Pathophysiology of Primary Coenzyme Q10 Deficiencies. Molecular Characterisation of COQ4 gene.

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Experimentación en Enfermedades RarasClave Programa: DBICódigo Línea: 14Las deficiencias primarias de coenzima Q10 (CoQ10) son un grupo de enfermedades raras genéticas causadas por mutaciones bialélicas recesivas en uno de los genes COQ requeridos en la ruta de biosíntesis de CoQ10, a nivel enzimático o regulador. Las manifestaciones clínicas asociadas son muy heterogéneas y afectan principalmente al sistema nervioso central y periférico, a los riñones, al músculo esquelético y al corazón. La primera parte de esta tesis se centra en el estudio del síndrome de la deficiencia primaria en CoQ10. En primer lugar, hemos realizado una revisión actualizada y exhaustiva de todas las manifestaciones clínicas asociadas a cada una de las variantes patogénicas en los genes COQ descritas en la literatura. En ella, describimos patrones de síntomas relacionados con la edad de aparición de la enfermedad para cada gen COQ e incluso, para cada mutación, cuando es posible. Con estos patrones, hemos intentado establecer correlaciones genotipo-fenotipo para la deficiencia primaria en CoQ10. En segundo lugar, hemos descrito nuevos casos de deficiencia primaria de CoQ10 debido a mutaciones en los genes COQ4 y COQ7, ampliando los fenotipos y genotipos asociados a estos trastornos. Hemos trabajado con diferentes modelos celulares in vitro con mutaciones en COQ4, COQ6 y COQ7, que acumulan intermediarios diagnósticos, específicos para cada defecto COQ. Además, demostramos que el tratamiento con análogos del 4-hidroxibenzoato (4-HB) es eficaz para mejorar la deficiencia de CoQ10 y el defecto respiratorio en nuestros modelos celulares humanos: el ácido 2,4-dihidroxibenzoico (2,4-dHB) para la deficiencia en COQ7 y el ácido vainillínico (VA) para los defectos en COQ6. En la segunda parte de la tesis, nos hemos centrado en la proteína COQ4 humana, que tiene un papel esencial en la biosíntesis de CoQ10. En nuestro trabajo, demostramos que la proteína COQ4 humana es esencial para la biosíntesis de CoQ10 en las células. Hemos generado una línea celular humana KO en COQ4, que tiene una gran deficiencia en CoQ10 y en la respiración mitocondrial. Estos defectos son rescatados con un tratamiento con CoQ10 o con la expresión del gen COQ4 WT. Además, la falta de proteína COQ4 produce la acumulación de un intermediario específico de la ruta de síntesis. También encontramos que la mayoría de las mutaciones descritas en pacientes son hipomórficas, capaces de recuperar completamente la biosíntesis de CoQ10 en las células COQ4 KO cuando son sobreexpresadas. Con estudios de proteómica, hemos podido identificar todas las proteínas COQ (excepto COQ8A) copurificando con COQ4, lo que es una evidencia más de la existencia del complejo de síntesis de CoQ en mamíferos. Además, observamos que la falta de proteínas COQ4 o COQ6 altera los niveles de otras proteínas COQs, probablemente modificando la estabilidad del complejo biosintético. Se ha descrito que las enzimas modificadoras de la cabeza de CoQ en levadura resuelven en loci discretos, denominados dominios CoQ, que se encuentran adyacentes a los sitios de contacto entre el retículo endoplásmico (ER) y las mitocondrias 1,2. Además, el mantenimiento y la distribución del ADN mitocondrial (mtDNA) depende de estos contactos ER-mitocondria 3, y los niveles de colesterol en estos sitios parecen ser clave para ello 4. En la última parte de la tesis, hemos explorado estas relaciones en nuestro modelo COQ4 KO. La falta de COQ4 produce una ligera disminución del número de copias de mtDNA, sin efecto sobre la transcripción de mtDNA y un efecto muy sutil sobre la traducción de proteínas codificadas por el mtDNA. Con respecto a la replicación del mtDNA, la falta de COQ4 o COQ6 induce una tasa de recuperación más rápida del mtDNA después de una depleción inducida. Para encontrar el mecanismo molecular de este sorprendente fenotipo, hemos estudiado el contenido de colesterol y la distribución de proteínas de los nucleoides en gradientes de densidad. Los niveles de colesterol total parecen no estar alterados en las mitocondrias de las células COQ4 KO. Sin embargo, hemos encontrado diferencias sutiles entre COQ4 KO y las células control en la distribución de proteínas de los nucleoides (TFAM y ATAD3) en estos gradientes. Este hecho podría estar detrás de la rápida recuperación del mtDNA después de la depleción inducida. Sin embargo, todavía queda mucho trabajo por hacer para comprender mejor la relación entre la biosíntesis de CoQ y el metabolismo del mtDNA.Universidad Pablo de Olavide de Sevilla. Departamento de Fisiología, Anatomía y Biología Celula

Role of COQ4 on mitochondrial DNA maintenance

Resumen del póster presentado en Mitochondrial Medicine, celebrado en Hinxton (Inglaterra) del 09 al 11 de mayo de 2018.Coenzyme Q (CoQ) is a lipidic molecule composed by a hydroquinone head and an isoprenoid chain. Since its discovery, several functions have been assigned to CoQ, being the transfer of electrons from complexes I and II to complex III in the mitochondrial respiratory chain the best known. CoQ also receives electrons from other dehydrogenases involved in different cellular processes and it is a potent membrane antioxidant. CoQ is endogenously synthesized by a set of enzymes forming a biosynthetic complex in the mitochondrial inner membrane, which has been mostly studied in yeast models. Defects in any of the genes coding for these proteins result in reduced levels of CoQ and, consequently, defects in energy production. COQ4 is one of the proteins involved in CoQ biosynthesis, but its exact enzymatic activity is still unknown. COQ4 KO HEK 293T-Rex/Flp-In cells generated by CRISPR/Cas9, as well as patient fibroblasts carrying mutations in COQ4 show the accumulation of a yet uncharacterised biosynthetic intermediate that lacks redox activity. Two candidate molecules have emerged from mass spectrometry analysis performed to identify this intermediate. On the other hand, the KO cells show a surprising phenotype related to mtDNA metabolism which may be due either to the lack of de novo synthesis of CoQ, to the biosynthetic complex instability itself, to the presence of the intermediate, or to a different and yet not characterized role of COQ4. Altogether, these results indicate a possible double function of the COQ4 protein

Primary Coenzyme Q deficiencies: A literature review and online platform of clinical features to uncover genotype-phenotype correlations

Primary Coenzyme Q (CoQ) deficiencies are clinically heterogeneous conditions and lack clear genotypephenotype correlations, complicating diagnosis and prognostic assessment. Here we present a compilation of all the symptoms and patients with primary CoQ deficiency described in the literature so far and analyse the most common clinical manifestations associated with pathogenic variants identified in the different COQ genes. In addition, we identified new associations between the age of onset of symptoms and different pathogenic variants, which could help to a better diagnosis and guided treatment. To make these results useable for clinicians, we created an online platform (https://coenzymeQbiology.github. io/clinic-CoQ-deficiency) about clinical manifestations of primary CoQ deficiency that will be periodically updated to incorporate new information published in the literature. Since CoQ primary deficiency is a rare disease, the available data are still limited, but as new patients are added over time, this tool could become a key resource for a more efficient diagnosis of this pathology.Centro Andaluz de Biología del DesarrolloCIBERE

Study of the genotype-phenotype correlation in fibroblasts of patients with mutations in COQ4

Motivation: Coenzyme Q10 (CoQ10), or ubiquinone, has a crucial role in the energetic metabolism due to its redox capacity in the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. Lack of this essential component leads to mitochondrial disorders characterized by a rare condition with a huge spectrum of different phenotypes and different genetic mutations. To date, specific genotype-phenotype correlations do not exist because the link between specific genetic defects and phenotypes is unclear. In this way, the diagnosis and treatment of this patients is so complicated. The diagnosis on time is extremely important to start the treatment in order to avoid the fulminant course of the disease with an irreversible damage and a fatal outcome. In a previous study reported in our lab it was described that CoQ10-deficient fibroblasts (independently from the etiology) showed a common transcriptomic profile. The expression of certain genes was modified in the same way, that it to say, they were always increased or decreased. COQ4 is one of the genes involved in CoQ10 biosynthesis. It has been also demonstrated that COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency. The aim of this work is to find a possible correlation between different COQ4 mutations, the pathological phenotype, the clinical severity of the disease and the level of markers genes expression.Methods: To achieve this goal, we have used two fibroblast cell lines as control, and four mutant fibroblast cell lines from patients with different COQ4 mutations. We have performed different genetic and biochemical assays such as analysis of the expression profile by microarray, Seahorse or flow cytometry.Results: Fibroblasts from subjects with COQ4 mutations show similar profiles between them, and at the same time, they show a different profile when we compared them to control fibroblasts. In adittion, a more differentiated profile is observed according as the severity of the symptoms increases. Taken together, these results suggest a correlation between the phenotype and the clinical severity.Conclusions: Cells with COQ4 mutations look for an adaptative biological response to face the mithocondrial damage due to the CoQ10 deficiency. And interestingly, our results suggest an association between genothype and phenotype in these patients

Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells

We thank the core facilities at GENYO for excellent technical support. We also thank the genomics unit at the CRG for assistance with RNA-seq and ChIP-seq experiments. The Landeira lab is supported by the Spanish ministry of science and innovation (PID2019-108108-100, EUR2021- 122005), the Andalusian regional government (PIER-0211-2019, PY20_00681) and the University of Granada (A-BIO-6-UGR20) grants. Research in the Klose lab is supported by the Wellcome Trust (209400/ Z/17/Z) and the European Research Council (681440). A.F. was sup- ported by a Sir Henry Wellcome Post-doctoral fellowship (110286/Z/15/ Z). Work in the Rada-Iglesias lab is funded by the Ministerio de Ciencia e Innovación, the Agencia Española de Investigación and the European Regional Development Fund (PGC2018-095301-B-I00 and RED2018- 102553-T); by the European Research Council (862022); and by the European Commission (H2020-MSCA-ITN-2019-860002).The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-35859-9The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimu- lation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to develop- mental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.Ministry of Science and Innovation, Spain (MICINN) Spanish Government PID2019-108108-100, EUR2021-122005Andalusian regional government PIER-0211-2019, PY20_00681University of Granada A-BIO-6-UGR20Wellcome Trust 209400/Z/17/ZEuropean Research Council (ERC) European Commission 862022Wellcome Trust PGC2018-095301-B-I00Ministry of Science and Innovation, Spain (MICINN) Instituto de Salud Carlos III Spanish GovernmentEuropean Commission RED2018-102553-T, H2020-MSCA-ITN-2019-860002European Commission European Commission Joint Research Centre 681440Agencia Española de Investigación110286/Z/15/

Secondary CoQ10 deficiency, bioenergetics unbalance in disease and aging

Coenzyme Q10 (CoQ10) deficiency is a rare disease characterized by a decreased accumulation of CoQ10 in cell membranes. Considering that CoQ10 synthesis and most of its functions are carried out in mitochondria, CoQ10 deficiency cases are usually considered a mitochondrial disease. A relevant feature of CoQ10 deficiency is that it is the only mitochondrial disease with a successful therapy available, the CoQ10 supplementation. Defects in components of the synthesis machinery caused by mutations in COQ genes generate the primary deficiency of CoQ10. Mutations in genes that are not directly related to the synthesis machinery cause secondary deficiency. Cases of CoQ10 deficiency without genetic origin are also considered a secondary deficiency. Both types of deficiency can lead to similar clinical manifestations, but the knowledge about primary deficiency is deeper than secondary. However, secondary deficiency cases may be underestimated since many of their clinical manifestations are shared with other pathologies. This review shows the current state of secondary CoQ10 deficiency, which could be even more relevant than primary deficiency for clinical activity. The analysis covers the fundamental features of CoQ10 deficiency, which are necessary to understand the biological and clinical differences between primary and secondary CoQ10 deficiencies. Further, a more in-depth analysis of CoQ10 secondary deficiency was undertaken to consider its origins, introduce a new way of classification, and include aging as a form of secondary deficiency.Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, Grant/Award Numbers: UPO-1259581, UPO-126247, UPO-1265673; Instituto de Salud Carlos III, Grant/Award Number: PI17/01286; Ministerio de Educación, Cultura y Deporte, Grant/Award Numbers: FPU14/04873, FPU16/0326

Fisiopatología de las Deficiencias Primarias de Coenzima Q10. Caracterización Molecular del Gen COQ4

Tesis doctoral.-- Universidad Pablo de Olavide. Programa de Doctorado en Biotecnología, Ingeniería y Tecnología Química (RD: 99/2011)[EN]: Primary Coenzyme Q10 (CoQ10) deficiencies are a group of rare conditions genetically caused by autosomal recessive biallelic mutations in one of the COQ genes required in CoQ10 biosynthesis pathway. The associated clinical manifestations are highly heterogeneous and mainly affect central and peripheral nervous system, kidney, skeletal muscle and heart. The first part of this thesis is focused on studying the primary CoQ10 deficiency syndrome. Firstly, we present an updated and comprehensive review of all the clinical manifestations associated to each of the pathogenic variantsin the COQgenes described in the literature. With this, we describe patterns of symptoms related with the age of onset of the disease for each COQ gene and even, when possible, for each mutation involved in primary CoQ10 deficiency, as an attempt to establish genotype-phenotype correlations.[ES]: Las deficiencias primarias de coenzima Q10 (CoQ10) son un grupo de enfermedades raras genéticas causadas por mutaciones bialélicas recesivas en uno de los genes COQ requeridos en la ruta de biosíntesis de CoQ10, a nivel enzimático o regulador. Las manifestaciones clínicas asociadas son muy heterogéneas y afectan principalmente al sistema nervioso central y periférico, a los riñones, al músculo esquelético y al corazón. La primera parte de esta tesis se centra en el estudio del síndrome de la deficiencia primaria en CoQ10

Clinical syndromes associated with Coenzyme Q10 deficiency

Primary Coenzyme Q deficiencies represent a group of rare conditions caused by mutations in one of the genes required in its biosynthetic pathway at the enzymatic or regulatory level. The associated clinical manifestations are highly heterogeneous and mainly affect central and peripheral nervous system, kidney, skeletal muscle and heart. Genotype–phenotype correlations are difficult to establish, mainly because of the reduced number of patients and the large variety of symptoms. In addition, mutations in the same COQ gene can cause different clinical pictures. Here, we present an updated and comprehensive review of the clinical manifestations associated with each of the pathogenic variants causing primary CoQ deficiencies.MA-F is a predoctoral research fellow from the Spanish Ministry of Education, Culture and Sports (FPU14/04873). ET is supported by Grant number CPDA140508/14 from University of Padova

Coenzyme Q biosynthesis and its role in the respiratory chain structure

Coenzyme Q (CoQ) is a unique electron carrier in the mitochondrial respiratory chain, which is synthesized on-site by a nuclear encoded multiprotein complex. CoQ receives electrons from different redox pathways, mainly NADH and FADH from tricarboxylic acid pathway, dihydroorotate dehydrogenase, electron transfer flavoprotein dehydrogenase and glycerol-3-phosphate dehydrogenase that support key aspects of the metabolism. Here we explore some lines of evidence supporting the idea of the interaction of CoQ with the respiratory chain complexes, contributing to their superassembly, including respirasome, and its role in reactive oxygen species production in the mitochondrial inner membrane. We also review the current knowledge about the involvement of mitochondrial genome defects and electron transfer flavoprotein dehydrogenase mutations in the induction of secondary CoQ deficiency. This mechanism would imply specific interactions coupling CoQ itself or the CoQ-biosynthetic apparatus with the respiratory chain components. These interactions would regulate mitochondrial CoQ steady-state levels and function.The Institute of Health Carlos III of the Spanish Ministry of Health has funded this work (FIS grant PI14-01962). M A-F is a recipient of a predoctoral award from the Spanish Ministry of Education, Culture and Sports.Peer Reviewe

Coenzyme Q Biosynthesis Disorders

Coenzyme Q (CoQ) is a lipidic molecule that transfers electrons between complexes I and II to complex III in the mitochondrial respiratory chain. It is also essential for processes mediated by other mitochondrial dehydrogenases, such as those involved in pyrimidine nucleotides biosynthesis, beta-oxidation and sulfide biosynthesis. A nuclear-encoded multiprotein complex at the inner mitochondrial membrane drives CoQ biosynthesis, which requires at least 13 proteins, leastways in yeasts. Mutations in the genes (COQ genes) coding for these proteins cause a decrease of CoQ biosynthesis rate leading to primary CoQ deficiency, a very heterogeneous group of mitochondrial diseases affecting different tissues and organs, and showing variable severity and age of onset. In general, this primary condition shows a good response to the supplementation with high doses of CoQ, but early diagnosis is compulsory to limit tissue damage. However, sometimes effectiveness is reduced, possibly due to its low bioavailability and, probably, difficulties crossing the blood-brain barrier. Secondary CoQ deficiency is a more common condition, in which defects of diverse mitochondrial processes induce an adaptive CoQ decrease. Secondary deficiency can be caused by oxidative phosphorylation (OXPHOS) defects, such as complex III dysfunction or mitochondrial DNA (mtDNA) depletion, or even non-OXPHOS mitochondrial defects. Here, we review the current knowledge of CoQ biosynthesis pathway, the genetic defects leading to primary deficiency and those conditions in which mitochondrial defects cause secondary deficiency