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1.
Mol Cell ; 81(4): 830-844.e13, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33453168

RESUMO

The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elongação da Transcrição Genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
Gene ; 764: 145100, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32877748

RESUMO

Adipocyte differentiation is an essential part of adipose tissue development, and is closely related to obesity and obesity-related diseases. In this study, we found that the expression of PPARγ, RUVBL2 and Adiponectin were concurrently obviously increased in the 5th-7th day of 3T3-L1 cell differentiation. PPARγ overexpression or the PPARγ activator facilitated Adiponectin trafficking and secretion and upregulated RUVBL2 expression as well as AS160 phosphorylation during adipogenic differentiation of 3T3-L1 cells. Consistently RUVBL2 overexpression also enhanced the polymerization and secretion of Adiponectin, in contrast, RUVBL2 knockdown reduced Adiponectin secretion. Further, PPARγ significantly enhanced RUVBL2 promoter activity and transcription. The progressive deletions and mutations of RUVBL2 promoter for PPARγ binding sites suggested that the PPARγ binding motif situated at -804/-781 bp is an essential component required for RUVBL2 promoter activity. Chromatin immunoprecipitation (ChIP) assays determined that PPARγ can directly interact with the RUVBL2 promoter DNA. Taken together, these data suggest that PPARγ promotes the expression, polymerization and secretion of Adiponectin by activating RUVBL2 transcriptionally, which accelerates 3T3-L1 cell differentiation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Adipócitos/fisiologia , Adipogenia/genética , Adiponectina/metabolismo , DNA Helicases/genética , PPAR gama/metabolismo , Células 3T3-L1 , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Clonagem Molecular , DNA Helicases/metabolismo , Camundongos , Mutação , Regiões Promotoras Genéticas/genética , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional , Regulação para Cima
3.
Proc Natl Acad Sci U S A ; 117(40): 25104-25115, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958650

RESUMO

Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteoma/genética , Receptores Notch/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Arginina/genética , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais/genética
4.
Int J Oral Sci ; 12(1): 22, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737282

RESUMO

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Saco Dentário/citologia , Displasia da Dentina/genética , Displasia da Dentina/fisiopatologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Osteogênese/genética , Processamento de RNA/genética , Estudos de Casos e Controles , Diferenciação Celular/genética , Células Cultivadas , Displasia da Dentina/patologia , Humanos , Mutação/genética
5.
Nat Commun ; 11(1): 3839, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737294

RESUMO

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA2/genética , Cromatina/química , Proteínas de Ligação a DNA/genética , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Reparo de DNA por Recombinação , ATPases Associadas a Diversas Atividades Celulares/deficiência , Animais , Apoptose/genética , Proteína BRCA2/deficiência , Divisão Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(31): 18459-18469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694211

RESUMO

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Mol Cell ; 79(4): 615-628.e5, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32668200

RESUMO

Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.


Assuntos
Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
8.
Am J Hum Genet ; 107(1): 15-23, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32473092

RESUMO

Normal oocyte meiosis is a prerequisite for successful human reproduction, and abnormalities in the process will result in infertility. In 2016, we identified mutations in TUBB8 as responsible for human oocyte meiotic arrest. However, the underlying genetic factors for most affected individuals remain unknown. TRIP13, encoding an AAA-ATPase, is a key component of the spindle assembly checkpoint, and recurrent homozygous nonsense variants and a splicing variant in TRIP13 are reported to cause Wilms tumors in children. In this study, we identified homozygous and compound heterozygous missense pathogenic variants in TRIP13 responsible for female infertility mainly characterized by oocyte meiotic arrest in five individuals from four independent families. Individuals from three families suffered from oocyte maturation arrest, whereas the individual from the fourth family had abnormal zygote cleavage. All displayed only the infertility phenotype without Wilms tumors or any other abnormalities. In vitro and in vivo studies showed that the identified variants reduced the protein abundance of TRIP13 and caused its downstream molecule, HORMAD2, to accumulate in HeLa cells and in proband-derived lymphoblastoid cells. The chromosome mis-segregation assay showed that variants did not have any effects on mitosis. Injecting TRIP13 cRNA into oocytes from one affected individual was able to rescue the phenotype, which has implications for future therapeutic treatments. This study reports pathogenic variants in TRIP13 responsible for oocyte meiotic arrest, and it highlights the pivotal but different roles of TRIP13 in meiosis and mitosis. These findings also indicate that different dosage effects of mutant TRIP13 might result in two distinct human diseases.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Infertilidade Feminina/genética , Mutação de Sentido Incorreto/genética , Oócitos/patologia , Adulto , Alelos , Linhagem Celular Tumoral , Feminino , Células HeLa , Homozigoto , Humanos , Meiose/genética , Fenótipo , Zigoto/patologia
9.
Hum Genet ; 139(10): 1325-1343, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32399598

RESUMO

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Aminoacil-tRNA Sintetases/genética , Proteínas de Ligação a DNA/genética , Dimetilaliltranstransferase/genética , Endopeptidase Clp/genética , Farnesiltranstransferase/genética , Predisposição Genética para Doença , Geraniltranstransferase/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Sequência de Bases , Criança , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ovário/metabolismo , Ovário/patologia , Linhagem , Peroxissomos/metabolismo , Peroxissomos/patologia
10.
Nucleic Acids Res ; 48(9): 4928-4939, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32297953

RESUMO

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Antineoplásicos/farmacologia , Dano ao DNA , Proteínas de Ligação a DNA/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Galinhas , Cromatina/enzimologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Mutagênicos/toxicidade , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo
13.
Ann Neurol ; 88(1): 18-32, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32219868

RESUMO

OBJECTIVE: Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed. METHODS: We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts. RESULTS: Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells. INTERPRETATION: This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.


Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , GTP Fosfo-Hidrolases/genética , Atrofia Óptica/genética , Doenças do Nervo Óptico/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
14.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013259

RESUMO

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Membrana/genética , Sinais de Orientação para Peroxissomos/genética , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/deficiência , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Macroautofagia , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Peroxinas/genética , Peroxinas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Biomed Pharmacother ; 125: 109964, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32044716

RESUMO

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumor in children and young adults. RNA N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA, which is involved in tumorigenesis and tumor progression. It has been reported that methyltransferase-like 3 (METTL3), the first reported m6A "writer", plays critical roles in cancer progression. However, its role and molecular mechanism in osteosarcoma is poor studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the progression of osteosarcoma. METHODS: We detected the mRNA expression of METTL3 in osteosarcoma cell lines, and immunofluorescence assay was performed to observe the location of METTL3. Cell lines with METTL3 gene overexpression or knockdown were established by pcDNA3.1-METTL3 or siRNA interferences in order to determine the function of METTL3 in osteosarcoma in vitro. Transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3 in osteosarcoma. RESULTS: We found that METTL3 localized in cytoplasm and nucleus of osteosarcoma cells. Silencing METTL3 in SAOS-2 and MG63 cells significantly inhibited the m6A methylation level, proliferation, migration, and invasion abilities, as well as promoted cell apoptosis. However, up-regulation of METTL3 had no significant effect on the biological behaviors of U2OS cells. Further mechanism analysis suggested that METTL3 knockdown inhibited the expression of ATPase family AAA domain containing 2 (ATAD2). Moreover, ATAD2 knockdown inhibited the proliferation and invasion of SAOS-2 and MG63 cells, while its overexpression showed a significant increase in cell proliferation and invasion. Furthermore, METTL3 knockdown abrogated the promoting effects of ATAD2 overexpression on osteosarcoma cells proliferation and invasion. CONCLUSION: Overall, our study revealed that METTL3 functions as an oncogene in the growth and invasion of osteosarcoma by regulating ATAD2, suggesting a potential therapeutic target for osteosarcoma treatment.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Metiltransferases/metabolismo , Osteossarcoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Inativação Gênica , Humanos , Metiltransferases/genética , Regulação para Cima
16.
Cancer Biomark ; 27(4): 445-460, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32065787

RESUMO

To characterize the somatic alterations of papillary thyroid carcinomas (PTC) in Chinese patients, we performed the next-generation-sequencing (NGS) study of the tumor-normal pairs of DNA and RNA samples extracted from 16 Chinese PTC patients. The whole genome sequencing (WGS) and transcriptome sequencing (RNA-seq) were conducted for 6 patients who were either current or former smokers and the whole exome sequencing (WES) and RNA-seq were conducted for another 10 patients who were never smokers. The NGS data were analyzed to identify somatic alteration events that may underlie PTC in Chinese patients. We identified a number of PTC driver genes harboring somatic driver mutations with significant functional impact such as COL11A1, TP53, PLXNA4, UBA1, AHNAK, CSMD2 and TTLL5 etc. Significant driver pathways underlying PTC were found, namely, the metabolic pathway, the pathway in cancer, the olfactory transduction pathway and the calcium signaling pathway. In addition, this study revealed genes with significant somatic copy number aberrations and corresponding somatic gene expression changes in PTC tumors, the most promising ones being BRD9, TRIP13, FZD3, and TFDP1 etc. We also identified several structural variants of PTCs, especially the novel in-frame fusion proteins such as TRNAU1AP-RCC1, RAB3GAP1-R3HDM1, and ENAH-ZSWIM5. Our study provided a list of novel PTC candidate genes with somatic alterations that may function as biomarkers for PTC in Chinese patients. The follow-up mechanism studies may be conducted based on the findings from this study.


Assuntos
Biomarcadores Tumorais/genética , Mutação , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ciclo Celular/genética , China/epidemiologia , Colágeno Tipo XI/genética , Biologia Computacional/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Câncer Papilífero da Tireoide/epidemiologia , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/epidemiologia , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do Exoma/métodos , Sequenciamento Completo do Genoma/métodos , Adulto Jovem
17.
Am J Hum Genet ; 106(2): 272-279, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004445

RESUMO

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Colesterol/metabolismo , Duplicação Gênica , Recombinação Homóloga , Proteínas de Membrana/genética , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , ATPases Associadas a Diversas Atividades Celulares/química , Sequência de Aminoácidos , Encefalopatias/etiologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Opacidade da Córnea/etiologia , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Variações do Número de Cópias de DNA , Feminino , Rearranjo Gênico , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Hipotonia Muscular/etiologia , Hipotonia Muscular/metabolismo , Hipotonia Muscular/patologia , Mutação , Conformação Proteica , Convulsões/etiologia , Convulsões/metabolismo , Convulsões/patologia , Homologia de Sequência
18.
Proc Natl Acad Sci U S A ; 117(10): 5386-5393, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079723

RESUMO

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.


Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Imunoprecipitação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
19.
Nat Commun ; 11(1): 322, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949167

RESUMO

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1lp.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cybp.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1lp.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc1 complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Epistasia Genética/genética , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/genética , Doenças Mitocondriais/genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Citocromos b , DNA Mitocondrial , Complexo III da Cadeia de Transporte de Elétrons/química , Metabolismo Energético , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Oxirredução
20.
Biochem Biophys Res Commun ; 523(4): 916-923, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31959473

RESUMO

Stomach cancer is a difficult-to-treat disease. Lack of detection markers and limited understanding of the disease mechanisms contribute to the aggressive nature of stomach cancer cells (SCCs). Recently, an ATPase, ATAD2 has been found to be highly expressed in stomach cancer contributing to increased malignancy. However, nothing is known about the mechanism of ATAD2 upregulation and its involvement in stomach carcinogenesis. Since hypoxic microenvironment plays a crucial role in the progression of solid tumors like stomach cancer; we have examined the regulation and function of ATAD2 expression in hypoxic SCCs. ATAD2 is induced in hypoxia-treated SCCs. Stomach adenocarcinoma and metastatic tissues with high HIF1α level also show enhanced ATAD2 expression. In the absence of hypoxia-inducible factor HIF1α, ATAD2 protein level is found to be less indicating towards a potential correlation between them. We identify the presence of HIF1α-binding site (HBS) and HIF1α ancillary site (HAS) in the ATAD2 promoter. Using both in vitro and in vivo binding studies, we confirm that HIF1α binds with the ATAD2 promoter in hypoxic condition. ATAD2 upregulation promotes proliferation and migration of SCCs exposed to hypoxia. Thus, we identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances tumor-promoting functions in hypoxic SCCs. Therefore, we propose ATAD2 as a promising therapeutic target for stomach cancer.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Regulação para Cima/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ativação Transcricional/genética
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