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1.
Mol Cell ; 79(6): 1051-1065.e10, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32877643

RESUMO

Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética
2.
Proc Natl Acad Sci U S A ; 117(37): 23165-23173, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868448

RESUMO

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.


Assuntos
Genes Bacterianos/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Substituição de Aminoácidos/genética , Escherichia coli/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Poliproteínas/genética , Proteômica/instrumentação
3.
Mol Cell ; 79(5): 708-709, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888436

RESUMO

The collaborative work of two HHMI groups, one at the University of Washington and the other at the Broad Institute of MIT and Harvard, led to the development of a novel molecular tool to edit single bases in the mtDNA (Mok et al., 2020).


Assuntos
Citidina Desaminase , DNA Mitocondrial , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mitocôndrias/genética
4.
Elife ; 92020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32959780

RESUMO

Experiments on mitochondrial DNA in worms highlight that cheating does not always pay off.


Assuntos
Genoma Mitocondrial , DNA Mitocondrial , Mitocôndrias/genética , Dinâmica Mitocondrial , Nutrientes
5.
Nucleic Acids Res ; 48(15): 8704-8723, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738044

RESUMO

Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3' to 5' progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mitocondrial/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Proteínas de Protozoários/genética , Edição de RNA/genética , RNA Guia/genética , Trypanosoma brucei brucei/metabolismo
6.
Nat Commun ; 11(1): 4281, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855416

RESUMO

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.


Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
PLoS Genet ; 16(7): e1008923, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735630

RESUMO

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1LV1, RCC1LV2 and RCC1LV3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1LV1 and RCC1LV3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1LV2. Overexpression and silencing of RCC1LV1 or RCC1LV3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas Mitocondriais/genética , Isoformas de Proteínas/genética , Proteínas Ribossômicas/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Biossíntese de Proteínas/genética , RNA/genética , Proteínas de Ligação a RNA/genética
8.
Nat Commun ; 11(1): 4056, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792483

RESUMO

Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid ß-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia/metabolismo , Mitocôndrias/metabolismo , Animais , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citometria de Fluxo , Humanos , Leucemia/genética , Leucemia Mieloide Aguda/patologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipogênese/genética , Lipogênese/fisiologia , Camundongos , Mitocôndrias/genética , Oxirredução , Fosforilação Oxidativa
9.
Am J Hum Genet ; 107(3): 514-526, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32791035

RESUMO

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento Completo do Exoma
10.
Gene ; 761: 145047, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32783993

RESUMO

Mitochondrial DNA (mtDNA) copy number and mitochondrial DNA haplogroups have been associated with different types of cancer, including breast cancer, because they alter cellular energy metabolism. However, whether mtDNA copy number or haplogroups are predictors of oxidative stress-related risks in human breast cancer tissue in Mexican patients remains to be determined. Using quantitative real-time PCR assays and sequencing of the mtDNA hypervariable region, analysis of mtDNA copy numbers in 82 breast cancer tissues (BCT) and matched normal adjacent tissues (NAT) was performed to determine if copy number correlated with clinical features and Amerindian haplogroups (A2, B2, B4, C1 and D1) . The results showed that the mtDNA copy number was significantly decreased in BCT compared with NAT (p = 0.010); it was significantly decreased in BCT and NAT in women > 50 years of age, compared with NAT in women < 50 years of age (p = 0.032 and p = 0.037, respectively); it was significantly decreased in NAT and BCT in the postmenopausal group and in BCT in the premenopausal group compared with NAT in the premenopausal group (p = 0.011, p = 0.010 and, p = 0.018; respectively); and it was also significantly decrease in members of the BCT group classified as having invasive ductal carcinoma I-III (IDC-I, IDC-II and IDC-III) and IDC-II for NAT compared to IDC-I of NAT (p = 0.025, p = 0.022 and p = 0.031 and p = 0.020; respectively). The mtDNA copy number for BCT from patients with haplogroup B2 was decreased compared to patients with haplogroup D1 (p = 0.01); for BCT from patients with haplogroup C1 was also decreased compare with their NAT counterpart (p = 0.006) and with BCT patients belonging to haplogroups A2 and D1 (p = 0.01 and p = 0.03; respectively). In addition, the mtDNA copy number was decrease in the sequences with three deletions relative to the rCRS at nucleotide positions A249del, A290del and A291del, or C16327T polymorphism with the same p = 0.019 for all four variants. Contrary, the copy number increased in sequences containing C16111T, G16319A or T16362C polymorphisms (p = 0.021, =0.048, and = 0.001; respectively). In conclusion, a decrease in the copy number of mtDNA in BCT compared with NAT was shown by the results, which suggests an imbalance in oxidative phosphorylation (OXPHOS) that can affect the apoptosis pathway and cancer progression. It was also observed an increase of the copy number in samples with specific polymorphisms, which may be a good sign of favourable prognosis.


Assuntos
Neoplasias da Mama/genética , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Adulto , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Haplótipos/genética , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Mitocôndrias/genética
11.
Nucleic Acids Res ; 48(15): 8645-8662, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32614436

RESUMO

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.


Assuntos
Polinucleotídeo Adenililtransferase/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Poliadenilação/genética , Proteínas de Protozoários/química , Edição de RNA/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Protozoário/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
12.
Arch Cardiovasc Dis ; 113(8-9): 564-571, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32680738

RESUMO

The mitochondria produce specific peptides-mitochondrial-derived peptides-that mediate the transcriptional stress response by their translocation into the nucleus and interaction with deoxyribonucleic acid. Mitochondrial-derived peptides are regulators of metabolism. This class of peptides comprises humanin, mitochondrial open reading frame of the 12S ribosomal ribonucleic acid type c (MOTS-c) and small humanin-like peptides (SHLPs). Humanin inhibits mitochondrial complex 1 activity and limits the level of oxidative stress in the cell. Data show that mitochondrial-derived peptides have a role in improving metabolic diseases, such as type 2 diabetes. Perhaps humanin can be used as a marker for mitochondrial function in cardiovascular disease or as a pharmacological strategy in patients with endothelial dysfunction. The goal of this review is to discuss the newly emerging functions of humanin, and its biological role in cardiovascular disorders.


Assuntos
Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Estresse Oxidativo , Transdução de Sinais , Regulação para Cima
13.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
14.
Gene ; 758: 144962, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32687946

RESUMO

Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by the autoimmune inflammation, demyelination, and neurodegeneration. This complex disease develops in genetically predisposed individuals under adverse environmental factors. To date, a large number of MS-associated polymorphic loci of the nuclear genome have been identified; however, their total variability can explain only about 48% of the observed inheritance of MS. Polymorphic variants of the mitochondrial genome and interactions of mitochondrial and nuclear genes (mitonuclear interactions) may be the possible sources of the "missing heritability". We analyzed the association with MS of 10 mitochondrial DNA polymorphisms (m.1719, m.4216, m.4580, m.4917, m.7028, m.9055, m.10398, m.12308, m.13368, m.13708) in DNA of 540 MS patients and 406 healthy individuals. The allele m.9055*G was the only mitochondrial variant associated with MS (Pf = 0.027). To evaluate interactions of mitochondrial and nuclear genomes, we searched for biallelic combinations containing one of 10 mitochondrial variants and one of 35 variants of immune-related nuclear genes. Carriership of mitochondrial variants m.4216, m.4580, or m.13708 in biallelic combinations with variants of nuclear genes IL7R, CLEC16A, CD6, CD86 or PVT1 was associated with MS (Pf = 0.0036-0.00030). We identified epistatic interaction between components of a combination (m.13708*A + PVT1 rs4410871*T). The existence of epistatic biallelic combination can reflect the genuine mitonuclear epistasis.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Predisposição Genética para Doença/genética , Genoma Mitocondrial/genética , Esclerose Múltipla/genética , Adulto , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Antígeno B7-2/genética , Feminino , Estudos de Associação Genética , Humanos , Subunidade alfa de Receptor de Interleucina-7/genética , Lectinas Tipo C/genética , Masculino , Mitocôndrias/genética , Proteínas de Transporte de Monossacarídeos/genética , Polimorfismo de Nucleotídeo Único/genética , RNA Longo não Codificante/genética
15.
Nat Commun ; 11(1): 3479, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661250

RESUMO

Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Ativação Plaquetária/fisiologia , Trombose/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Masculino , Espectrometria de Massas , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Ativação Plaquetária/genética , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Trombose/genética
16.
PLoS One ; 15(7): e0232559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658922

RESUMO

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.


Assuntos
Códon de Terminação/genética , Perfilação da Expressão Gênica , Mitocôndrias/genética , Mutação , Presenilina-2/genética , Ribossomos/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Contagem de Células , Homozigoto , Hipóxia/genética , Neurônios/citologia , Estabilidade de RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
17.
PLoS Biol ; 18(7): e3000745, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667908

RESUMO

Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.


Assuntos
Envelhecimento/genética , Mamíferos/genética , Mitocôndrias/genética , Mutação/genética , Oócitos/metabolismo , Especificidade de Órgãos/genética , Animais , Análise Mutacional de DNA , DNA Mitocondrial/genética , Feminino , Frequência do Gene/genética , Deriva Genética , Células Germinativas/metabolismo , Padrões de Herança/genética , Modelos Logísticos , Masculino , Camundongos , Modelos Genéticos , Taxa de Mutação , Nucleotídeos/genética , Linhagem
18.
Cancer Invest ; 38(7): 375-393, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32673136

RESUMO

mtDNA is the closed circular, ds-DNA present in mitochondria of eukaryotic cells and are inherited maternally. Besides being the power house of the cell, mitochondria are also responsible for the regulation of redox homeostasis, signaling, metabolism, immunity, survival and apoptosis. Lack of a 'Systematic Review' on mtDNA variations and cancers encouraged us to perform the present study. Pubmed', 'Embase' and 'Cochrane Library' databases were searched using keywords 'Mitochondrial DNA' OR 'mtDNA' OR 'mDNA' AND 'polymorphism' AND 'cancer' AND 'risk' to retrieve literature. Polymorphisms occupy first rank among mtDNA variations followed by CNV, MSI, mutations and hold a great potential to emerge as key predictors for human cancers.


Assuntos
DNA Mitocondrial/genética , Neoplasias/genética , Polimorfismo Genético , Feminino , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação
19.
Proc Natl Acad Sci U S A ; 117(32): 19266-19275, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703809

RESUMO

Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria-lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria-lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria-lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria-lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria-lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria-lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.


Assuntos
Cálcio/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mucolipidoses/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Transporte Biológico , Humanos , Lisossomos/genética , Mitocôndrias/genética , Dinâmica Mitocondrial , Mucolipidoses/genética , Canais de Receptores Transientes de Potencial/genética
20.
Am J Hum Genet ; 107(2): 364-373, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707086

RESUMO

We report bi-allelic pathogenic HPDL variants as a cause of a progressive, pediatric-onset spastic movement disorder with variable clinical presentation. The single-exon gene HPDL encodes a protein of unknown function with sequence similarity to 4-hydroxyphenylpyruvate dioxygenase. Exome sequencing studies in 13 families revealed bi-allelic HPDL variants in each of the 17 individuals affected with this clinically heterogeneous autosomal-recessive neurological disorder. HPDL levels were significantly reduced in fibroblast cell lines derived from more severely affected individuals, indicating the identified HPDL variants resulted in the loss of HPDL protein. Clinical presentation ranged from severe, neonatal-onset neurodevelopmental delay with neuroimaging findings resembling mitochondrial encephalopathy to milder manifestation of adolescent-onset, isolated hereditary spastic paraplegia. All affected individuals developed spasticity predominantly of the lower limbs over the course of the disease. We demonstrated through bioinformatic and cellular studies that HPDL has a mitochondrial localization signal and consequently localizes to mitochondria suggesting a putative role in mitochondrial metabolism. Taken together, these genetic, bioinformatic, and functional studies demonstrate HPDL is a mitochondrial protein, the loss of which causes a clinically variable form of pediatric-onset spastic movement disorder.


Assuntos
Encefalopatias/genética , Proteínas Mitocondriais/genética , Doenças Neurodegenerativas/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Criança , Feminino , Humanos , Masculino , Mitocôndrias/genética , Linhagem , Fenótipo , Adulto Jovem
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