1,364 research outputs found
Yeast models for the study of mitochondrial genetic defects and other metabolic disorders
No full textYeast is a versatile tool to study the function of genes involved in mitochondrial processes and in metabolic pathways. A large number of human genes involved in such pathways have yeast orthologues; moreover, yeast is easy to manipulate and it can switch from a fermentative to a respiratory metabolism, thus permitting to study mitochondrial phenotypes. For all these reasons, we employed S. cerevisiae for the functional characterization of different genes involved in inherited metabolic and mitochondrial disorders.
Gyrate atrophy of choroid and retina (GA) is an autosomal recessive disorder caused by mutations at the level of the ornithine aminotransferase (OAT), a mitochondrial matrix protein involved in the ornithine metabolism. We identified a number of missense mutations in OAT and we proved their pathogenicity in a model of S. cerevisiae deleted for CargB, the homologue of OAT. Moreover, analysis of protein stability and residual enzymatic activity permitted us to elucidate the mechanism by which the aminoacid substitution affects the protein function. However, these data did not allow to establish any genotype-phenotype correlation, suggesting that other factors than the specific OAT genotypes are responsible for the phenotypic variability present in the patients.
Yeast Cox23p is a mitochondrial protein with a twin CX9C domain, involved in the COX assembly and possibly in copper homeostasis. In our lab we identified using a bioinformatics approach its human homologue, hCOX23 and characterized its function. We demonstrated using mass spectrometry that the recombinant protein binds Cu(I), providing the first direct evidence of its copper binding. Upon silencing of COX23 we could not observe a phenotype in HeLa cells, but expression of human COX23 in S. cerevisiae deleted for the corresponding gene, showed that it can vicariate for the function of yCox23, confirming its involvement in COX biogenesis. We then demonstrated that it exerts its function in the intermembrane space (IMS) and that Cmc4p, another twin CX9C protein, has overlapping functions. Since the majority of primary COX deficiencies have not a known cause, the characterization of genes involved in the COX biogenesis is of primary importance to find new possible candidates in these disorders.
Coq6p is a monooxygenase involved in the synthesis of CoQ6 in yeast. Recently, point mutations in its human homologue have been associated with steroid resistant nephrotic syndrome (SRNS). We modelled the missense mutations on a yeast strain deleted for Coq6 and we demonstrated that all the human mutations reduce the ability of the human gene to rescue the phenotype of the deleted yeast. Moreover, we mutated the corresponding aminoacid on the yeast gene and we proved that all these allelic combinations retain some residual activity, supporting the notion that complete lack of CoQ biosynthesis is embryonically lethal.
OPA1 is a dynamin related protein mutated in dominant optic atrophy (ADOA), the most common inherited optic neuropathy. It is involved in different processes such as mitochondrial fusion and apoptosis. In human OPA1 is present with 8 different splicing variants, each of them processed to originate a long form, attached to the inner mitochondrial membrane (IMM) and a short, soluble form, localized in the IMS. The processing of OPA1 is strictly regulated, because the ratio between the two forms is important for the different protein functions. Several proteases have been implicated in the processing. To better characterise this mechanism and to understand the role of the specific splicing isoforms, we decided to employ a model of S. cerevisiae deleted for Mgm1, the homologue of OPA1. Expression of single OPA1 splicing variants cannot rescue the phenotype of the deleted strain while a hybrid form of the protein, containing the mitochondrial targeting and the processing sequences of Mgm1, restores the growth of ΔMgm1. These data indicate that the function of the active core of OPA1 is conserved among evolution and the lack of complementation of OPA1 is probably due to a different mechanism of processing in the two organisms. The hybrid gene will represent a simple tool to study the pathogenicity of missense OPA1 mutations identified in patients with ADOA and ADOA plus.
All together these data demonstrate that yeast represent a simple and effective system to characterize the function and to study the pathogenicity of a broad spectrum of conserved proteins such as the ones involved in mitochondrial respiration, mitochondrial morphology or other metabolic pathways.S. cerevisiae è un sistema molto versatile per studiare la funzione dei geni coinvolti in numerose vie mitocondriali e metaboliche. La maggior parte dei geni umani coinvolti in tali processi presentano ortologhi in lievito. Inoltre, questo organismo è facile da manipolare ed è in grado di produrre ATP sia attraverso la glicolisi che attraverso la catena respiratoria, sulla base della fonte di carbonio fornita; tale caratteristica permette lo studio fenotipi mitocondriali.
Per tutte queste ragioni, abbiamo impiegato S. cerevisiae per la caratterizzazione funzionale di geni coinvolti in alcune malattie ereditarie metaboliche e mitocondriali.
L’atrofia girata della retina e della coroide (GA) è una malattia autosomica recessiva causata da mutazioni a livello dell’enzima ornitina aminotransferasi (OAT), una proteina della matrice mitocondriale coinvolta nel metabolismo dell’ornitina. Abbiamo individuato una serie di mutazioni missenso nel gene OAT e ne abbiamo dimostrato la patogenicità in un modello di S. cerevisiae deleto per il gene CargB, l'omologo di OAT. Ulteriori studi sull’analisi della stabilità della proteina e la misurazione dell’attività enzimatica residua hanno permesso di chiarire il meccanismo attraverso il quale le differenti mutazioni missenso influiscono sulla funzione dell’enzima. Tuttavia questi dati non permettono di stabilire alcuna correlazione genotipo-fenotipo, suggerendo che altri fattori oltre la specifica variazione aminoacidica sono responsabili per la variabilità fenotipica osservata nei pazienti.
Cox23p è una proteina di lievito localizzata nei mitocondri e coinvolta nell’ assemblaggio della COX, il complesso IV della catena respiratoria. Possiede il dominio twin CX9C, presente in altre proteine coinvolte nel trasporto del rame. Nel nostro laboratorio abbiamo identificato mediante un approccio bioinformatico il suo omologo umano, hCOX23 e ne abbiamo caratterizzato la funzione. Abbiamo dimostrato con tecniche di spettrometria di massa che la proteina ricombinante lega Cu (I), fornendo la prima prova diretta della sua abilità di legare il rame. Il silenziamento di COX23 in cellule HeLa non ha evidenziato alcun fenotipo. Al contrario, l’espressione del gene umano in un ceppo di lievito deleto per il gene corrispondente, ha dimostrato che COX23 può complementare il fenotipo, confermando il suo coinvolgimento nel processo di assemblaggio della COX. Abbiamo inoltre dimostrato che yCox23p è localizzato nello spazio intermembrana (IMS) e che Cmc4p, un'altra proteina contenente il dominio twin CX9C , ha funzioni rindondanti. Dal momento che la maggior parte dei deficit primari di COX non hanno ancora una causa nota, la caratterizzazione dei geni coinvolti nella biogenesi COX è di primaria importanza per trovare nuovi possibili candidati responsabili di queste patologia.
Coq6p è una monoossigenasi coinvolta nella sintesi di CoQ6 in lievito. Recentemente mutazioni puntiformi nel suo omologo umano sono state associate con la sindrome nefrosica steroido-resistente (SRNS). Abbiamo espresso le mutazioni missenso in un ceppo di lievito deleto per il gene Coq6 e abbiamo dimostrato che tutte le mutazioni riducono o aboliscono la capacità del gene umano di complementare il fenotipo del lievito deleto. Le mutazioni umane sono state successivamente introdotte nei rispettivi residui conservati del gene di lievito. Questo ha permesso di dimostrare che tutte queste combinazioni alleliche presentano una certa attività residua. Tali dati supportano l’ipotesi che la mancanza totale di CoQ biosintesi è letale a livello embrionale.
OPA1 è una proteina mitocondriale coinvolta in diversi processi cellulari tra cui la fusione mitocondriale ed l’apoptosi. Mutazioni a livello di questa proteina causano l’atrofia ottica dominante (ADOA), la più comune neuropatia ottica ereditaria. Nell’uomo il gene OPA1 è presente in 8 differenti varianti di splicing, ognuna delle quali può originare una forma lunga, attaccata alla membrana mitocondriale interna (IMM) e una forma solubile, localizzata nel IMS. Il processamento di OPA1 è strettamente regolato, in quanto il rapporto tra le due forme è importante per le funzioni della stessa. Numerose proteasi sono state indicate come coinvolte in tale processo. Per caratterizzare questo meccanismo e per comprendere il ruolo specifico di ciascuna delle isoforme di splicing, abbiamo deciso di impiegare un modello di S. cerevisiae deleto per Mgm1, l'omologo di OPA1. L’espressione delle singole varianti di splicing non è in grado di ripristinare la crescita del ceppo deleto mentre una forma ibrida della proteina, contenente la sequenza di import mitocondriale e di processamento di Mgm1, permette il recupero del fenotipo di ΔMgm1. Questi dati indicano che la funzione di OPA1 è conservata e la mancanza di complementazione di OPA1 è probabilmente dovuta ad un differente meccanismo di processamento nel lievito rispetto all’uomo. Il gene ibrido rappresenterà un semplice strumento per studiare la patogenicità di missenso OPA1 mutazioni identificate nei pazienti con ADOA e ADOA plus.
Nel complesso questi dati dimostrano che il lievito rappresenta un sistema semplice ed efficace per caratterizzare la funzione e per studiare la patogenicità di un ampio spettro di proteine coinvolte nei processi di respirazione, morfologia mitocondriale e in altre vie metaboliche
Molecular Genetics of Argininosuccinic Aciduria
No full textArgininosuccinic aciduria is an autosomal recessive disorder of the urea cycle caused by mutations in argininosuccinate
lyase (ASL). Two main clinical phenotypes are reported: an acute neonatal form characterised by severe hyperammonaemia and coma, and a subacute, late-onset form which may present with relatively milder neurological
symptoms.
More than 120 ASL mutations have been reported so far: the majority are missense, but virtually all types of point mutations are found. Large rearrangements are rare and standard genomic deoxyribonucleic acid (DNA) analysis has a high diagnostic yield. Genotype–phenotype correlations have been difficult to establish as standard biochemical techniques are not sufficiently sensitive to measure residual activity, and other factors such as intragenic complementation, overexpression of nonfunctional ASL transcripts and environmental factors may modulate the phenotype.
Clinical manifestations result from the block in the urea cycle and also from impairment of nitric oxide biosynthesis, and the therapy is aimed at restoring these two functions
mtDNA replication, maintenance, and nucleoid organization
No full textPart of the genetic information in human cells resides in the mitochondria. Faithful maintenance of mitochondrial deoxyribonucleic acid (mtDNA) is crucial for the oxidative phosphorylation system that produces the majority of the cellular ATP, and therefore to life. This chapter provides an introduction into the characteristics of human mtDNA and summarizes the processes and factors required for the replication and maintenance of this small but essential genome. We also describe the organization of mtDNA in specialized nucleoprotein structures called nucleoids. Where applicable, we refer to human disease states that are caused by defects in the described factors or processes.</p
Yeast complementation is sufficiently sensitive to detect the residual activity of ASL alleles associated with mild forms of argininosuccinic aciduria
No full textDemonstration that functional complementaiton in yeast can detect residual activity in hypomorphic ASL alleles and provide phenotype-genotype correlations for this disorder
Primary Coenzyme Q10 Deficiency
No full textCLINICAL CHARACTERISTICS:
Primary coenzyme Q10 (CoQ10) deficiency is usually associated with multisystem involvement, including neurologic manifestations such as fatal neonatal encephalopathy with hypotonia; a late-onset slowly progressive multiple-system atrophy-like phenotype (neurodegeneration with autonomic failure and various combinations of parkinsonism and cerebellar ataxia, and pyramidal dysfunction); and dystonia, spasticity, seizures, and intellectual disability. Steroid-resistant nephrotic syndrome (SRNS), the hallmark renal manifestation, is often the initial manifestation either as isolated renal involvement that progresses to end-stage renal disease (ESRD), or associated with encephalopathy (seizures, stroke-like episodes, severe neurologic impairment) resulting in early death. Hypertrophic cardiomyopathy (HCM), retinopathy or optic atrophy, and sensorineural hearing loss can also be seen.
DIAGNOSIS/TESTING:
The diagnosis of primary CoQ10 deficiency in a proband is established by identification of biallelic pathogenic variants in one of the nine genes encoding proteins directly involved in the synthesis of coenzyme Q10 or by detection of reduced levels of CoQ10 (ubiquinone) in skeletal muscle or reduced activities of complex I+III and II+III of the mitochondrial respiratory chain on frozen muscle homogenates.
MANAGEMENT:
Treatment of manifestations: In individuals with primary CoQ10 deficiency early treatment with high-dose oral CoQ10 supplementation (ranging from 5 to 50 mg/kg/day) can limit disease progression and reverse some manifestations; however, established severe neurologic and/or renal damage cannot be reversed. ACE inhibitors may be used in combination with CoQ10 supplementation in persons with proteinuria; renal transplantation is an option for those with ESRD. Treatment of hypertrophic cardiomyopathy, retinopathy, and sensorineural hearing loss is per usual practice. Prevention of primary manifestations: Supplementation with high-dose oral CoQ10 can prevent progression of the renal disease and onset of neurologic manifestations. Surveillance: Periodic neurologic evaluation, urine analysis (for proteinuria) and renal function tests, ophthalmologic evaluation, and audiometry. Evaluation of relatives at risk: Presymptomatic diagnosis for the purpose of early treatment with CoQ10 supplementation is warranted for relatives at risk.
GENETIC COUNSELING:
Primary coenzyme Q10 deficiency is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk relatives, prenatal testing for pregnancies at increased risk, and preimplantation genetic diagnosis are possible if the pathogenic variants in a family are known
Genetics of coenzyme q10 deficiency.
No full textCoenzyme Q10 (CoQ10) is an essential component of eukaryotic cells and is involved in crucial biochemical reactions such as the production of ATP in the mitochondrial respiratory chain, the biosynthesis of pyrimidines, and the modulation of apoptosis. CoQ10 requires at least 13 genes for its biosynthesis. Mutations in these genes cause primary CoQ10 deficiency, a clinically and genetically heterogeneous disorder. To date mutations in 8 genes (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) have been associated with CoQ10 deficiency presenting with a wide variety of clinical manifestations. Onset can be at virtually any age, although pediatric forms are more common. Symptoms include those typical of respiratory chain disorders (encephalomyopathy, ataxia, lactic acidosis, deafness, retinitis pigmentosa, hypertrophic cardiomyopathy), but some (such as steroid-resistant nephrotic syndrome) are peculiar to this condition. The molecular bases of the clinical diversity of this condition are still unknown. It is of critical importance that physicians promptly recognize these disorders because most patients respond to oral administration of CoQ10
Genetic bases and clinical manifestations of coenzyme Q10 (CoQ 10) deficiency.
No full textCoenzyme Q(10) is a remarkable lipid involved in many cellular processes such as energy production through the mitochondrial respiratory chain (RC), beta-oxidation of fatty acids, and pyrimidine biosynthesis, but it is also one of the main cellular antioxidants. Its biosynthesis is still incompletely characterized and requires at least 15 genes. Mutations in eight of them (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) cause primary CoQ(10) deficiency, a heterogeneous group of disorders with variable age of onset (from birth to the seventh decade) and associated clinical phenotypes, ranging from a fatal multisystem disease to isolated steroid resistant nephrotic syndrome (SRNS) or isolated central nervous system disease. The pathogenesis is complex and related to the different functions of CoQ(10). It involves defective ATP production and oxidative stress, but also an impairment of pyrimidine biosynthesis and increased apoptosis. CoQ(10) deficiency can also be observed in patients with defects unrelated to CoQ(10) biosynthesis, such as RC defects, multiple acyl-CoA dehydrogenase deficiency, and ataxia and oculomotor apraxia.Patients with both primary and secondary deficiencies benefit from high-dose oral supplementation with CoQ(10). In primary forms treatment can stop the progression of both SRNS and encephalopathy, hence the critical importance of a prompt diagnosis. Treatment may be beneficial also for secondary forms, although with less striking results.In this review we will focus on CoQ(10) biosynthesis in humans, on the genetic defects and the specific clinical phenotypes associated with CoQ(10) deficiency, and on the diagnostic strategies for these conditions
Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina
No full textGyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs
The ethical and moral-based dimension of leadership in CSR-oriented strategies and sustainable entrepreneurship
No full textThis chapter addresses the theme of leadership and its influence on corporate social
responsibility (CSR) and sustainability-oriented strategies. Specifically, it aims to analyse,
through a deductive and literature-based approach, the relevance of the ethical and moral
leadership models with respect to the diffusion of CSR and sustainable entrepreneurship. Linking ethical and moral-based leadership to the CSR and sustainable entrepreneurship discourse allows us to point out the relevance of an authentic orientation in supporting
change and fostering sustainable entrepreneurship The theoretical construct of responsible and sustainable leadership derives from the
intersection of the moral-based leadership concepts with those of CSR and sustainable
entrepreneurship..Drawing from these premises, this chapter seeks to identify leadership models and
attributes consistent with (and necessary to develop) an authentic CSR-oriented strategy
and able to foster sustainable entrepreneurship.
Accordingly, in this chapter, we argue that leadership represents a key aspect that
warrants more research within both CSR and sustainable entrepreneurship studies. The
research questions that orients this study can be summarised as follows: Which leadership
models favour authentic CSR practices? Are moral, ethical-based and virtues-based models
of leadership effective in developing CSR and fostering sustainable entrepreneurship?
These questions guide the critical review of the different leadership approaches, bringing
attention to the models that are most coherent in regard to the actual socio-economic
context which requires managers and entrepreneurs to govern the internal and external
complexity and actively contribute to sustainability. The methodological approach is
mainly based on a literature review that surveys critical points in current literature that is
relevant to the topic.
The work is structured as follows. First, we offer an analysis of the leadership theoretical
framework in the context of the CSR debate. We begin with a brief methodological
note, followed by an analysis of the antecedents of CSR and sustainable entrepreneurship
in terms of values and virtues.
Second, a review of relevant literature on leadership approaches and models consistent
with the CSR and sustainable entrepreneurship discourse is presented, emphasising the relationship between transformational, moral and virtues-based leadership and
CSR-oriented strategies and sustainable entrepreneurship. Drawing from the analysis,
four main propositions are introduced. The final sections illustrate the propositions and
summarise the implications and limitations of the study
A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
No full textThe role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1
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