Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis, by Adrenodoxin Reductase/Ferredoxin.

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in-depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by decreasing NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis-Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half-reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH-AdxR-Yah1p-Coq6p, where Yah1p shuttles electrons between AdxR and Coq6p. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.

FDXR Mutations Cause Sensorial Neuropathies and Expand the Spectrum of Mitochondrial Fe-S-Synthesis Diseases.

Hearing loss and visual impairment in childhood have mostly genetic origins, some of them being related to sensorial neuronal defects. Here, we report on eight subjects from four independent families affected by auditory neuropathy and optic atrophy. Whole-exome sequencing revealed biallelic mutations in FDXR in affected subjects of each family. FDXR encodes the mitochondrial ferredoxin reductase, the sole human ferredoxin reductase implicated in the biosynthesis of iron-sulfur clusters (ISCs) and in heme formation. ISC proteins are involved in enzymatic catalysis, gene expression, and DNA replication and repair. We observed deregulated iron homeostasis in FDXR mutant fibroblasts and indirect evidence of mitochondrial iron overload. Functional complementation in a yeast strain in which ARH1, the human FDXR ortholog, was deleted established the pathogenicity of these mutations. These data highlight the wide clinical heterogeneity of mitochondrial disorders related to ISC synthesis.

ARH1 in Health and Disease.

Arginine-specific mono-adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD)+-dependent, reversible post-translational modification involving the transfer of an ADP-ribose from NAD+ by bacterial toxins and eukaryotic ADP-ribosyltransferases (ARTs) to arginine on an acceptor protein or peptide. ADP-ribosylarginine hydrolase 1 (ARH1) catalyzes the cleavage of the ADP-ribose-arginine bond, regenerating (arginine)protein. Arginine-specific mono-ADP-ribosylation catalyzed by bacterial toxins was first identified as a mechanism of disease pathogenesis. Cholera toxin ADP-ribosylates and activates the α subunit of Gαs, a guanine nucleotide-binding protein that stimulates adenylyl cyclase activity, increasing cyclic adenosine monophosphate (cAMP), and resulting in fluid and electrolyte loss. Arginine-specific mono-ADP-ribosylation in mammalian cells has potential roles in membrane repair, immunity, and cancer. In mammalian tissues, ARH1 is a cytosolic protein that is ubiquitously expressed. ARH1 deficiency increased tumorigenesis in a gender-specific manner. In the myocardium, in response to cellular injury, an arginine-specific mono-ADP-ribosylation cycle, involving ART1 and ARH1, regulated the level and cellular distribution of ADP-ribosylated tripartite motif-containing protein 72 (TRIM72). Confirmed substrates of ARH1 in vivo are Gαs and TRIM72, however, more than a thousand proteins, ADP-ribosylated on arginine, have been identified by proteomic analysis. This review summarizes the current understanding of the properties of ARH1, e.g., bacterial toxin action, myocardial membrane repair following injury, and tumorigenesis.

Genetic epidemiology of autosomal recessive hypercholesterolemia in Sicily: Identification by next-generation sequencing of a new kindred.

BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) is a rare inherited lipid disorder. In Sardinia, differently from other world regions, the mutated allele frequency is high. It is caused by mutations in the low-density lipoprotein receptor adaptor protein 1 gene. Fourteen different mutations have been reported so far; in Sardinia, 2 alleles (ARH1 and ARH2) explain most of the cases. Four ARH patients, all carriers of the ARH1 mutation, have been identified in mainland Italy and 2 in Sicily. OBJECTIVE: The objectives of the study were to improve the molecular diagnosis of familial hypercholesterolemia (FH) and to estimate the frequency of the ARH1 allele in 2 free-living Sicilian populations. METHODS: We sequenced by targeted next-generation sequencing 20 genes related to low-density lipoprotein metabolism in 50 hypercholesterolemic subjects. Subjects from 2 free-living populations from Northern (Ventimiglia Heart Study, 848 individuals) and Southern Sicily (Zabut Zabùt Aging Project, 1717 individuals) were genotyped for ARH1 allele. RESULTS: We identified 1 homozygous carrier of the ARH1 mutation among the 50 hypercholesterolemic outpatients. Population-based genotyping of ARH1 in 2565 subjects allowed the identification of 1 heterozygous carrier. The overall estimated allele frequency of ARH1 in Sicily was 0.0002 (0.02%). CONCLUSIONS: The identification of a new case of ARH in Sicily among 50 clinically diagnosed FH highlights the importance of next-generation sequencing analysis as tool to improve the FH diagnosis. Our results also indicate that ARH1 carrier status is present in ∼1:2500 of Sicilian inhabitants, confirming that ARH is extremely rare outside Sardinia.

Mono-ADP-Ribosylhydrolase Assays.

Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.