小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子.利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因.该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74%的同源性.在多肽链的N-端具有在真核生物中高度保守的UFD1结构域.我们将该基因定位在小麦的第六染色体群并将其命名为TUFD1.South-ern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的.无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源.除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域.TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达.     

泛素融合降解蛋白UFD1的结构与功能

泛素-蛋白酶体途径是细胞内蛋白质选择性降解的主要途径,参与多种真核生物细胞生理过程,与细胞的生理功能和病理状态有着密切的关系。该途径中UFD1作为泛素识别因子介导泛素化的靶蛋白至26S蛋白酶体降解。该文在概述泛素-蛋白酶体途径作用机制的基础上,对哺乳动物和酵母UFD1蛋白的结构及其在细胞周期调控、转录调控、内质网相关蛋白降解中的功能进行了综述。     

渗透胁迫下小麦叶片蛋白质合成与降解的示踪研究

渗透胁迫降低了叶片、特别是生长叶片蛋白质中固定~(14)CO_2及由根系吸收的~(14)C-Gly的掺入率,但同等程度胁迫处理,抗旱品种的掺入率降低幅度小于敏感品种;轻度胁迫后复水,抗旱品种生长叶蛋白质的放射性高于对照,而敏感品种仍低于对照。Poly(A~+)-mRNA的体外翻译测定证明,胁迫时蛋白质合成能力降低的主要原因是Poly(A~+)-mRNA翻译活性的降低。渗透胁迫也促进了叶片蛋白质降解,但与蛋白质合成不同,在成熟叶片中表现得更突出。     

Specific Degradation of D1 Protein during Exposure of Thylakoid Membranes to High Temperature in the Dark

Exposure of thylakoid membranes to high temperature in dark leads to the degradation of D1 protein. Maximum degradation of D1 protein occurred at 45 °C. Using N-terminal specific D1 antibody, a 23 kDa fragment of D1 protein was detected. The degradation of D1 protein could be prevented both by radical scavengers and inhibitors of serine protease and metallo-protease. These results suggest that degradation of D1 protein during exposure of thylakoid membranes to high temperature in dark is catalyzed by protease. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosynthesis and Degradation of a Wheat Embryo Cytokinin-Binding Protein during Embryogenesis and Germination

The accumulation and degradation of a wheat (Triticum durum) embryo cytokinin-binding protein (CBF-1) was followed during embryo development and germination by its N⁶-benzyladenine (BA) binding activity and immunological reactivity (rocket immunoelectrophoresis and Western blotting). Both BA binding activity and CBF-1 appeared at 2 weeks post-anthesis and rose sharply between 2 to 4 weeks before leveling off to approximately 47 micrograms per embryo (9% of the soluble embryo protein at maturity). In vitro translation of polyadenylated RNA from 20-day-old embryos yielded a polypeptide which was immunoprecipitable with anti-CBF-1 IgG and migrated closely to the 54-kilodalton CBF-1 polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon germination, both the amount of CBF-1 and BA binding activity dropped to low levels within 3 days. The data are discussed in relation to the possible role of CBF-1 as a regulator of cytokinin availability, and comparisons are drawn between the structural and biosynthetic similarities found between CBF-1 and the vicilin storage proteins of legumes. An improved method for isolating undegraded CBF-1 from whole seeds is also presented.     

Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : I. Interaction with Senescence Processes

Photosynthetic capacity decreases rapidly when temperate species are exposed to heat stress during reproductive development. We investigated whether injury in wheat (Triticum aestivum L.) resulted from general acceleration of senescence processes or specific heat-induced lesions. In situ photosynthetic capacity of leaf discs and thylakoid reactions were measured using flag leaf tissue from two cultivars maintained at 20 and 35°C during maturation. Photosynthetic rates of leaf discs decreased faster at 35 than at 20°C and were more photolabile in cv Len than in cv Waverly at high temperature. Patterns of thylakoid breakdown also differed in the two wheat genotypes at 20°C: intersystem electron transport and photosystem II activity decreased linearly during postanthesis development in Len wheat, whereas coupling of photophosphorylation to electron transport declined late during senescence in Waverly wheat. Heat stress induced early loss of intersystem electron transport followed sequentially by decreased silicomolybdic acid, + 3-(3,4-dichlorophenyl)-1-dimethylurea-mediated photosystem II activity and 2,5-dichloro-p-benzoquinone-mediated photosystem II activity in Len. Stress accelerated the uncoupling process, but loss of intersystem electron transport and photosystem II activities was slower in Waverly than in Len. We conclude that high temperature initially accelerated thylakoid component breakdown, an effect similar to normal senescence patterns. Thylakoid breakdown may induce a destabilizing imbalance between component reaction rates; an imbalance between photosystem II and cytochrome f/b₆-mediated activities would be particularly damaging during heat stress.     

Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrP^Sc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.     

Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : II. Interaction with Source and Sink Processes

High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20°C) and stress (35°C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b₆-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Developmental Differences in Embryos of High and Low Protein Wheat Seeds during Germination

Developmental patterns of embryos from high and low protein wheat (Triticum aestivum) grain produced under varied fertilizer conditions were compared. High protein grain produced seedlings 25% heavier with 25% more total RNA, 30% more DNA, 40% more amino acids, 60% more ribosomes, and 80% more soluble protein content than that of low protein seed. Consistently higher glutamine synthetase and α-amylase and lower acid phosphatase activities were observed in high protein seeds, though the isozyme pattern of α-amylase was not different in the two kinds of seeds. The high total ribosomes and particularly, polysome content observed in high protein seeds may be responsible for the rapid growth and high yield of these seeds.     

渗透胁迫下小麦根系渗透调节与根冠淀粉水解的研究

用不同浓度的PEG—600对抗旱性不同的小麦幼苗进行渗透胁迫处理,研究了小麦幼苗根系的淀粉酶活性、可溶性糖含量、渗透势、渗透调节能力和根冠淀粉的水解状况。结果表明,随着渗透胁迫程度的加重,抗旱性强的小麦品种昌乐5号和北农2号根系渗透势和饱和渗透势的降低程度大于抗旱性弱的小麦品种鲁麦5号和921842,并且抗旱性强的小麦品种根系的渗透调节能力大于抗旱性弱的小麦品种。随着渗透胁迫程度的加重．各品种小麦根冠淀粉粒均有不同程度的减少。而抗旱性强的品种根冠淀粉粒的减少程度小于抗旱性弱的品种;抗旱性强的小麦品种根系淀粉酶活性显著高于抗旱性弱的小麦品种,但是,随着渗透胁迫程度的加重,抗旱性弱的品种淀粉酶活性增加的幅度远高于抗旱性强的品种。可溶性糖含量的变化趋势与淀粉酶活性的变化趋势一致．即渗透胁迫下根冠淀粉水解程度大的小麦品种,可溶性糖的含量高。但根冠淀粉水解在根系的渗透调节以及在小麦适应水分胁迫中的作用还有待于进一步探讨。     

内质网压力响应相关的蛋白质降解

内质网相关蛋白质降解途径(ERAD),即蛋白质分泌过程中错误折叠或未折叠的蛋白质在内质网中被识别并逆向运输到细胞质经聚泛素化后由蛋白酶体降解的过程.自从发现该途径后对其机制的阐明一直处于不断探索的阶段.近年来,对ERAD底物识别、逆向运输和泛素化新组分的发现以及新技术的应用,使得该途径的具体分子机制更加清晰.本文全面梳理并综述了内质网应激响应、ERAD降解过程与机理的最新进展,并对模式蛋白底物和最新研究方法进行了总结,以期展示该领域的研究概况.     

Tissue Tensions during Cell Elongation in Wheat Roots and a Comparison with Contractile Roots

A study has been made of tissue tensions in growing Triticum roots. After the initial phase of elongation with both a lateral and an exponential longitudinal growth, the tissues are under strong longitudinal stress. Plasmolysis in situ or splitting of the root in the pericyclic region causes an increase in length of the perivascular part. Freed from the stele the perivascular part contracts on plasmolysis. During the following only longitudinal growth, the stress decreases under decreased root diameter. When elongation has ceased, the root in situ contracts on plasmolysis. It is suggested that during radial growth longitudinal stresses are built up, which are released when the stele catches up with the growth of the cortex. The similarity to conditions in contractile roots of Arum (Lamant and Heller 1967) is pointed out. A difference is found in the polarity of growth and thus in the mode of release of the stresses. The cause of the tensions and methodical difficulties are discussed.     

Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of Barley Roots

Root growth of barley (Hordeum vulgare L., cv. Akashinriki)was inhibited by 200 raM NaCl, when 1 mM CaCl₂ was present inthe hydroponic culture solution. Increasing the CaCl₂ up to10 mM partially prevented this inhibition. However, inhibitionalso occurred with 100 mM NaCl in the presence of 0.1 mM CaCl₂.The nuclei of meristematic cells in roots in which growth hadbeen inhibited by salt stress were studied after staining withDAPI (4',6-diamino-2-phenylindol). Nuclear deformation of thecells occurred with 12 h of salt stress with 500 mM NaCl, andwas followed by degradation. The nuclear degradation was alsoobserved when the roots were exposed to more than 300 mM NaClfor 24 h. Biochemical analysis revealed that nuclear degradationwas accompanied by apoptosis-like DNA fragmentation. The intracellularmechanisms of nuclear degradation in cells after salt stressare discussed. ¹Emertius professor, Okayama University.     

The Ubiquitin Ligase Itch Regulates Apoptosis by Targeting Thioredoxin-interacting Protein for Ubiquitin-dependent Degradation

Thioredoxin interacting protein (TXNIP) was originally characterized as an endogenous inhibitor of thioredoxin, a key regulator in cellular redox homeostasis. TXNIP is also known to play important roles in tumor growth and metastasis, glucose and lipid metabolism. TXNIP expression is induced by various stress stimuli. However, it has been unclear how TXNIP is down-regulated. Here, we report that TXNIP undergoes proteasomal degradation in cells. We identify Itch as the E3 ubiquitin ligase for TXNIP. We demonstrate that Itch mediates polyubiquitination of TXNIP both in vitro and in vivo. Overexpression of Itch leads to TXNIP proteasomal degradation. Knockdown of Itch by small interfering RNA causes an accumulation of the steady-state level of TXNIP. We also show that the PPXY motifs of TXNIP and the WW domains of Itch mediate their interaction. Furthermore, the Itch-TXNIP interaction regulates intracellular reactive oxygen species levels and apoptosis. These findings establish a new mechanism for the negative regulation of TXNIP by Itch and shed new light on the regulation of cellular redox homeostasis.     

渗透胁迫对小麦幼苗根系呼吸的影响

用PEG—6000调节培养液的渗透势,研究了渗透胁迫对小麦幼苗根系呼吸作用的影响。在-0.5 MPa的溶液中根总呼吸强度显著降低,不同苗龄根的反应差异明显;随胁迫加强呼吸强度随之降低;根系ATP含量减少。在胁迫初期呼吸废物对呼吸强度的降低无补偿作用,而在后期(72 h后)则可提高呼吸强度。 中度水分胁迫下,HMP支路活性上升,EMP-TCAO途径活性降低;抗氰呼吸活性增大,而对氰敏感的系统活性减低;细胞色素氧化酶活性显著低于对照。