The dihydrolipoyl acetyltransferase (E2) component of the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in which E2's inner domain forms a dodecahedron shaped structure surrounded by its globular outer domains that are connected to each other and the inner domain by 2-3-kDa mobile hinge regions. Two of the outer domains are approximately 10 kDa lipoyl domains, an NH2-terminal one, E2L1, and, after the first hinge region a second one, E2L2. The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a molecule of kinase tightly bound to the E2 core can rapidly phosphorylate 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombinant constructs consisting of the entire lipoyl domain region or the individual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively bind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs containing the E2L2 domain rather than to ones containing only the E2L1 domain. GST-E2L2-kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 by treatment with lipoamidase eliminated kinase binding supporting a direct role of the lipoyl prosthetic group in this association. Transfer to and selective binding of the kinase by E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 increased the activity of resolved kinase by up to 43%. We conclude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic group. We further suggest that transfer of tightly bound kinase between E2L2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented).(ABSTRACT TRUNCATED AT 400 WORDS)

SummaryThe extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial humoral response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V3 usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the El-and E2-specific components. We also examined the phenotype, lymphokine profile, and V3 usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon 3' production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Thl-and Th2-1ike clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCIk) V3 usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V~ repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TClk V3 repertoire is heterogenous.

Micromolar Ca2؉ facilitates ϳ10-fold enhancement of pyruvate dehydrogenase phosphatase (PDP) activity by aiding the association of PDP with the dihydrolipoyl acetyltransferase (E2) component. Connected by linker regions, E2 consists of two lipoyl domains, the NH 2 -lipoyl domain (L1) and the interior lipoyl domain (L2), and a pyruvate dehydrogenase component binding domain surrounding a 60-mer inner core. Using recombinant constructs of L1 or L2, E2-enhanced PDP activity was markedly decreased by L2 but not by L1, effectively competing with intact E2 in Ca 2؉-dependent binding of PDP (half-maximal reduction at 2.0 M L2 versus 6.7 M E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca 2؉-dependent binding of PDP to L2 (K d , ϳ1.7 M L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca 2؉ chelation. The large activity enhancement of PDP by E2 was eliminated by enzymatic removal of lipoates from E2 and restored by their enzymatic reintroduction. The critical role of the L2 lipoate is not in binding of PDP to E2, since PDP was still bound by delipoylated L2, and delipoylated L2 inhibited E2-enhanced PDP activity, although lipoylated L2 was more effective in each of these tests. Thus, pyruvate dehydrogenase complex activity is increased by enhanced availability of PDP to its E2-bound, phosphorylated pyruvate dehydrogenase substrate as a consequence of the Ca 2؉ -facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate. Mammalian pyruvate dehydrogenase complex (PDC)1 is organized around a 60-subunit dihydrolipoyl acetyltransferase (E2) structure, which consists of four independently folded domains connected to each other by mobile linker regions 20 -30 amino acids in length (1-3). Association of 20 trimer units of the COOH-terminal domain of E2 produces a central cavity in the shape of a dodecahedron. Exterior to this inner core assemblage, after the first linker region, each E2 has a 5-kDa domain that binds the pyruvate dehydrogenase (E1) component (4, 5); 20 -30 E1 tetramers (␣ 2 ␤ 2 ) bind per E2 60 . Then, set off by two more linker regions are two ϳ10-kDa lipoyl domains, an interior one (L2), and an NH 2 -terminal one (L1). An E3-binding protein (E3BP) is similar to E2 in consisting of three linker connected domains (6) in which the distinct inner domain of E3BP binds the inner domain of E2 (7, 8) apparently inside the dodecahedron cavity 2 and connects by a linker region to an exterior E3 binding domain (10), followed by a linker-connected lipoyl domain.PDC is regulated by interconversion of E1 between a nonphosphorylated, active form and a phosphorylated, inactive form (E1b). Pyruvate dehydrogenase phosphatase (PDP) catalyzes the Mg 2ϩ -requiring, Ca 2ϩ -stimulated dephosphorylation and activation of E1 (11)(12)(13)(14). Removal of phosphates from the ␣ subunit of E1 can occur with resolved E1b but is enhanced manyfold when E1b and PDP associate with th...

Considerable resources are spent within the biopharmaceutical industry to perform viral clearance studies, which are conducted for widely used unit operations that are known to have robust and effective retrovirus clearance capability. The collaborative analysis from the members of the BioPhorum Development Group Viral Clearance Working Team considers two common virus reduction steps in biopharmaceutical processes: low-pH viral inactivation and viral filtration. Analysis included eight parameters for viral inactivation and nine for viral filtration. The extensive data set presented in this paper provides the industry with a reference point for establishing robust processes in addition to other protocols available in the literature (e.g., ASTM Std. E2888-12 for low-pH inactivation). In addition, it identifies points of weakness in the existing data set and instructs the design and interpretation of future studies. Included is an abundance of data that would have been difficult to generate individually but collectively will help support modular viral clearance claims.

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