Characterizing the transcriptional regulation of let-721, a Caenorhabditis elegans homolog of human electron flavoprotein dehydrogenase

Abstract

LET-721 is the Caenorhabditis elegans ortholog of electron-transferring flavoprotein dehydrogenase (ETFDH). We are studying this protein in C. elegans in order to establish a tractable model system for further exploration of ETFDH structure and function. ETFDH is an inner mitochondrial membrane localized enzyme that plays a key role in the beta-oxidation of fatty acids and catabolism of amino acids and choline. ETFDH accepts electrons from at least twelve mitochondrial matrix flavoprotein dehydrogenases via an intermediate dimer protein and transfers the electrons to ubiquinone. In humans, ETFDH mutations result in the autosomal recessive metabolic disorder, multiple acyl-CoA dehydrogenase deficiency. Mutants of let-721 in C. elegans are either maternal effect lethals or semi-sterile. let-721 is transcribed in the pharynx, body wall muscle, hypoderm, intestine and somatic gonad. In addition, the subcellular localization of LET-721 agrees with predictions that it is localized to mitochondria. We identified and confirmed three cis-regulatory sequences (pha-site, rep-site, and act-site). Phylogenetic footprinting of each site indicates that they are conserved between four Caenorhabditis species. The pha-site mapped roughly 1,300 bp upstream of let-721's translational start site and is necessary for expression in pharyngeal tissues. The rep-site mapped roughly 830 bp upstream of the translational start site and represses expression of LET-721 within pharyngeal tissues. The act-site mapped roughly 800 bp upstream of the translational start site and is required for expression within spermatheca, body wall muscle, pharynx, and intestine. Taken together, we find that LET-721 is a mitochondrially expressed protein that is under complex transcriptional controls.

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad‐1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission

“…A phenotypical aspect correlated to altered beta-oxidation in C. elegans is a defect in fertility. 23,27 Consistently, in flad-1 silenced animals we registered an impaired ability to lay eggs in both fed 21 and starved conditions (Figure 3). To prove that the phenotypical defect induced by flad-1 silencing consists in eggs production, rather than in eggs deposition, we analysed the response of both control and F I G U R E 1 Silencing of flad-1 gene.…”

“…The suggested neuronal neurotransmitter deprivation is, nevertheless, superimposable to beta-oxidation impairments, predictably affecting muscular energy production, fertility, and egg laying. 23 Consistently, in interfered animals, externally added acetylcholine produces beneficial effects similar to that of flavins on locomotion behavior.…”

“…A defect in neuro‐mediator synthesis/release, rather than muscular reception, was definitively assessed on the bases of a retained sensitivity to levamisole of both egg laying and locomotion behavior. The suggested neuronal neurotransmitter deprivation is, nevertheless, superimposable to beta‐oxidation impairments, predictably affecting muscular energy production, fertility, and egg laying 23 …”

“…Among the potential “cognates” of FAD synthase, of pivotal importance in bioenergetics, the product of human ETFDH gene associated to MADD deserves deep attention. Interestingly enough, most of the residues mutated in human ETFDH gene in MADD are conserved in the C. elegans orthologue let‐721 (WBGene00002855), 23 whose only product LET‐721 (CE29662, Wormbase), shows 63% identity and 77% similarity (performed by Blastp) with the human corresponding mitochondrial precursor protein (Q16134‐1, in Uniprot). Low levels of flavin cofactors may cause ETFDH instability or altered folding, in turn triggering mitochondrial protein unfolding response (mtUPR) in mammals 24,25 …”

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy

“…Caenorhabditis elegans let-721 let-721 mutants are maternal effects either lethal or semi-sterile. [192]…”