Plakins in development and disease

Plakins are large multi-domain molecules that have various functions to link cytoskeletal elements together and to connect them to junctional complexes. Plakins were first identified in epithelial cells where they were found to connect the intermediate filaments to desmosomes and hemidesmosomes [Ruhrberg, C., and Watt, F.M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 7, 392-397.]. They were subsequently found to be important for the integrity of muscle cells. Most recently, they have been found in the nervous system, where their functions appear to be more complex, including cross-linking of microtubules (MTs) and actin filaments [Leung, C.L., Zheng, M., Prater, S.M., and Liem, R.K. (2001). The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 154, 691-697., Leung, C.L., Sun, D., Zheng, M., Knowles, D.R., and Liem, R.K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147, 1275-1286.]. These plakins have also indicated their relationship to the spectrin superfamily of proteins and the plakins appear to be evolutionarily related to the spectrins, but have diverged to perform different specialized functions. In invertebrates, a single plakin is present in both Drosophila melanogaster and Caenorhabditis elegans, which resemble the more complex plakins found in mammals [Roper, K., Gregory, S.L., and Brown, N.H. (2002). The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families. J Cell Sci 115, 4215-4225.]. In contrast, there are seven plakins found in mammals and most of them have alternatively spliced forms leading to a very complex group of proteins with potential tissue specific functions [Jefferson, J.J., Leung, C.L., and Liem, R.K. (2004). Plakins: goliaths that link cell junctions and the cytoskeleton. Nat Rev Mol Cell Biol 5, 542-553.]. In this review, we will first describe the plakins, desmoplakin, plectin, envoplakin and periplakin and then describe two other mammalian plakins, Bullous pemphigoid antigen 1 (BPAG1) and microtubule actin cross-linking factor 1 (MACF1), that are expressed in multiple isoforms in different tissues. We will also describe the relationship of these two proteins to the invertebrate plakins, shortstop (shot) in Drosophila and VAB-10 in C. elegans. Finally, we will describe an unusual mammalian plakin, called epiplakin.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

乳腺癌易感蛋白2的结构和功能

乳腺癌易感蛋白2是由乳腺癌易感基因2编码的一种在维持哺乳动物细胞染色体的稳定及DNA损伤生物应答中发挥重要作用的蛋白质。文章通过介绍近几年来对乳腺癌易感蛋白2的结构研究,阐述其在双链DNA损伤修复中的作用模型及其在肿瘤抑制中的功能。     

An Immunochromatographic Assay of 2,4-Dichlorophenoxyacetic Acid and Simazine Using Monoclonal Antibodies Labeled with Colloidal Gold

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,3,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3–7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds.     

Genetic diversity and environmental associations of wild barley,Hordeum spontaneum (Poaceae), in Iran

Genetic diversity and structure of populations of the wild progenitor of barleyHordeum spontaneum in Iran was studied by electrophoretically discernible allozymic variation in proteins encoded by 30 gene loci in 509 individuals representing 13 populations of wild barley. The results indicate that: a)Hordeum spontaneum in Iran is extremely rich genetically but, because of predominant self-pollination, the variation is carried primarily by different homozygotes in the population. Thus, genetic indices of polymorphismP-1% = 0.375, range = 0.267–0.500, and of genetic diversity,He = 0.134, range = 0.069–0.198, are very high. b) Genetic differentiation of populations includes clinal, regional and local patterns, sometimes displaying sharp geographic differentiation over short distances. The average relative differentiation among populations isGst = 0.28, range = 0.02–0.61. c) A substantial portion of the patterns of allozyme variation in the wild gene pool is significanctly correlated with the environment and is predictable ecologically, chiefly by combinations of temperature and humidity variables. d) The natural populations studied, on the average, are more variable than two composite crosses, and more variable than indigenous land races of cultivated barely,Hordeum vulgare, in Iran. — The spatial patterns and environmental correlates and predictors of genetic variation ofH. spontaneum in Iran indicate that genetic variation in wild barley populations is not only rich but also at least partly adaptive. Therefore, a much fuller exploitation of these genetic resources by breeding for disease resistance and economically important agronomic traits is warranted.     

西藏块菌属的分类研究

首次报道产于西藏的块菌属３种,包括２个新种和１个新记录种。新种是刘氏块菌ＴｕｂｅｒｌｉｕｉＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ．和西藏块菌ＴｕｂｅｒｘｉｚａｎｇｅｎｓｅＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ,新记录种是少孢块菌Ｔｕｂｅｒｏｌｉｇｏｓｐｅｒｍｕｍ（Ｔｕｌ．＆Ｃ．Ｔｕｌ．）Ｔｒａｐｐｅ．标本全部保存于西藏高原生态研究所标本室（ＨＸＺＥ）。     

The Presence of (+)-S-Adenosyl-l-Methionine in the Rat Brain and Its Lack of Effect on Phenylethanolamine N-Methyltransferase Activity

Abstract: (+)-S-Adenosyl- l -methionine [(+)-SAM] was isolated from rat brain and was quantified by HPLC followed by UV spectrophotometric measurements and by ¹H-NMR. Its estimated ratio in brain is 3% of total SAM. Because of its commercial unavailability, (+)-SAM was also prepared from chemically synthesized SAM by separation of the two diastereoisomers on a preparative reverse-phase Nucleosil C8 column. The (+) diastereoisomer thus obtained was then assayed in vitro both as an inhibitor and a substrate of phenylethanolamine N -methyltransferase. Enzymatic activity was measured by HPLC analysis. It was shown that (+)-SAM has no effect on phenylethanolamine N -methyltransferase activity; therefore, it is unlikely that (+)-SAM plays any possible role in regulation of adrenaline synthesis in the brain.     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.