ELECTRON TRANSFER BETWEEN HEME PROTEINS

蛋白质电子转移反应在代谢过程中起着关键作用 从合成到能量转换。现时的建议 继续在这方面的研究，侧重于两个系统。第一 着重于细胞色素c：细胞色素c过氧化物酶偶联物（缩写为 (Cc：Ccp），其已成为生理蛋白质的范例， 蛋白质电子转移我们将映射域进行绑定， 反应性在这个系统中，使用特定地点的替代品， 细胞色素c。第二个研究系统是血红蛋白-血红蛋白 还原酶系统（简称Hb/b5），维持活性，还原Hb 在红细胞中。与Cc：Ccp系统相比， 了解这种反应。详细的研究将检查依赖 反应自由能（AG）、温度和蛋白质组成的速率 （通过检查自然物种变异以及现场进行更改- 具体的替代品）。关键解决方案变量的影响，即pH 和离子强度也将被评估。具体的作用 识别和/或结合的机制。采取 这些研究将为详细的 了解另一个典型的生理蛋白质电子 对人类健康至关重要的转移反应。 R01GM35174 拟议的工作旨在定义物理化学和生物化学 人B的亲和力依赖性阳性和阴性选择的基础 细胞通过mIgM信号通路。这将通过使用 一组充分表征的具有不同结合的鼠抗IgM mAb 位点亲和力和实验操纵的化合价，作为模型 可以与所有IgM +人B细胞结合的抗原。 <1>研究 将评估交联形成速率，或者替代地， 是亲和力的限制参数- 在限制性条件下依赖于mIgM介导的信号转导 mIgM受体密度，如发生在B细胞激活的晚期， mIgM介导的信号是完整细胞周期进程所必需的。这 将涉及初始解离动力学的比较 配体的单价接合（k1）、交联形成的速率，以及 交联解离速率，早期酪氨酸 激酶活化和蛋白酪氨酸磷酸化。 <2>研究 将评估不同的信号通路是否可能是差异性的， 受mIgM配体亲和力和配体价态的影响，通过 评估早期酪氨酸磷酸化的程度， 不同的蛋白质受到这些参数的不同影响。 <3> 这些研究将评估是否存在物理化学结合 诱导B细胞进入S期的要求与那些 需要上调bcl-2，并增加合成的一些 或所有显示调节G1到S期转变的蛋白质， 其它真核细胞，即细胞周期蛋白和CDK激酶。 <4>这项工作将 评估接受不足的mIgM介导的成熟B细胞是否 G1到S相变的信号被引导到激活中- 相关的凋亡或无反应性。 <5>mIgM：配体结合要求 用于诱导未成熟B淋巴细胞的无反应性和凋亡， 使用B细胞表达膜的转基因小鼠进一步评估 人类μ链的形式 <6>作为最后一个主要目标，这些研究将 评价B细胞mIgM和其他B细胞粘附是否共连接 分子（即CD 21，CD 22和VLA-4）通过相同的分子或细胞 底物的亲和力和/或化合价要求， 诱导B细胞增殖。如果是这样，进一步的研究将确定 共连接是否导致增强的配体结合动力学;增强的 或修饰的受体近端信号转导; 细胞凋亡; bcl-2水平升高;和/或一些或 所有负责G1到S相变的蛋白质。采取 总之，拟议的研究应该提供相当多的新见解 mIgM和其他抗体的定量和定性占有率 B细胞表面的辅助受体转化为信号， B细胞无反应性、B细胞缺失或B细胞克隆增殖。因为 B细胞的亲和力依赖性选择在（a）免疫中是重要的 对入侵病原体和故意接种疫苗的反应， (b)B细胞对自身抗原的自身免疫，（c） 某些B细胞恶性肿瘤，这些见解应导致增强 对上述现象进行监管干预。

Protein electron transfer reactions play key roles in metabolism ranging from synthesis through energy transduction. The present proposal continues studies in this vein, focusing on two systems. The first focuses on the cytochrome c:cytochrome c peroxidase couple (abbreviated (Cc:Ccp), which has become a paradigm for physiological protein to protein electron transfer. We will map the domain for binding and reactivity within this system, using site-specific replacements of cytochrome c. The second system for study is the hemoglobin-hemoglobin reductase system (abbreviated Hb/b5), which maintains active, reduced Hb in the erythrocytes. By comparison with the Cc:Ccp system, must less is known about this reaction. Detailed studies will examine the dependence of rate on reaction free energy (AG), temperature and protein composition (varied by examination of natural species variance, as well as by site- specific replacements). The effects of key solution variables, i.e., pH and ionic strength, will also be assessed. The role of specific recognition and/or binding in the mechanism will be probed. Taken together these studies will provide the foundation for detailed understanding of another paradigmatic physiological protein electron transfer reaction that is central to human health. R01GM35174 The proposed work seeks to define the physicochemical and biochemical basis for affinity-dependent positive and negative selection of human B cells via the mIgM signaling pathway. This will be accomplished by using a set of well-characterized murine anti-IgM mAbs with differing binding site affinities, and experimentally manipulated valencies, as model antigens which can engage with all IgM + human B cells. <1> The studies will evaluate whether rate of crosslink formation, or alternatively, rate of crosslink dissociation, is the limiting parameter in affinity- dependent, mIgM-mediated signal transduction under conditions of limiting mIgM receptor density, such as occurs late in B cell activation when new mIgM-mediated signals are required for full cell cycle progression. This will involve comparisons of the dissociation kinetics for initial monovalent engagement of ligand (k1), rates for crosslink formation, and rates for crosslink dissociation, with the kinetics of early tyrosine kinase activation and protein tyrosine phosphorylation. <2> The studies will evaluate whether distinct signaling pathways may be differentially affected by ligand affinity for mIgM and ligand valency, through assessing the degree to which the early tyrosine phosphorylation of distinct proteins is differentially affected by these parameters. <3> The studies will evaluate whether the physicochemical binding requirements for inducing B cell S phase entry are identical to those required for upregulation of bcl-2, and for increased synthesis of some or all of the proteins shown to regulate the G1 to S phase transition in other eukaryotic cells, i.e. cyclins and cdk kinases. <4> The work will evaluate whether mature B cells which receive insufficient mIgM-mediated signals for the G1 to S phase transition are channeled into activation- related apoptosis or anergy. <5> The mIgM : ligand binding requirements for inducing anergy and apoptosis in immature B lymphocytes will be further assessed using transgenic mice whose B cells express the membrane form of human mu chain. <6> As a final major aim, the studies will evaluate whether co-ligation,of B cell mIgM and other B cell adhesion molecules (i.e. CD21, CD22, & VLA-4) by the same molecular or cellular substrate, can reduce the affinity and/or valency requirements for inducing B cell proliferation. If so, additional studies will determine whether co-ligation results in enhanced ligand binding kinetics; enhanced or modified receptor-proximal signal transduction; diminished levels of apoptosis; enhanced levels of bcl-2; and/or enhanced levels of some or all the proteins responsible for she G1 to S phase transition. Taken together, the proposed studies should provide considerable new insights into how the quantitative and qualitative occupancy of mIgM and other ancillary receptors on the surface of B cells translates into signals for B cell anergy, B cell deletion, or B cell clonal proliferation. Because affinity-dependent selection of B cells is important in (a) immune responses to intruding pathogens and deliberately administered vaccines, (b) B cell autoimmunity to self antigens, (c) the clonal evolution of certain B cell malignancies, these insights should lead to enhanced regulatory intervention of the above phenomena.