PROTEIN SYNTHESIS INITIATION IN RED BLOOD CELLS

本研究包括两个相关的项目：l.研究P67。A 67 KDa 糖蛋白p67保护eIF-2 α-亚单位免受抑制性 通过eIF-2激酶（HRI和PKR）磷酸化，因此， 蛋白质合成所必需的。我们的研究表明p67活性 在mRNA水平上受到调节，也受到蛋白质糖基化的调节- 去糖基化有迹象表明，p67特异性结合到 eIF-2 γ亚基，并利用其糖基残基保护eIF-2 α- 亚单位具体目标是：（A）研究p67合成的调控， 降解 p67去糖基化酶活性将从 网织红细胞裂解物及其在调节p67活性中的作用将被 研究了p67在mRNA水平的调节将使用“径流”进行研究。 实验(B)p67在蛋白调节中的作用研究 在正常和病毒感染的细胞中的合成。 p67的作用 去糖基化和改变p67水平对蛋白质合成的影响， 在不同的生理条件下， 如血清饥饿、促分裂原刺激和病毒感染。 p67水平 通过调控p67-cDNA或p67反义DNA的表达， 在转染的细胞中。(C)定位p67上的活性位点。尝试将 对p67上的糖基化位点以及p67上的位点进行定位 负责p67与eIF-2 γ亚基结合并保护eIF-2 抑制性磷酸化的α亚基。体外诱变p67 cDNA将在杆状病毒系统中表达。突变的蛋白质 然后进行分离和分析。二.肽链起始机制。 需要三种蛋白因子eIF-2B、Co-eIF-2A和eIF-3来促进 通过eIF-2形成三元复合物。 eIF-3中的180 kDa多肽 蛋白质复合物可能负责elF-3活性，以促进 三元络合物形成 具体目标是：（A）研究 p180、Co-eIF-2A和其他蛋白因子在三元和Met- tRNAf.40S.mRNA复合物的形成，也在整个蛋白质合成。 将尝试广泛纯化不同的蛋白质因子， 重组有效的三元和Met-tRNAf. 40 S.mRNA复合物的形成。 (B)编码Co-eIF-2A和（C）的cDNA的克隆和表征 Co-eIF-2A反义基因序列的制备及其在大肠杆菌中表达的研究 Co-eIF-2A在蛋白质合成中的需求。标准实验 程序将用于特定目标B和C。本研究是 希望能更好地了解分子机制 在翻译水平上的基因表达。 一个有趣的可能性是 我们将能够通过调节p67水平来控制病毒感染， 细胞。这将为我们提供一种对抗病毒性疾病的工具。

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.