Biology subjects

Rua, A. J.

Publications and source records attributed to Rua, A. J..

3 recordsLinked to original sources

bioRxiv2024

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a {Delta}taz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a {Delta}crd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and -ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

biophysics↗

bioRxiv2024

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal-ligand H23 found in odd-numbered zinc-fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711 it has been thought that Z7 is probably non-functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal ligand. The Z7 domain adopts a stable tertiary structure upon metal binding. The NMR structure of Zn2+-bound Z7 shows the classical {beta}{beta}-fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal-binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring-flipping of phenyalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the Kd values for Zn2+ and Co2+ and retain the high thermal stability of the WT domain above 80 {degrees}C. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4-nitophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors. Our findings that Z7 can fold with only a subset of three metal ligands suggests the recent view that most everything about protein structure can be predicted through homology modeling might be premature for at least the resilient and versatile zinc-finger motif.

biophysics↗

bioRxiv2023

NMR structure verifies the eponymous degenerate zinc finger domain of transcription factor ZNF750

ZNF750 is a nuclear transcription factor that activates skin differentiation and has tumor suppressor roles in several cancers. Unusually, ZNF750 has only a single zinc-finger (ZNF) domain, Z*, with an amino acid sequence that differs markedly from the CCHH family consensus. Because of its sequence differences Z* is classified as degenerate, presumed to have lost the ability to bind the zinc ion required for folding. AlphaFold predicts an irregular structure for Z* with low confidence. Low confidence predictions are often inferred to be intrinsically disordered regions of proteins, which would be the case if Z* did not bind Zn2+. We use NMR and CD spectroscopy to show that a 25-51 segment of ZNF750, corresponding to the Z* domain, folds into a well-defined antiparallel {beta}{beta} tertiary structure with a pM dissociation constant for Zn2+, and a thermal stability >80 {degrees}C. Of three alternative Zn2+ ligand sets, Z* uses a CCHC rather than the expected CCHH motif. The switch in the last ligand maintains the folding topology and hydrophobic core of the classical ZNF motif. CCHC ZNFs are associated with protein-protein interactions but Z* binds DNA. Since the metal chelating site is on the other side of the molecule, it suggests functional preferences are a result of divergent evolution rather than physical constraints on the structure. The structure of Z* provides a context for understanding the domains DNA-binding properties and mutations associated with cancers. We expect other ZNFs currently classified as degenerate, are CCHC-type structures like Z*.

molecular biology↗