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.
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