Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Biochemistry; Molecular medicineBioquímica; Medicina molecularBioquímica; Medicina molecularImbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459)

Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit

Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance

Mitochondrial DNA D-loop variants correlate with a primary open-angle glaucoma subgroup

IntroductionPrimary open-angle glaucoma (POAG) is a characteristic optic neuropathy, caused by degeneration of the optic nerve-forming neurons, the retinal ganglion cells (RGCs). High intraocular pressure (IOP) and aging have been identified as major risk factors; yet the POAG pathophysiology is not fully understood. Since RGCs have high energy requirements, mitochondrial dysfunction may put the survivability of RGCs at risk. We explored in buffy coat DNA whether mtDNA variants and their distribution throughout the mtDNA could be risk factors for POAG.MethodsThe mtDNA was sequenced from age- and sex-matched study groups, being high tension glaucoma (HTG, n=71), normal tension glaucoma patients (NTG, n=33), ocular hypertensive subjects (OH, n=7), and cataract controls (without glaucoma; n=30), all without remarkable comorbidities.ResultsNo association was found between the number of mtDNA variants in genes encoding proteins, tRNAs, rRNAs, and in non-coding regions in the different study groups. Next, variants that controls shared with the other groups were discarded. A significantly higher number of exclusive variants was observed in the D-loop region for the HTG group (~1.23 variants/subject), in contrast to controls (~0.35 variants/subject). In the D-loop, specifically in the 7S DNA sub-region within the Hypervariable region 1 (HV1), we found that 42% of the HTG and 27% of the NTG subjects presented variants, while this was only 14% for the controls and OH subjects. As we have previously reported a reduction in mtDNA copy number in HTG, we analysed if specific D-loop variants could explain this. While the majority of glaucoma patients with the exclusive D-loop variants m.72T>C, m.16163 A>G, m.16186C>T, m.16298T>C, and m.16390G>A presented a mtDNA copy number below controls median, no significant association between these variants and low copy number was found and their possible negative role in mtDNA replication remains uncertain. Approximately 38% of the HTG patients with reduced copy number did not carry any exclusive D-loop or other mtDNA variants, which indicates that variants in nuclear-encoded mitochondrial genes, environmental factors, or aging might be involved in those cases.ConclusionIn conclusion, we found that variants in the D-loop region may be a risk factor in a subgroup of POAG, possibly by affecting mtDNA replication

Caracterítzació fisiològica de derivats de Citreicella aestuarii 357 degradadors de naftalé i salicilat

[cat] Els microorganismes degradadors d’hidrocarburs tenen una importància cabdal en problemes de bioremediació d’ambients contaminats per cru de petroli i altres derivats, com per exemple els vessaments marins de petroli. L’explotació de microorganismes amb capacitat degradadora d’hidrocarburs que viuen majoritàriament en ambients marins, com els inclosos en el grup Roseobacter, obri un ventall de possibilitats en la bioremediació d’ambients contaminats per hidrocarburs de diverses estructures. Un exemple d’espècie amb capacitat degradadora és Citreicella aestuarii 357, un microorganisme aïllat a partir d’una mostra de sorra contaminada per petroli obtinguda de la platja de Praia da Seda (Galícia, Espanya) després de l’abocament del petrolier Prestige. 357 presenta un operó catabòlic per a degradació d’hidrocarburs amb l’absència d’un gen regulador que controli aquest metabolisme. En aquest estudi es recerca la millora en la degradació d’hidrocarburs aromàtics mitjançant soques derivades de Citreicella aestuarii 357 modificades genèticament, a les que s’ha introduït una seqüència amb el gen regulador nahR, el promotor Sal i un gen per a resistència a la kanamicina. És recerca la capacitat de degradació millorada de composts com el naftalè i el salicilat, dos composts que li presenten dificultats a la soca salvatge. L’anàlisi dels fenotips de les diferents soques evidencia la possibilitat d’una millora en certes condicions de cultiu en medi Marine Broth i Medi Mineral Marí en diferents quantitats de salicilat i naftalè. Encara així, 357 no es veu millorada significativament respecte a capacitat de creixement i degradació per cap de les 6 soques derivades estudiades, fet que es remarca al genotip, ja que els gens on s’ha produït la inserció no tenen relació significativa amb el metabolisme d’hidrocarburs aromàtics