DNA transcription, replication, and repair are regulated by histone acetylation, a process that requires the generation of acetyl-coenzyme A (CoA). Here, we show that all the subunits of the mitochondrial pyruvate dehydrogenase complex (PDC) are also present and functional in the nucleus of mammalian cells. We found that knockdown of nuclear PDC in isolated functional nuclei decreased the de novo synthesis of acetyl-CoA and acetylation of core histones. Nuclear PDC levels increased in a cell-cycle-dependent manner and in response to serum, epidermal growth factor, or mitochondrial stress; this was accompanied by a corresponding decrease in mitochondrial PDC levels, suggesting a translocation from the mitochondria to the nucleus. Inhibition of nuclear PDC decreased acetylation of specific lysine residues on histones important for G1-S phase progression and expression of S phase markers. Dynamic translocation of mitochondrial PDC to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation.
P ulmonary arterial hypertension (PAH) is a pulmonaryselective vascular remodeling disease in which cells within the vessel wall, including pulmonary artery smooth muscle cells (PASMCs), are characterized by a proproliferative and antiapoptotic diathesis. Pulmonary arterial remodeling occludes the vessel lumen that leads to right ventricular failure and premature death, with the median survival of untreated patients limited to 3 years. 1 However, even in those receiving standard therapies, prognosis remains poor. Clinical Perspective on p 125Although the pathology is restricted to the pulmonary vasculature, sparing the systemic vessels, all approved PAH therapies were originally developed as systemic vasodilators. Moreover, in contrast to the earlier belief that vasoconstriction plays a central role in PAH pathogenesis, it is now accepted that PAH is a result of proliferative remodeling with vasoconstriction playing a limited role. 4 An additional challenge is that, despite the recent groupings of several conditions that share similar lung histology to idiopathic PAH under a "PAH umbrella," the pathogenesis of PAH is multifactorial, suggesting that therapies that target 1 molecular abnormality in 1 form of PAH may not be as effective in other forms of the disease. For example, PAH is associated with inflammatory conditions like scleroderma, viral infections with HIV or herpes simplex virus, hypoxia, or loss-of-function mutations in the bone morphogenetic protein receptor 2 (BMPRII). [4][5][6] In PAH, like in cancer, many different molecular abnormalities can be active in a patient. Thus, an ideal PAH therapy should target common features of all these diverse biological processes in a manner that remains relatively selective to the pulmonary Background-Evidence suggestive of endoplasmic reticulum (ER) stress in the pulmonary arteries of patients with pulmonary arterial hypertension has been described for decades but has never been therapeutically targeted. ER stress is a feature of many conditions associated with pulmonary arterial hypertension like hypoxia, inflammation, or loss-offunction mutations. ER stress signaling in the pulmonary circulation involves the activation of activating transcription factor 6, which, via induction of the reticulin protein Nogo, can lead to the disruption of the functional ER-mitochondria unit and the increasingly recognized cancer-like metabolic shift in pulmonary arterial hypertension that promotes proliferation and apoptosis resistance in the pulmonary artery wall. We hypothesized that chemical chaperones known to suppress ER stress signaling, like 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid, will inhibit the disruption of the ER-mitochondrial unit and prevent/reverse pulmonary arterial hypertension. Methods and Results-PBA in the drinking water both prevented and reversed chronic hypoxia-induced pulmonary hypertension in mice, decreasing pulmonary vascular resistance, pulmonary artery remodeling, and right ventricular hypertrophy and improving functional capacity w...
Most solid tumors are characterized by a metabolic shift from glucose oxidation to glycolysis, in part due to actively suppressed mitochondrial function, a state that favors resistance to apoptosis. Suppressed mitochondrial function may also contribute to the activation of hypoxia-inducible factor 1α (HIF1α) and angiogenesis. We have previously shown that the inhibitor of pyruvate dehydrogenase kinase (PDK) dichloroacetate (DCA) activates glucose oxidation and induces apoptosis in cancer cells in vitro and in vivo. We hypothesized that DCA will also reverse the 'pseudohypoxic' mitochondrial signals that lead to HIF1α activation in cancer, even in the absence of hypoxia and inhibit cancer angiogenesis. We show that inhibition of PDKII inhibits HIF1α in cancer cells using several techniques, including HIF1α luciferase reporter assays. Using pharmacologic and molecular approaches that suppress the prolyl-hydroxylase (PHD)-mediated inhibition of HIF1α, we show that DCA inhibits HIF1α by both a PHD-dependent mechanism (that involves a DCA-induced increase in the production of mitochondria-derived α-ketoglutarate) and a PHD-independent mechanism, involving activation of p53 via mitochondrial-derived H(2)O(2), as well as activation of GSK3β. Effective inhibition of HIF1α is shown by a decrease in the expression of several HIF1α regulated gene products as well as inhibition of angiogenesis in vitro in matrigel assays. More importantly, in rat xenotransplant models of non-small cell lung cancer and breast cancer, we show effective inhibition of angiogenesis and tumor perfusion in vivo, assessed by contrast-enhanced ultrasonography, nuclear imaging techniques and histology. This work suggests that mitochondria-targeting metabolic modulators that increase pyruvate dehydrogenase activity, in addition to the recently described pro-apoptotic and anti-proliferative effects, suppress angiogenesis as well, normalizing the pseudo-hypoxic signals that lead to normoxic HIF1α activation in solid tumors.
The Dsb family of enzymes catalyzes disulfide bond formation in the gram-negative periplasm, which is required for folding and assembly of many secreted proteins. Pertussis toxin is arguably the most complex toxin known: it is assembled from six subunits encoded by five genes (for subunits S1 to S5), with 11 intramolecular disulfide bonds. To examine the role of the Dsb enzymes in assembly and secretion of pertussis toxin, we identified and mutated the Bordetella pertussis dsbA, dsbB, and dsbC homologues. Mutations in dsbA or dsbB resulted in decreased levels of S1 (the A subunit) and S2 (a B-subunit protein), demonstrating that DsbA and DsbB are required for toxin assembly. Mutations in dsbC did not impair assembly of periplasmic toxin but resulted in decreased toxin secretion, suggesting a defect in the formation of the Ptl secretion complex.Pertussis toxin is a major virulence factor of the gram-negative bacterium Bordetella pertussis, which is the causative agent of whooping cough (34,43). The toxin is a member of the AB 5 family of toxins, which includes Shiga toxin, cholera toxin, and Escherichia coli heat-labile toxin. It plays a crucial role in virulence by mediating ADP-ribosylation of host GTP-binding proteins (G i , G o , and G t ), thereby disrupting normal host cellular regulation (19,26). The systemic effects on the infected host include blocking of antimicrobial activity in a number of immune effector cells, resulting in a less effective immune response by the host (20, 42).Five structural toxin genes (S1 to S5) and the nine ptl (for pertussis toxin liberation) genes (ptlA to ptlI), encoding the secretion complex, are cotranscribed from a single operon (28, 45) that is positively regulated by the Bvg two-component regulatory system (22). S1 is the enzymatic A subunit of the toxin, while the B subunit or B pentamer binds mammalian cells and delivers the toxin into the mammalian cytoplasm (25,40). Pertussis toxin is a somewhat atypical AB 5 toxin. The B pentamer is not a homo-oligomer but rather consists of subunits S2, S3, S4, and S5, in a 1:1:2:1 ratio (30, 31, 39), which associate with the C terminus (2, 49) of the S1, or A, subunit. Each toxin subunit is translated with its own signal sequence (30, 31), and the subunits are secreted to the periplasm presumably via the Bordetella equivalent of the E. coli Sec machinery. Once in the periplasm, the subunits are assembled into the 105-kDa holotoxin that is recognized by the Ptl type IV secretion complex (10,15,35), which then moves the holotoxin through the outer membrane into the extracellular milieu (12,14,45). Additionally, it has been demonstrated that only the holotoxin is targeted for secretion, as expression of B subunit (S2 to S5) or A subunit (S1) in isolation did not result in extracellular secretion of toxin subunits (15). A region of the S1 subunit which might act as a recognition domain for this interaction of holotoxin with the Ptl secretion apparatus has been described (10).Interestingly, AB 5 toxins have been described only for gramn...
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