Characterization Of The Yeast Cysteine Desulfurase Complex Within The Mitochondrial Fe-S Cluster Biogenesis

Disrupted iron homeostasis within the human body materializes as various disorders. Pathophysiology of many of them relates to iron induced oxidative damage to key cellular components caused by iron accumulation within the tissues. Pertaining to the growing occurrence, cost of patient care and devastating burden associated with these diseases, the call for understanding the role of iron homeostasis within these disorders becomes inevitable. Being an abundant iron containing cofactor, the role of Fe-S clusters in cellular iron homeostasis is indisputable in the case of Friedreich’s ataxia, a disease caused by a deficiency in the protein frataxin that is indispensable during Fe-S cluster assembly. Friedreich’s ataxia and similar disorders associated with defective Fe-S cluster assembly accentuate the need to unravel the structural and functional aspects of this fundamental biochemical pathway. Effective disease treatment has been hampered by the lack of a molecular level understanding of the individual roles of the key proteins in Fe-S cluster formation. The work presented within this dissertation diminish this knowledge gap significantly by providing a comprehensive biophysical characterization of the key proteins involved in the sulfur mobilization step during the yeast mitochondrial Fe-S cluster synthesis process. The role of the accessory protein “Isd11” within the catalytic role of the cysteine desulfurase “Nfs1” in delivering sulfane sulfur for cluster assembly has been studied. The effect of Yfh1 on sulfur mobilization by the Nfs1-Isd11 complex has also been evaluated in detail. Our results suggest a possible regulatory function of Isd11 and possibly rationalize the evolutionary requirement for its role as an additional cofactor introduced in eukaryotes. Frataxin’s effect on cluster assembly was evident to materialize the most at the overall assembled complex level rather than at the individual protein level. This work, therefore, provides novel and significant insight into how the cluster assembly proteins function, and sets the groundwork for which additional experiments that need to be designed to further unravel the mechanistic details of sulfur mobilization during Fe-S cluster bioassembly. These molecular level details will assist in future drug design strategies directed at treating the diseases outlined above

Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2-/- mice

Background aims: Secretin (SCT) and secretin receptors (SR, only expressed in cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction (DR) and liver fibrosis in bile duct ligated and Mdr2-/- (primary sclerosing cholangitis, PSC, model) mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2-/- mice. Approach results: In vivo studies were performed in male wild-type (WT) and Mdr2-/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. The biliary SCT/SR expression and SCT serum levels increased in Mdr2-/- mice and late-stage PSC patients compared to controls. There was a significant increase in DR, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-β1/VEGF-A axis, and biliary phosphorylation of PKA and ERK1/2 in Mdr2-/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2-/- compared to WT mice, which was reversed by long-term SCT 5-27 treatment. In vitro, SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-β1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Down-regulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. Conclusions: Modulating the SCT/SR axis may be critical for managing PSC

Knockout of secretin ameliorates biliary and liver phenotypes during alcohol-induced hepatotoxicity

Abstract Background Alcohol-related liver disease (ALD) is characterized by ductular reaction (DR), liver inflammation, steatosis, fibrosis, and cirrhosis. The secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes) regulates liver phenotypes in cholestasis. We evaluated the role of Sct signaling on ALD phenotypes. Methods We used male wild-type and Sct−/− mice fed a control diet (CD) or ethanol (EtOH) for 8 wk. Changes in liver phenotypes were measured in mice, female/male healthy controls, and patients with alcoholic cirrhosis. Since Cyp4a10 and Cyp4a11/22 regulate EtOH liver metabolism, we measured their expression in mouse/human liver. We evaluated: (i) the immunoreactivity of the lipogenesis enzyme elongation of very-long-chain fatty acids 1 (Elovl, mainly expressed by hepatocytes) in mouse/human liver sections by immunostaining; (ii) the expression of miR-125b (that is downregulated in cholestasis by Sct) in mouse liver by qPCR; and (iii) total bile acid (BA) levels in mouse liver by enzymatic assay, and the mRNA expression of genes regulating BA synthesis (cholesterol 7a-hydroxylase, Cyp27a1, 12a-hydroxylase, Cyp8b1, and oxysterol 7a-hydroxylase, Cyp7b11) and transport (bile salt export pump, Bsep, Na+-taurocholate cotransporting polypeptide, NTCP, and the organic solute transporter alpha (OSTa) in mouse liver by qPCR. Results In EtOH-fed WT mice there was increased biliary and liver damage compared to control mice, but decreased miR-125b expression, phenotypes that were blunted in EtOH-fed Sct−/− mice. The expression of Cyp4a10 increased in cholangiocytes and hepatocytes from EtOH-fed WT compared to control mice but decreased in EtOH-fed Sct−/− mice. There was increased immunoreactivity of Cyp4a11/22 in patients with alcoholic cirrhosis compared to controls. The expression of miR-125b decreased in EtOH-fed WT mice but returned at normal values in EtOH-fed Sct−/− mice. Elovl1 immunoreactivity increased in patients with alcoholic cirrhosis compared to controls. There was no difference in BA levels between WT mice fed CD or EtOH; BA levels decreased in EtOH-fed Sct−/− compared to EtOH-fed WT mice. There was increased expression of Cyp27a1, Cyp8b1, Cyp7b1, Bsep, NTCP and Osta in total liver from EtOH-fed WT compared to control mice, which decreased in EtOH-fed Sct−/− compared to EtOH-fed WT mice. Conclusions Targeting Sct/SR signaling may be important for modulating ALD phenotypes

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply