDPK protein was present in all tested tissues including foot, adductor muscle, digestive gland, gonad, mantle, gill, and hemolymph. It was up-regulated in hemolymph after bacterial challenge. Taken together, these results suggest that NDPK may play roles in the innate immune response of scallop.
Keywords: scallop; Chlamys farreri ; nucleoside diphosphate kinase (NDPK); expression profile; recombinant expression

pH-induced equilibrium unfolding of apomyoglobin: Substitutions at conserved Trp14 and Met131 and non-conserved Val17 positions by A. E. Dyuysekina; D. A. Dolgikh; E. N. Samatova; E. I. Tiktopulo; V. A. Balobanov; V. E. Bychkova (693-701).
A number of residues in globins family are well conserved but are not directly involved in the primary oxygen-carrying function of these proteins. A possible role for these conserved, non-functional residues has been suggested in promoting a rapid and correct folding process to the native tertiary structure. To test this hypothesis, we have studied pH-induced equilibrium unfolding of mutant apomyoglobins with substitutions of the conserved residues Trp14 and Met131, which are not involved in the function of myoglobin, by various amino acids. This allowed estimating their impact on the stability of various conformational states of the proteins and selecting conditions for a folding kinetics study. The results obtained from circular dichroism, tryptophan fluorescence, and differential scanning microcalorimetry for these mutant proteins were compared with those for the wild type protein and for a mutant with the non-conserved Val17 substituted by Ala. In the native folded state, all of the mutant apoproteins have a compact globular structure, but are destabilized in comparison to the wild type protein. The pH-induced denaturation of the mutant proteins occurs through the formation of a molten globule-like intermediate similar to that of the wild type protein. Thermodynamic parameters for all of the proteins were calculated using the three state model. Stability of equilibrium intermediates at pH ∼4.0 was shown to be slightly affected by the mutations. Thus, all of the above substitutions influence the stability of the native state of these proteins. The cooperativity of conformational transitions and the exposed to solvent protein surface were also changed, but not for the substitution at Val17.
Keywords: apomyoglobin; conserved residues; protein stability; protein unfolding; intermediate state

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay by De-Yu Chen; Li-Min Liu; Sheng-Jie Liu; Mao-Ying Zhu; Lan Xu; Tian-Hua Huang (702-710).
An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.
Keywords: prostaglandin D synthase (L-PGDS); single-chain antibody (ScFv); expression

Interaction of human α-lactalbumin with fatty acids: Determination of binding parameters by C. Barbana; M. D. Perez; C. Pocovi; L. Sanchez; Z. Wehbi (711-716).
The interaction of holo-and apo-forms of human α-lactalbumin with fatty acids was studied by a partition equilibrium method. Apo-α-lactalbumin, obtained by treatment with EDTA, displays one binding site for fatty acids, the association constants for oleic and palmitic acids being 1.9·106 and 4.2·105 M−1, respectively. However, holo-α-lactalbumin was unable to bind fatty acids as measured by this technique. Likewise, no fatty acids bound to holo-α-lactalbumin, isolated using nondenaturing conditions, were detected by gas chromatography. These results demonstrate that the conformational change induced in α-lactalbumin by the removal of calcium enables the protein to interact with fatty acids.
Keywords: human α-lactalbumin; fatty acids; binding; partition equilibrium

Activated protein C via PAR1 receptor regulates survival of neurons under conditions of glutamate excitotoxicity by L. R. Gorbacheva; T. P. Storozhevykh; V. G. Pinelis; O. N. Davydova; S. Ishiwata; S. M. Strukova (717-724).
The effect of an anticoagulant and cytoprotector blood serine proteinase—activated protein C (APC)—on survival of cultured hippocampal and cortical neurons under conditions of glutamate-induced excitotoxicity has been studied. Low concentrations of APC (0.01–10 nM) did not cause neuron death, but in the narrow range of low concentrations APC twofold and stronger decreased cell death caused by glutamate toxicity. High concentrations of APC (> 50 nM) induced the death of hippocampal neurons similarly to the toxic action of glutamate. The neuroprotective effect of APC on the neurons was mediated by type 1 proteinase-activated receptor (PAR1), because the inactivation of the enzyme with phenylmethylsulfonyl fluoride or PAR1 blockade by a PAR1 peptide antagonist ((Tyr1)-TRAP-7) prevented the protective effect of APC. Moreover, APC inhibited the proapoptotic effect of 10 nM thrombin on the neurons. Geldanamycin, a specific inhibitor of heat shock protein Hsp90, completely abolished the antiapoptotic effect of 0.1 nM APC on glutamate-induced cytotoxicity in the hippocampal neurons. Thus, APC at low concentrations, activating PAR1, prevents the death of hippocampal and cortical neurons under conditions of glutamate excitotoxicity.
Keywords: activated protein C; glutamate toxicity; apoptosis; hippocampal and cortical neurons; proteinase-activated receptor; geldanamycin

Analysis of RNA cleavage by RNA polymerases from Escherichia coli and Deinococcus radiodurans by D. V. Pupov; N. A. Barinova; A. V. Kulbachinskiy (725-729).
RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase β′ subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20°C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2–3-fold) increase in the cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.
Keywords: RNA polymerase; RNA cleavage; catalysis rate

Mechanism of photoisomerization of the rhodopsin chromophore by N. S. Vassilieva-Vashakmadze; R. A. Gakhokidze; A. R. Gakhokidze (730-732).
This work is devoted to the problem connected with rhodopsin activation. Electrostatic forces involved in photoisomerization of retinal are considered. It is suggested that the repulsion force and rotating moment between electric dipole moments of methyl groups on the C5 and C13 atoms of retinal can promote isomerization upon light absorption because the π-π* transition is accompanied by a decrease in the potential barrier for torsional rotations around the C11–C12 bond.
Keywords: visual receptor; diamagnetism; dipole moment; rotating moment; isomerization