A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress

Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.     

冬播麦区Glu-1和Glu-3位点变异及1B/1R易位与小麦加工品质性状的关系

 贮藏蛋白组成是决定小麦加工品质的重要因素。本文调查了我国冬播麦区251份主栽品种和高代品系的高分子量麦谷蛋白亚基（HMW-GS）、低分子量麦谷蛋白亚基（LMW-GS）和1B/1R易位的分布状况，研究了它们与加工品质性状的关系。结果表明，品质较差的HMW-GS N、7+9、2+12和LMW-GS Glu-A3a与Glu-B3j（1B/1R易位）在冬播麦区分布较广，频率分别为39.4%、45.0%、59.8%、37.1%和44.6%。HMW-GS和LMW-GS等位变异对籽粒蛋白质含量影响较小，对SDS沉降值、和面时间与耐揉性的加性和互作效应达1%的显著水平。按位点对加工品质性状的贡献大小，Glu-D1>Glu-B3>Glu-B1>Glu-A3>Glu-A1；就单个亚基而言，Glu-A1位点，1>2*>N；Glu-B1位点，7+8>14+15>7+9；Glu-D1位点，5+10>4+12>2+12；Glu-A3位点，Glu-A3d>Glu-A3a>Glu-A3c>Glu-A3e，Glu-B3位点； Glu-B3d>Glu-B3b>Glu-B3f >Glu-B3j。1B/1R易位对SDS沉降值、和面时间和耐揉性等加工品质性状有显著负面效应。通过选择优质高低分子量麦谷蛋白亚基和淘汰1B/1R易位系，将有助于提高我国小麦的面筋质量。     

Structural roles for human translation factor eIF3 in initiation of protein synthesis

Protein synthesis in mammalian cells requires initiation factor eIF3, a approximately 750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5'-cap or an internal ribosome entry site (IRES). Cryo-electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5'-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.     

Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1

Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.     

Structure and allostery of the PKA RIIβ tetrameric holoenzyme

In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4-β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.     

植物蛋白激酶GCN2结构、功能及调控研究进展

植物对于生物和非生物胁迫的应答效率直接影响植物的生长发育。蛋白的磷酸化和去磷酸化修饰在植物环境胁迫应答中起到了重要作用。在真核生物蛋白翻译起始过程中,蛋白激酶GCN2能通过磷酸化真核翻译起始因子e IF2α来调控蛋白的翻译,进而对逆境胁迫进行应答。在植物中,GCN2通过磷酸化e IF2α抑制蛋白的合成,从而激活植物自身免疫防御以应答各种胁迫。从GCN2的结构、调控及其在植物中的新功能等方面综述了植物GCN2的研究进展,以期为揭示植物GCN2介导的胁迫应答机理提供参考。     

波斯小麦高分子量谷蛋白亚基组成分析

利用SDS-PAGE技术对来源于16个国家(地区)93份波斯小麦材料的高分子量谷蛋白亚基进行了检测。结果表明,在Glu-A1和Glu-B1两个位点共发现8种亚基类型,6种亚基组合。其中,在Glu-A1位点上null亚基频率高达96.77%,仅3份材料含有2*亚基。通过提取种子总蛋白和选择性提取高分子量谷蛋白两种方法在所有材料中均检测到一条介于普通小麦对照By8和Dy10亚基之间的条带,推测为表达的Ay亚基。在Glu-B1位点上7 8亚基频率高达95.70%,同时筛选出具有优质亚基17 18和14 15的材料各1份,为小麦品质育种提供了基础材料。     

小麦籽粒Waxy蛋白亚基动态表达规律的研究

[目的]对新疆自育的4个春小麦品种新春8号、新春9与、新春11号和新春14号的Waxy蛋白亚基在籽粒灌浆过程中的动态表达进行分析,研究扬花期后籽粒贮藏蛋白中Waxy蛋白亚基的动态累积规律,为小麦淀粉的品质改良提供理论依据.[方法]利用Waxy蛋白亚基特异分子标记,通过SDS-PAGE电泳进行Waxy蛋白亚基的检测.[结果]4个品种Waxy蛋白亚基的组成不尽相同,新春8号和新春11号Waxy蛋白3个亚基都存在,而新春9号和新春14号为Wx-B1亚基的缺失类型.新春8号亚基出现的时期较晚,花后20 d开始检测到;新春14号Wx-D1亚基的表达要晚于Wx-A1亚基;新春9号和11号亚基于花后15 d检测到,但在籽粒灌浆各时期表达强弱也不尽一致.[结论]Waxy蛋白亚基形成具有时间性,且各亚基在籽粒灌浆过程中的表达规律品种间也不尽一致.     

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I.     

Protein phosphatases: properties and role in cellular regulation

Protein phosphorylation is a principal regulatory mechanism in the control of almost all cellular processes. The nature of the protein phosphatases that participate in these reactions has been a subject of controversy. Four enzymes, termed protein phosphatases 1, 2A, 2B, and 2C, account for virtually all of the phosphatase activity toward phosphoproteins involved in controlling glycogen metabolism, glycolysis, gluconeogenesis, fatty acid synthesis, cholesterol synthesis, and protein synthesis. The properties, physiological roles, and mechanisms for regulating the four protein phosphatases are reviewed.     

Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) dissociate into guanosine triphosphate (GTP)-bound alpha subunits and a complex of beta and gamma subunits after interaction with receptors. The GTP-alpha subunit complex activates appropriate effectors, such as adenylyl cyclase, retinal phosphodiesterase, phospholipase C, and ion channels. G protein beta gamma subunits have been found to have regulatory effects on certain types of adenylyl cyclase. In the presence of Gs alpha, the alpha subunit of the G protein that activates adenylyl cyclase, one form of adenylyl cyclase was inhibited by beta gamma, some forms were activated by beta gamma, and some forms were not affected by beta gamma. These interactions suggest mechanisms for communication between distinct signal-transducing pathways.     

新疆小麦品种(系)高分子量麦谷蛋白亚基遗传多样性分析

[目的]更好的了解和有效利用新疆选育的小麦种质材料.[方法]采用SDS-PAGE技术对88份小麦育成品种与自育品系的高分子量麦谷白亚基(HMW-GS)组成进行了分析.[结果]参试材料中共有15种HMW-GS类型,Glu-A1位点上有、1和2~*,以亚基为主要类型(占57.96; );Giu-B1位点上有7+9、7+8、6+8、14+15、17+18、7、8和20八种类型,以7+9和7+8为主要亚基类型(分别占45.46;和39.77;);Glu-D1位点上有5+10、2+12、3+12和2+11四种类型,其中2+12亚基的频率高达59.09;;88份小麦品种(系)的亚基组合类型共有25种,其中主要亚基组合为”,7+8,2+12",占17.05; .通过对比新疆育成小麦品种和自育品系的优质亚基出现频率发现,品系中优质亚基1、14+15和5+10出现频率较育成品种有所增长,2~*、7+8、17+18优质亚基的出现频率下降.在参试材料中,32个含有5+10优质亚基,有21个含有1亚基,6个含2~*亚基,1个含14+15亚基,1个含17+18亚基,并有两个材料检测出优质亚基组合类型(”2~*、17+18、5+10",”1、7+8、5+10").[结论]这些优异种质资源可供优质小麦育种利用.     

A mutation in the catalytic subunit of cAMP-dependent protein kinase that disrupts regulation

A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.     

Three-dimensional structure of cholera toxin penetrating a lipid membrane

Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution. Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring. The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane. Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring.     

Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.     

Polypeptide composition of fraction 1 protein from parasexual hybrid plants in the genus Nicotiana

Analysis of the subunit polypeptide composition of Fraction 1 proteins gives information on the expression of both nuclear and chloroplast genomes; the large subunits of the protein are coded by chloroplast DNA, whereas the small subunits are coded by nuclear DNA. Fraction 1 protein isolated from the leaves of parasexual hybrid plants derived from the fusion of protoplasts of Nicotiana glauca and N. langsdorffii contains the small subunit polypeptides of both parent species and the large subunit polypeptides of only N. glauca. Fraction 1 protein isolated from the leaves of a hybrid plant obtained after the uptake of chloroplasts of N. suaveolens by protoplasts of white tissue of a variegating mutant of N. tabacum contains the large subunit polypeptides of both N. suaveolens and N. tabacum, as well as the small subunit polypeptides of both these species.     

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.