Biochemistry (Moscow) (v.82, #7)

Methylglyoxal and small heat shock proteins by M. V. Sudnitsyna; N. B. Gusev (751-759).
Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.
Keywords: small heat shock proteins; oligomeric structure; chaperone-like activity; dicarbonyl proteome; methylglyoxal

Design, synthesis, and some aspects of the biological activity of mitochondria-targeted antioxidants by G. A. Korshunova; A. V. Shishkina; M. V. Skulachev (760-777).
This review summarizes for the first time data on the design and synthesis of biologically active compounds of a new generation–mitochondria-targeted antioxidants, which are natural (or synthetic) p-benzoquinones conjugated via a lipophilic linker with (triphenyl)phosphonium or ammonium cations with delocalized charge. It also describes the synthesis of mitochondria-targeted antioxidants – uncouplers of oxidative phosphorylation – based on fluorescent dyes.
Keywords: mitochondria; antioxidants; uncouplers of oxidative phosphorylation; benzoquinones; phosphonium and ammonium cations

A new concept of action of hemostatic proteases on inflammation, neurotoxicity, and tissue regeneration by L. R. Gorbacheva; E. V. Kiseleva; I. G. Savinkova; S. M. Strukova (778-790).
Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.
Keywords: thrombin; activated protein C; protease-activated receptors; biased agonism; inflammation; tissue regeneration

EB-family proteins: Functions and microtubule interaction mechanisms by V. V. Mustyatsa; A. V. Boyakhchyan; F. I. Ataullakhanov; N. B. Gudimchuk (791-802).
Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.
Keywords: microtubules; dynamic microtubules; EB-proteins

Intracellular cargo transport by kinesin-3 motors by N. Siddiqui; A. Straube (803-815).
Intracellular transport along microtubules enables cellular cargoes to efficiently reach the extremities of large, eukaryotic cells. While it would take more than 200 years for a small vesicle to diffuse from the cell body to the growing tip of a one-meter long axon, transport by a kinesin allows delivery in one week. It is clear from this example that the evolution of intracellular transport was tightly linked to the development of complex and macroscopic life forms. The human genome encodes 45 kinesins, 8 of those belonging to the family of kinesin-3 organelle transporters that are known to transport a variety of cargoes towards the plus end of microtubules. However, their mode of action, their tertiary structure, and regulation are controversial. In this review, we summarize the latest developments in our understanding of these fascinating molecular motors.
Keywords: molecular motors; microtubule-based transport; kinesin; autoinhibition; intracellular transport; Unc104/KIF1; cargo trafficking

Coupling of translation initiation and termination does not depend on the mode of initiation by E. A. Sogorin; G. K. Selikhanov; S. Ch. Agalarov (816-820).
Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.
Keywords: translation initiation; translation termination; initiation factors; hippuristanol

Escherichia coli signal peptidase recognizes and cleaves archaeal signal sequence by Majida Atta Muhammad; Samia Falak; Naeem Rashid; Qurra-tul-Ann Afza Gardner; Nasir Ahmad; Tadayuki Imanaka; Muhammad Akhtar (821-825).
Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.
Keywords: α-amylase; Thermococcus kodakarensis ; signal peptide; purification; mass spectrometry; N-terminal sequencing

Dexamethasone is widely used in multiple myeloma (MM) for its cytotoxic effects on lymphoid cells. However, many MM patients are resistant to dexamethasone, although some can benefit from dexamethasone treatment. In this study, we noted that ω-3 polyunsaturated fatty acids (PUFAs) enhanced the dexamethasone sensitivity of MM cells by inducing cell apoptosis. q-PCR analysis revealed that miR-34a could be significantly induced by PUFAs in U266 and primary MM cells. Transfection with miR-34a antagonist or miR-34a agomir could restore or suppress the dexamethasone sensitivity in U266 cells. Both luciferase reporter assay and Western blot showed that Bcl-2 is the direct target of miR-34a in MM cells. In addition, we observed that PUFAs induced p53 protein expression in MM cells under dexamethasone administration. Furthermore, suppressing p53 by its inhibitor, Pifithrin-α, regulated the miR-34a expression and modulated the sensitivity to dexamethasone in U266 cells. In summary, these results suggest that PUFAs enhance dexamethasone sensitivity to MM cells through the p53/miR-34a axis with a likely contribution of Bcl-2 suppression.
Keywords: polyunsaturated fatty acids; eicosapentaenoic acid; docosahexaenoic acid; dexamethasone; apoptosis; miR-34a; p53; Bcl-2

Study of the lifespans of normal (non mutant) mice and growth delay mutants has shown that mortality rates for both kinds of animals exhibit reproducible fluctuations. In the case of the mutant mice, the positions of peaks on the differential mortality curves (mortality rate plotted against lifespan) coincided in different-sex groups of animals and in same-sex subgroups of animals. Differential mortality curves of the mutant mice also had a peak at 1 month of age that was absent from the differential mortality curves of the normal mice. In the case of normal animals, positions of most peaks were the same in the studied independent subgroups of males, and to a lesser extent – independent subgroups of females, which might be explained by a shift in mortality peak positions due to the reproductive activity of females. Similar positions of mortality rate peaks in the differential mortality curves for animals from independent groups and subgroups indicate the existence of increased risks of death at specific ages. The observed pattern could be due to the programming in the genome of both the periods of increased risk of death and the intermitting intervals of stable development.
Keywords: lifespan; mice; growth delay mutation; differential mortality curves; mortality peaks; Gompertz model

Effect of low temperature on globin expression, respiratory metabolic enzyme activities, and gill structure of Litopenaeus vannamei by Meng Wu; Nan Chen; Chun-Xiao Huang; Yan He; Yong-Zhen Zhao; Xiao-Han Chen; Xiu-Li Chen; Huan-Ling Wang (844-851).
Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.
Keywords: Litopenaeus vannamei ; low temperature; Cygb ; Ngb ; respiratory enzymes; gill structure

Expression, purification, and activity of ActhiS, a thiazole biosynthesis enzyme from Acremonium chrysogenum by Zhihui Song; Jie Pan; Liping Xie; Guihua Gong; Shu Han; Wei Zhang; Youjia Hu (852-860).
Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthia-zole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.
Keywords: Acremonium chrysogenum ; ActhiS; ADT; thiazole biosynthesis