Oxygen-dependent proinflammatory functions of polymorphonuclear leukocytes (PMNs) reflect synergy between granule contents and reactive oxygen species. Myeloperoxidase (MPO), a heme protein in the azurophilic granules, catalyzes the generation of HOCI in the presence of H2O2. The NADPH-dependent oxidase contains flavocytochrome b558, a heterodimeric membrane component that like MPO is a hemeprotein. Since these two hemeproteins are essential for optimal PMN activity, the biosynthesis of each in the endoplasmic reticulum (ER) is finely controlled to optimize correct folding and acquisition of heme. The applicants have identified two ER resident proteins, calreticulin (CRT) and calnexin (CLN), as participants in the biosynthesis of normal MPO and in the retrieval and degradation of mutated MPO. During the past year they have also initiated studies to characterize features of normal flavocytochrome b558 biosynthesis.Currently unknown are the means by which heme is inserted into apoproteins synthesized in the ER or, in the case of flavocytochrome b558, the determinants of subunit association. Information is also limited on the role of molecular chaperones in monitoring quality control during myeloid protein synthesis or on the precise peptide motifs recognized by CRT and CLN that target a given protein as """"""""unfolded."""""""" Using MPO and flavocytochrome b558 as representative myeloid proteins, the applicants aim to test the hypotheses: (a) that heme acquisition and/or heterodimer formation are events essential for normal biosynthetic maturation of flavocytochrome b558; (b) that heme acquisition by apoforms is facilitated by ER-resident molecular chaperones; and (c) that CRT and CLN recognize specific peptide motifs in MPO precursors. The applicants will test these hypotheses by pursuing three specific aims: (1) to characterize critical steps in flavocytochrome b558 biosynthesis, specifically heme acquisition and heterodimer formation; (2) to define the relative roles of CRT/CLN in heme acquisition and ER """"""""quality control"""""""" during MPO biosynthesis; and (3) to determine the relative importance of heme acquisition and heterodimer formation to the biogenesis of other members of the NOX protein family. The applicants believe that these will define principles that apply not only to these specific myeloid proteins but to proteins in general that acquire heme in the ER.
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