PROTEIN SYNTHESIS INITIATION IN RED BLOOD CELLS

本研究包括两个相关课题：L。p67研究。A 67 KDa 糖蛋白p67保护eIF-2α亚基免受抑制 由eIF-2激酶(HRI和PKR)磷酸化，因此， 是蛋白质合成所必需的。我们的研究表明p67的活动 在信使核糖核酸水平和蛋白质糖基化水平上进行调节- 去糖基化。有迹象表明，p67特异性地结合到 EIF-2γ亚基，并利用其糖基残基保护eIF-2α- 亚单位。具体目标是：(A)研究p67合成和调控 退化。P67脱糖基酶活性将从 网织红细胞裂解物及其在调节p67活性中的作用将是 学习。P67--在信使核糖核酸水平上的调节将用“径流”来研究。 实验。(B)研究p67在蛋白质调控中的作用 正常细胞和病毒感染细胞的合成。P67的作用 脱糖和改变p67水平对蛋白质合成的影响 使用动物细胞在不同的生理条件下进行研究 如血清饥饿、有丝分裂原刺激和病毒感染。P67水平 将通过调节p67-cDNA或p67反义DNA的表达而改变 在转基因细胞中。(C)绘制p67上的活性部位图。尝试将 绘制p67上的糖基化位点和p67上的位点 负责p67与eIF-2γ亚基的结合及对eIF-2的保护作用 抑制磷酸化所致的α亚基。体外诱变的p67基因 该基因在杆状病毒系统中得到表达。突变的蛋白质将 然后被隔离和分析。二、肽链启动的机理。 需要三种蛋白质因子eIF-2B、Co-eIF-2A和eIF-3来促进 EIF-2形成三元络合物。EIF-3中的180 kDa多肽 蛋白复合体可能是ELF-3活性促进的原因 三元络合物的形成。具体目标是：(A)研究 P180、Co-EIF-2A等蛋白质因子在三元和蛋氨酸中的表达 TRNAf.40S.mRNA复合体的形成以及整体蛋白质的合成。 将尝试广泛纯化不同的蛋白质因子和 重组有效的三元和Met-tRNAf.40S.mRNA复合体。 (B)编码Co-eIF-2A和(C)的cDNA的克隆和鉴定 反义Co-eIF-2A基因序列的制备及其生物学活性的研究 Co-eIF-2A在蛋白质合成中的需求。标准实验 程序将用于具体目标B和C。这项研究是 可望提供更好的理解分子机制的基础。 翻译水平上的基因表达。一个耐人寻味的可能性是 我们将能够通过调节p67水平来控制病毒感染 这些细胞。这将为我们提供抗击病毒性疾病的工具。

This research includes two related projects: l. Studies of p67. A 67 KDa glycoprotein, p67 protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases (HRI and PKR) and is, therefore, necessary for protein synthesis. Our work has indicted that p67 activity is regulated at the mRNA level and also by protein glycosylation- deglycosylation. There are indications that p67 binds specifically to the eIF-2gamma subunit and uses its glycosyl residues to protect eIF-2 alpha- subunit. Specific aims are: (A) Studies of regulation of p67 synthesis and degradation. A p67 deglycosylase activity will be purified from reticulocyte lysates and its roles in regulation of p67 activity will be studied. p67-regulation at the mRNA level will be studied using "run-off" experiments. (B) Studies of the roles of p67 in regulation of protein synthesis in normal and viral infected cells. Effects of p67 deglycosylation and changing p67 levels on protein synthesis will be studied using animal cells under different physiological conditions such as serum starvation, mitogen stimulation and viral infection. p67 level will be changed by regulated expression of p67-cDNA or p67 antisense DNA in transfected cells. (C) Mapping the active sites on p67. Attempts will be made to map the glycosylation sites on p67 and also the sites on p67 responsible for p67 binding to eIF-2gamma subunit and protection of eIF-2 alpha-subunit from inhibitory phosphorylation. In vitro mutagenized p67 cDNA will be expressed in baculovirus system. The mutated proteins will then be isolated and analyzed. II. Mechanism of peptide chain initiation. Three protein factors eIF-2B, Co-eIF-2A and eIF-3 are required to promote ternary complex formation by eIF-2. The 180 kDa polypeptide in eIF-3 protein complex is possibly responsible for elF-3 activity to promote ternary complex formation. Specific aims are: (A) Studies of the roles of p180, Co-eIF-2A and other protein factors in ternary and Met- tRNAf.40S.mRNA complex formation and also in over-all protein synthesis. Attempts will be made to extensively purify different protein factors and reconstitute efficient ternary and Met-tRNAf.40S.mRNA complex formation. (B) Cloning and characterization of a cDNA encoding Co-eIF-2A and (C) Preparation of anti sense Co-eIF-2A gene sequences and studies of the requirement of Co-eIF-2A in protein synthesis. Standard experimental procedures will be used for specific aims B and C. This research is expected to provide a better understanding of the molecular mechanism of gene expression at the translation level. An intriguing possibility is that we will be able to control viral infection by regulating p67 level in the cells. This will provide us with a tool to combat viral diseases.