The Institute of Protein Research of the Russian Academy of Sciences Is 50 Years Old.

Here I introduce collection of review articles written by members of the Institute of Protein Research of the Russian Academy of Sciences. This collection commemorates the 50th anniversary of the Institute. The review articles cover a broad range of problems concerning the spatial structure of protein molecules, including the state of the molten globule, protein-RNA interactions, polysome and ribosome structure, the molecular colony method, and the original methods for studying the structure of proteins. Several of the reviews consider the practical use of knowledge about the structure of proteins and protein polymers. They reflect both the long experience of the authors and contemporary scientific data.

Interactions between the Translation Machinery and Microtubules.

Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.

Logistics in the cell: cargoes and transportation.

Eukaryotic cells are large and thus require a vesicular transport system. The system involves the formation of membrane transport containers, their short- and long-distance movements, recognition of destination points, and fusion with other membranes. Understanding the molecular mechanisms of these processes is of theoretical and practical significance. This special issue of Biochemistry (Moscow) collects surveys and experimental articles describing various aspects of vesicular transport.

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors.

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles - transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.

Cellular acidosis inhibits assembly, disassembly, and motility of stress granules.

Stress granules (SGs) are large ribonucleoprotein (RNP)-containing particles that form in cytoplasm in response to a variety of acute changes in the cellular environment. One of the general parameters of the cell environment is pH. In some diseases, as well as in muscle fatigue, tissue acidosis occurs, leading to decrease in intracellular pH. Here we studied whether decrease in pH causes the formation of SGs in cultured animal cells, whether it affects the formation of the SGs under the action of arsenite and, if such effects occur, what are the mechanisms of the influence of acidosis. Acidosis was simulated by decreasing the pH of the culture medium, which acidified the cytoplasm. We found that medium acidification to pH 6.0 in itself did not cause formation of SGs in cells. Moreover, acidification prevented the formation of SGs under treatment with sodium arsenite or sodium arsenite together with the proteasome inhibitor MG132, and it inhibited the dissociation of preformed SGs under the influence of cycloheximide. We established that pH decrease did not affect the phosphorylation of eIF2α that occurs under the action of sodium arsenite, and even caused such phosphorylation by itself. We also found that the velocity of SG motion in cytoplasm at acidic pH was very low, and the mobile fraction of SG-incorporated PABP protein revealed by FRAP was decreased. We suppose that acidic pH impairs biochemical processes favoring assembly of RNPs in stress conditions and RNP dissociation on the termination of stress. Thus, in acidosis the reaction of the cellular translation apparatus to stress is modified.

[Stress granules in the cells with intact and discrupted microtubules: analysis with new algorithm of image processing].

Stress granules--temporary RNP structures that are formed in cells under stress. They are studied mainly by means of fluorescence microscopy with the quantitative analysis of cell images. We have developed a new algorithm for automatic detection of stress granules in the cytoplasm of cultured animal cells having non-uniform cytoplasmic background. Using this approach, we have found that visible stress granules are formed in cells as "all or nothing", and their number in cells is rather constant. We also show that disruption of cellular microtubules lead to a decrease in the average size of stress granules and an increase in their number in the cell.

[Is the microtubule disruption-induced alteration of peroxide concentration a factor inhibiting the assembly of ribonucleoprotein stress granules?].

It has been examined whether the destruction of cell microtubules affects the increase in the intracellular hydrogen peroxide concentration caused by sodium arsenite, which induces the formation of stress ribonucleoprotein granules. As expected, sodium arsenite caused a 50% increase in hydrogen peroxide concentration in HeLa cells; on the other hand, another stress granule inducer tert-buthylhydroquinone did not affect the peroxide concentration. The disruption of microtubules by nocodazole or vinblastine also resulted in some increase in the intracellular peroxide concentration,y taxol did not affect it. The combined treatment of cells with and the microtubule stabilization by taxol did not affect it. The combined treatment of cells with arsenite and antimicrotubule drugs caused an additive effect, and the peroxide concentration increased twice or more. Thus, the inhibition of stress granule formation after microtubule disruption cannot be explained by a decrease in peroxide concentration as compared with the affect of arsenite.

[MAST2-like protein kinase from grape vine Vitis vinifera: cloning of catalytic domain cDNA].

The aim of our work is the identification of protein kinases phosphorylating microtubule proteins in plant cells. Using bioinformatic approach, we found genes of putative homologues of microtubule-associated mammalian protein kinase MAST2 in higher plant genomes. The gene of closest MAST2 homologue, putative protein, named GMLK (Grape MAST2-Like Kinase, A7NTE9_VITVI), was found in grape Vitis vinifera. We report here the cloning of cDNA of GMLK (A7NTE9) from Pinot Noir grape vine leaves.

Dynamics of nonmembranous cell components: role of active transport along microtubules.

Here we discuss some common mechanisms of microtubule-dependent active transport of nonmembranous components in animal cells. We summarize data about mRNA, cytoskeletal elements, structural proteins, and signaling complexes transport. We also characterize the series of molecular interactions that connect nonmembranous cargoes and microtubules and describe the regulatory pathways for these interactions.

[Protein kinase LOSK regulates the network of microtubules and cell locomotion].

It has been found that the inhibition of the activity of protein kinase LOSK reduces the ability of cells to the directed movement over the substrate and changes the parameters of the interaction of cells with the substrate. It is suggested that the chaotization of microtubules leads to the stabilization of cell contacts with the substrate and, consequently, to a slowing down of locomotion.

[Bioinformatic search of plant protein kinases, participating in microtubule protein phosphorylation and cell division regulation].

Bioinformatic search of plant homologues of human protein kinases SLK, PAK6, PAK7, MARK1, MAST2, TTBK1, TTBK2, AURKA, PLK1, PLK2 and PASK participating in microtubular protein phosphorylation and cell division regulation is carried out. The homologues of protein kinases SLK, MAST2 and AURKA were identified. It is found that closest homologue of human AURKA protein kinase is a protein with unknown function A7PY12_VITVI (STALK--Serine-Threonine Aurora-Like Kinase) from grape (Vitis vinifera). Reconstruction and analysis of three-dimensional structure of STALK protein confirmed its relation to the group of AURKA-like protein kinases.

[Bioinformatic search for plant homologs of Ste20-like serine/threonine protein kinases].

Eleven plant homologs of animal and yeast Ste20-like protein kinases were identified. It was shown that the nearest plant homologs of the Ste20-like protein kinases are the unknown proteins A9RVK0 from Physcomitrella patens ssp. patens and A7P2E2 from Vitis vinifera. Cladistic analysis showed a protein kinase dstl from Dictyostelium discoideum as the closest protein to the newly found plant homologs. A predicted spatial structure of the A9RVK0 from P. patens ssp. patens catalytic domain is presented.

[Disturbance of the radial system of interphase microtubules in the presence of excess serum in cell culture medium].

The role of the orderliness of microtubules in the cell has been studied. For this purpose, a population of Vero cells with chaotically arranged microtubules was used, which was obtained after the addition of excess serum to cell culture medium. An increase in the total area and a slight dispersion of the Golgi apparatus were found; however, the rate of culture growth as a whole remained normal. Thus, the radial arrangement of microtubules is not vital even for cells where it is usually well pronounced.

Microtubules' interaction with cell cortex is required for their radial organization, but not for centrosome positioning.

Microtubules in interphase mammalian cells usually form a radial array with minus-ends concentrated in the central region and plus-ends placed at the periphery. This is accepted as correct, that two factors determinate the radial organization of microtubules - the centrosome, which nucleate and anchor the microtubules minus-ends, and the interaction of microtubules with cortical dynein, which positions centrosome in the cell center. However, it looks as if there are additional factors, affecting the radial structure of microtubule system. We show here that in aged Vero cytoplasts (17 h after enucleation) microtubule system lost radial organization and became chaotic. To clear up the reasons of that, we studied centrosome activity, its position in the cytoplasts and microtubule dynamics. We found that centrosome in aged cytoplasts was still active and placed in the central region of the cytoplasm, while after total disruption of the microtubules it was displaced from the center. Microtubules in aged cytoplasts were not stabilized, but they lost their ability to stop to grow near cell cortex and continued to grow reaching it. Aged cytoplast lamellae was partially depleted with dynactin though Golgi remained compact indicating dynein activity. We conclude that microtubule stoppage at cell cortex is mediated by some (protein) factors, and these factors influence radial structure of microtubule system. It seems that the key role in centrosome positioning is played by dynein complexes anchored everywhere in the cytoplasm rather than anchored in cell cortex.

The centrosome keeps nucleating microtubules but looses the ability to anchor them after the inhibition of dynein-dynactin complex.

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.

[Stochastic computer model of cellular microtubule dynamics].

A computer model of the system of microtubules has been developed to study the mechanisms of action of various factors on this system. The model describes the process of polymerization/depolymerization of microtubules as a set of chemical reactions with certain rate constants using a stochastic approach. Microtubules are visualized in the program field, which makes the model visual. The program imitates the dynamics and structure of the system of cellular microtubules with great, reliability. The parameters generated by the model correlate with the corresponding parameters of microtubules in living cells. We are going to develop this approach to modeling microtubules and similar structures to bring them into a better accord with living systems and to study the influence of various factors on these systems.

[Dynein and dynactin as organizers of the system of cell microtubules].

A review of the role of the microtubule motor dynein and its cofactor dynactin in the formation of a radial system of microtubules in the interphase cells and of mitotic spindle. Deciphering of the structure, functions, and regulation of activity of dynein and dynactin promoted the understanding of mechanisms of cell and tissue morphogenesis, since it turned out that these cells help the cell in finding its center and organize microtubule-determined anisotropy of intracellular space. The structure of dynein and dynactin molecules has been considered, as well as possible pathways of regulation of the dynein activity and the role of dynein in transport of cell components along the microtubules. Attention has also been paid to the functions of dynein and dynactin not related directly to transport: their involvement in the formation of an interphase radial system of microtubules. This system can be formed by self-organization of microtubules and dynein-containing organelles or via organization of microtubules by the centrosome, whose functioning requires dynein. In addition, dynein and dynactin are responsible for cell polarization during its movement, as well as for the position of nucleus, centrosomes, and mitotic spindle in the cell.

[Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization].

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.

[Large subunit of translation initiation factor--3 p170 contains potentially functional nuclear localization signals]. / Signaly iadernoi lokalizatsii v bol'shoi sub''edintse faktora initsiatsii transliatsii 3--belke p170.

Eukaryotic translation factors and their subunits can have independent cellular functions, including regulation of nuclear events. We analyzed primary structure of p170 large subunit of human translation initiation factor eIF3 and found four potential bipartite nuclear localization signals (NLS). Then we studied whether these NLS were functional, that is were able to direct protein to cell nucleus. Complementary DNA of p170 fragments were expressed in cultured CV-1 and Cos-1 green monkey cells, and localization of fused with GFP proteins was determined by fluorescent microscopy. We established that p170 molecule possessed at least two functional NLS which determined nuclear localization of p170 fragments. At the same time more long p170 fragments containing the same functional NLS could be retained in cytoplasm. We speculate that either using specific factors or after limited proteolysis p170 can enter cell nucleus and participate in genome expression regulation. Also we do not exclude the possibility that functioning of p170 in cytoplasm can be regulated by reversible binding of importins to its NLS.