Antimitochondrial autoantibodies are characteristically present in sera of patients with primary biliary cirrhosis. The antimitochondrial autoantibodies recognize four major antigens from beef heart ...mitochondria at relative molecular weights of 74, 56, 52 and 48 kD. In the present study, we report that the 56 kD antigen is the protein X of pyruvate dehydrogenase complex and that it possesses cross‐reactive antimitochondrial autoantibody epitope(s) with the 74 kD antigen, the acetyltransferase (E2) of the pyruvate dehydrogenase complex. This was demonstrated by comparing the specificities of primary biliary cirrhosis sera with a protein X‐specific rabbit antiserum and by absorbing primary biliary cirrhosis sera with recombinant pyruvate dehydrogenase‐E2 fusion protein. In the two‐dimensional gel analysis, primary biliary cirrhosis sera and protein X‐specific rabbit antiserum reacted to the same two isoelectric point polypeptides at 56 kD molecular weight. The absorption of primary biliary cirrhosis sera with the human recombinant pyruvate dehydrogenase‐E2 removed reactivity toward both the 74 and 56 kD antigens. Furthermore, analysis of 82 antimitochondrial autoantibody‐positive primary biliary cirrhosis sera by immunoblotting did not reveal any sera which reacted solely against either the 74 or 56 kD antigen. Finally, primary biliary cirrhosis sera recognized protein X from human, bovine and porcine sources but not protein X from rat or mouse origin. The identification of protein X as another major target of the autoimmune response in primary biliary cirrhosis suggests that the pyruvate dehydrogenase complex may have a central role in the induction of this enigmatic disease.
Micromolar Ca2+ facilitates ∼10-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 NH2-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 Ca2+-dependent binding of PDP (half-maximal reduction at 2.0 μM L2 versus 6.7 μM E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca2+-dependent binding of PDP to L2 (Kd, ∼1.7 μM L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca2+ 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 Ca2+-facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate.
Previous studies documenting the existence of cross-reactivity between the lipoated (but not unlipoated) forms of the inner lipoyl domain (E2L2) of PDC-E2 the major autoantigen in Primary biliary ...cirrhosis (PBC) and trifluoroacetylated (TFA) proteins, led us to hypothesize that PBC may be due to an initial insult with an environmental agent that cross-reacts with TFA. Therefore, we performed a comparative study of the reactivity of rabbit anti-TFA antibody and anti-lipoic acid (LA) antibody against the mitochondrial autoantigens of human PBC and various TFA and LA conjugated proteins. Whereas both anti-TFA and anti-LA reacted with PDC-E2, the wild-type lipoated form of E2L2, OGDC-E2, E3-BP and LA-KLH, neither reacted with BCOADC-E2 or the non-lipoated form of E2L2. Of interest was that while anti-TFA reacted with PDC-E2, TFA-RSA and LA-KLH, it failed to inhibit PDC-E2 enzyme function. In contrast, anti-LA demonstrated cytoplasmic and mitochondrial staining, and inhibited PDC enzyme activity. Hence, although considerable cross reactivity exists between anti-TFA and anti-LA, the molecular nature of the interaction is clearly different. One of 14 PBC sera reacted weakly with TFA-albumin, whereas four of 14 PBC sera reacted with LA-KLH. Immunohistochemically, both anti-TFA and anti-LA antibodies reacted focally with periportal hepatocytes and bile ducts in both PBC and controls. However, anti-LA produced much stronger focalized staining of the bile ducts of diseased liver. This study suggests that while anti-TFA antibody recognizes lipoic acid-linked enzymes and proteins, the epitope recognized differs from that of anti-LA antibody and PBC autoantibodies. It is unlikely that a response to TFA is the triggering event in PBC. Anti-LA antibodies share a higher degree of similarity to PBC sera providing suggestive evidence that anti-LA antibodies or anti-LA like antibodies (mimotopes) may help define the initiator of the autoimmune response.
In most organisms, the pyruvate dehydrogenase complex catalyzes the pivotal irreversible reaction that leads to the consumption of glucose in the aerobic, energy-generating pathways. A combination of ...biochemical and molecular biology studies have greatly expanded our understanding of the overall structural organization of this multicomponent system, delineated the locations and elucidated the functions of structural domains of the catalytic components, and revealed significant evolutionary changes. Important to this progress was the deduction of the primary amino acid sequences from cDNA clones for each of the catalytic components from several species. The greatest detail is available for the FAD-containing dihydrolipoamide dehydrogenase component, which is the only component for which tertiary structure information has recently emerged. For the dihydrolipoamide acetyltransferase core component, a similar but species-variable multidomain structure is established that is responsible for the distinct architectures of the inner cores, the peripheral binding of the other components, and the conveyance of reaction intermediates between distantly separated active sites. A second lipoyl-bearing component, protein X, has been shown to play a critical role in the organization and function of the complex from many higher organisms. Although much is known about the means of effector modulation of mammalian complex activity, identification of the signal eliciting its regulation by insulin still poses an exciting challenge.
Prior studies have suggested that loss of plasma cell CD56 expression in multiple myeloma defines a unique patient subset and that CD56 expression reliably discriminates between monoclonal gammopathy ...of undetermined significance (MGUS) and multiple myeloma (MM). We conducted a study of 68 untreated patients with MM from a single institution to define the clinicopathological correlates of CD56 expression. We find CD56 expression in 55% of MM. Lack of CD56 expression does not define a unique clinicopathological or prognostic entity in MM. Strong CD56 expression can also be found in MGUS and does not help to distinguish from MM.
Abstract
Abstract #3057
Introduction: Patients (pts) with ER/PR/HER2 negative advanced breast cancer (triple negative, TN, basal-like) have limited treatment options and a poor prognosis. Ixabepilone ...(ixa), the first in a new class of antineoplastic agents, showed clinical benefit in combination with capecitabine (C) in 2 large clinical trials in metastatic breast cancer (MBC) pts either resistant to (study 046; JCO, 2007) or pretreated with anthracycline (A) and taxanes (T) (study 048). In 048, ixa + C, compared to C alone, demonstrated significant increases in PFS (HR 0.79 0.69-0.90) and ORR (43 vs. 29%). A trend towards increased OS was seen in both 048 (HR 0.90 0.78-1.03) and 046 (HR 0.90 0.77-1.05), which did not reach statistical significance. Here we present a pooled analysis of efficacy endpoints (ORR, PFS and OS) in pts with TN disease from these 2 phase III studies. Methods: 1973 pts with MBC previously treated with A and T were randomized in 2 phase III trials (046 and 048) to receive either ixa (40 mg/m2 IV over 3h Q3w) + C (1000 mg/m2 PO BID x14d Q3w) or C alone (1250 mg/m2 PO BID x14d Q3w). Due to the similarity of the study populations, individual pt data from both studies was pooled to better evaluate treatment effect within pre-planned patient subgroups. Results: 443 pts had TN disease in the two studies combined. ORR and PFS were superior in pts receiving combination therapy compared to those on C alone with a trend towards improved survival that did not reach statistical significance. Conclusions: Ixa + C is the first combination to show statistically significant PFS benefit in pts with advanced TN breast cancer from a pooled analysis of 2 phase III trials. Clinical benefit was consistently seen pts with TN disease in the individual studies as well.
Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3057.
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