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1.
EMBO J ; 40(13): e106864, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33978233

RESUMO

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.


Assuntos
Cisteína Endopeptidases/genética , Síndrome de Klinefelter/genética , Mutação/genética , Adulto , Aneuploidia , Animais , Humanos , Masculino , Camundongos , Camundongos Knockout , Cromossomos Sexuais/genética , Espermatozoides/patologia , Adulto Jovem
2.
Nucleic Acids Res ; 51(14): 7357-7375, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37378420

RESUMO

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.


Assuntos
Meiose , Ribonuclease H , Humanos , Masculino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinases/genética , Espermatócitos/metabolismo , Ribonuclease H/metabolismo
3.
Nucleic Acids Res ; 51(8): 3855-3868, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36938872

RESUMO

Meiotic recombinases RAD51 and DMC1 mediate strand exchange in the repair of DNA double-strand breaks (DSBs) by homologous recombination. This is a landmark event of meiosis that ensures genetic diversity in sexually reproducing organisms. However, the regulatory mechanism of DMC1/RAD51-ssDNA nucleoprotein filaments during homologous recombination in mammals has remained largely elusive. Here, we show that SPIDR (scaffold protein involved in DNA repair) regulates the assembly or stability of RAD51/DMC1 on ssDNA. Knockout of Spidr in male mice causes complete meiotic arrest, accompanied by defects in synapsis and crossover formation, which leads to male infertility. In females, loss of Spidr leads to subfertility; some Spidr-/- oocytes are able to complete meiosis. Notably, fertility is rescued partially by ablation of the DNA damage checkpoint kinase CHK2 in Spidr-/- females but not in males. Thus, our study identifies SPIDR as an essential meiotic recombination factor in homologous recombination in mammals.


Assuntos
Proteínas de Ciclo Celular , Rad51 Recombinase , Animais , Masculino , Camundongos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico/genética , Reparo do DNA , Recombinação Homóloga/genética , Mamíferos/metabolismo , Meiose/genética , Camundongos Knockout , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
4.
Ann Hematol ; 102(3): 583-595, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36697954

RESUMO

Acute myeloid leukemia (AML) is a group of hematological malignancies characterized by clonal proliferation of immature myeloid cells. Lipid rafts are highly organized membrane subdomains enriched in cholesterol, sphingolipids, and gangliosides and play roles in regulating apoptosis through subcellular redistribution. Flotillin1 (FLOT1) is a component and also a marker of lipid rafts and had been reported to be involved in the progression of cancers and played important roles in cell death. However, the role of FLOT1 in AML remains to be explored. In this study, we found that increased expression of FLOT1 was correlated with poor clinical outcome in AML patients. Knockdown of FLOT1 in AML cells not only promoted cell death in vitro but also inhibited malignant cells engraftment in vivo. Mechanically, FLOT1 knockdown triggered apoptosis and pyroptosis. FLOT1 overexpression promoted AML cell growth and apoptosis resistance. Our findings indicate that FLOT1 is a prognostic factor of AML and may be a potential target for AML treatment.


Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Humanos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Leucemia Mieloide Aguda/patologia , Piroptose
5.
Nucleic Acids Res ; 48(12): 6624-6639, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463460

RESUMO

Repair of DNA double-strand breaks (DSBs) with homologous chromosomes is a hallmark of meiosis that is mediated by recombination 'bridges' between homolog axes. This process requires cooperation of DMC1 and RAD51 to promote homology search and strand exchange. The mechanism(s) regulating DMC1/RAD51-ssDNA nucleoprotein filament and the components of 'bridges' remain to be investigated. Here we show that MEIOK21 is a newly identified component of meiotic recombination bridges and is required for efficient formation of DMC1/RAD51 foci. MEIOK21 dynamically localizes on chromosomes from on-axis foci to 'hanging foci', then to 'bridges', and finally to 'fused foci' between homolog axes. Its chromosome localization depends on DSBs. Knockout of Meiok21 decreases the numbers of HSF2BP and DMC1/RAD51 foci, disrupting DSB repair, synapsis and crossover recombination and finally causing male infertility. Therefore, MEIOK21 is a novel recombination factor and probably mediates DMC1/RAD51 recruitment to ssDNA or their stability on chromosomes through physical interaction with HSF2BP.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Animais , Proteínas de Transporte/genética , Pareamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Técnicas de Inativação de Genes , Proteínas de Choque Térmico/genética , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética
6.
Mol Med ; 27(1): 57, 2021 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-34092215

RESUMO

BACKGROUND: Acetaminophen (APAP) overdose causes hepatotoxicity and even acute liver failure. Recent studies indicate that sterile inflammation and innate immune cells may play important roles in damage-induced hepatocytes regeneration and liver repair. The scavenger receptor CD36 has its crucial functions in sterile inflammation. However, the roles of CD36 in APAP induced acute liver injury remain unclear and warrant further investigation. METHODS: WT C57BL/6 J and CD36-/- mice were intraperitoneally injected with APAP (300 mg/kg) after fasting for 16 h. Liver injury was evaluated by serum alanine aminotransferase (ALT) level and liver tissue hematoxylin and eosin (H&E) staining. Liver inflammatory factor expression was determined by real-time polymerase chain reaction (PCR). The protein adducts forming from the metabolite of APAP and the metabolism enzyme cytochrome P450 2E1 (CYP2E1) levels were measured by Western blot. Liver infiltrating macrophages and neutrophils were characterized by flow cytometry. RNA sequencing and Western blot were used to evaluate the effect of damage-associated molecular patterns (DAMP) molecule high mobility group B1 (HMGB1) on WT and CD36-/- macrophages. Moreover, PP2, a Src kinase inhibitor, blocking CD36 signaling, was applied in APAP model. RESULTS: The expression of CD36 was increased in the liver of mice after APAP treatment. Compared with WT mice, APAP treated CD36-/- mice show less liver injury. There was no significant difference in APAP protein adducts and CYP2E1 expression between these two strains. However, reduced pro-inflammatory factor mRNA expression and serum IL-1ß level were observed in APAP treated CD36-/- mice as well as infiltrating macrophages and neutrophils. Moreover, CD36 deficiency impaired the activation of c-Jun N-terminal kinase (JNK) caused by APAP. Interestingly, the lack of CD36 reduced the activation of extracellular regulated protein kinases (Erk) and v-akt murine thymoma viral oncogene homolog (Akt) induced by HMGB1. RNA transcription sequencing data indicated that HMGB1 has a different effect on WT and CD36-/- macrophages. Furthermore, treatment with PP2 attenuated APAP induced mouse liver injury. CONCLUSION: Our data demonstrated that CD36 deficiency ameliorated APAP-induced acute liver injury and inflammatory responses by decreasing JNK activation. CD36 might serve as a new target to reduce acute liver injury.


Assuntos
Antígenos CD36/deficiência , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Suscetibilidade a Doenças , Acetaminofen/efeitos adversos , Animais , Biomarcadores , Biópsia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Predisposição Genética para Doença , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Quinases da Família src/metabolismo
7.
Cell Mol Life Sci ; 77(4): 719-733, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31302752

RESUMO

Cytidine base editors (CBEs) have been demonstrated to be useful for precisely inducing C:G-to-T:A base mutations in various organisms. In this study, we showed that the BE4-Gam system induced the targeted C-to-T base conversion in porcine blastocysts at an efficiency of 66.7-71.4% via the injection of a single sgRNA targeting a xeno-antigen-related gene and BE4-Gam mRNA. Furthermore, the efficiency of simultaneous three gene base conversion via the injection of three targeting sgRNAs and BE4-Gam mRNA into porcine parthenogenetic embryos was 18.1%. We also obtained beta-1,4-N-acetyl-galactosaminyl transferase 2, alpha-1,3-galactosyltransferase, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient pig by somatic cell nuclear transfer, which exhibited significantly decreased activity. In addition, a new CBE version (termed AncBE4max) was used to edit genes in blastocysts and porcine fibroblasts (PFFs) for the first time. While this new version demonstrated a three genes base-editing rate of 71.4% at the porcine GGTA1, B4galNT2, and CMAH loci, it increased the frequency of bystander edits, which ranged from 17.8 to 71.4%. In this study, we efficiently and precisely mutated bases in porcine blastocysts and PFFs using CBEs and successfully generated C-to-T and C-to-G mutations in pigs. These results suggest that CBEs provide a more simple and efficient method for improving economic traits, reducing the breeding cycle, and increasing disease tolerance in pigs, thus aiding in the development of human disease models.


Assuntos
Citidina/genética , Edição de Genes/métodos , Suínos/genética , Animais , Blastocisto/metabolismo , Sistemas CRISPR-Cas , Galactosiltransferases/genética , Vetores Genéticos/genética , Oxigenases de Função Mista/genética , Mutagênese , N-Acetilgalactosaminiltransferases/genética , RNA Guia de Cinetoplastídeos/genética , Suínos/embriologia
8.
Nucleic Acids Res ; 47(11): 5670-5683, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30949703

RESUMO

Meiosis is a specialized cell division for producing haploid gametes from diploid germ cells. During meiosis, synaptonemal complex (SC) mediates the alignment of homologs and plays essential roles in homologous recombination and therefore in promoting accurate chromosome segregation. In this study, we have identified a novel protein SCRE (synaptonemal complex reinforcing element) as a key molecule in maintaining the integrity of SC during meiosis prophase I in mice. Deletion of Scre (synaptonemal complex reinforcing element) caused germ cell death in both male and female mice, resulting in infertility. Our mechanistic studies showed that the synapses and SCs in Scre knockout mice were unstable due to the lack of the SC reinforcing function of SCRE, which is sparsely localized as discrete foci along the central elements in normal synaptic homologous chromosomes. The lack of Scre leads to meiosis collapse at the late zygotene stage. We further showed that SCRE interacts with synaptonemal complex protein 1 (SYCP1) and synaptonemal complex central element 3 (SYCE3). We conclude that the function of SCRE is to reinforce the integrity of the central elements, thereby stabilizing the SC and ensuring meiotic cell cycle progression. Our study identified SCRE as a novel SC fastener protein that is distinct from other known SC proteins.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Prófase Meiótica I , Proteínas Nucleares/fisiologia , Complexo Sinaptonêmico/fisiologia , Animais , Sistemas CRISPR-Cas , Segregação de Cromossomos , Proteínas de Ligação a DNA , Feminino , Células HEK293 , Humanos , Masculino , Meiose , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ligação Proteica , Recombinação Genética , Espermatócitos/metabolismo , Testículo/metabolismo
9.
PLoS Pathog ; 14(12): e1007193, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543715

RESUMO

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.


Assuntos
Antivirais , Peste Suína Clássica/prevenção & controle , Engenharia Genética/métodos , Animais , Animais Geneticamente Modificados , Vírus da Febre Suína Clássica , Suínos
10.
Nat Chem Biol ; 14(9): 876-886, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30120361

RESUMO

Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote ß-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor-arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of ß-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.


Assuntos
Regulação Alostérica , Arrestina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Células Cultivadas , Células HEK293 , Humanos
11.
J Cell Mol Med ; 23(1): 293-305, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30394687

RESUMO

Gastric cancer (GC) is a malignancy of the lining of the stomach and is prone to distant metastasis, which involves a variety of complex molecules. The S100 proteins are a family of calcium-binding cytosolic proteins that possess a wide range of intracellular and extracellular functions and play pivotal roles in the invasion and migration of tumour cells. Among these, S100A10 is known to be overexpressed in GC. Lysine succinylation, a recently identified form of protein post-translational modification, is an important regulator of cellular processes. Here, we demonstrated that S100A10 was succinylated at lysine residue 47 (K47), and levels of succinylated S100A10 were increased in human GC. Moreover, K47 succinylation of S100A10 was stabilized by suppression of ubiquitylation and subsequent proteasomal degradation. Furthermore, carnitine palmitoyltransferase 1A (CPT1A) was found to function as a lysine succinyltransferase that interacts with S100A10. Succinylation of S100A10 is regulated by CPT1A, while desuccinylation is regulated by SIRT5. Overexpression of a succinylation mimetic mutant, K47E S100A10, increased cell invasion and migration. Taken together, this study reveals a novel mechanism of S100A10 accumulation mediated by succinylation in GC, which promotes GC progression and is regulated by the succinyltransferase CPT1A and SIRT5-mediated desuccinylation.


Assuntos
Anexina A2/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas S100/metabolismo , Neoplasias Gástricas/patologia , Animais , Anexina A2/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Lisina/metabolismo , Masculino , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Proteínas S100/genética , Sirtuínas/metabolismo , Neoplasias Gástricas/metabolismo , Succinatos/metabolismo
12.
Int J Mol Sci ; 20(3)2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717351

RESUMO

Myostatin (MSTN) is a member of the TGF-ß superfamily that negatively regulates skeletal muscle growth and differentiation. However, the mechanism by which complete MSTN deletion limits excessive proliferation of muscle cells remains unclear. In this study, we knocked out MSTN in mouse myoblast lines using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system and sequenced the mRNA and miRNA transcriptomes. The results show that complete loss of MSTN upregulates seven miRNAs targeting an interaction network composed of 28 downregulated genes, including TGFB1, FOS and RB1. These genes are closely associated with tumorigenesis and cell proliferation. Our study suggests that complete loss of MSTN may limit excessive cell proliferation via activation of miRNAs. These data will contribute to the treatment of rhabdomyosarcoma (RMS).


Assuntos
MicroRNAs/genética , Células Musculares/metabolismo , Miostatina/deficiência , Animais , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Ordem dos Genes , Redes Reguladoras de Genes , Vetores Genéticos/genética , Camundongos , Anotação de Sequência Molecular , Interferência de RNA , RNA Mensageiro/genética , Reprodutibilidade dos Testes
13.
Transgenic Res ; 26(6): 799-805, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28993973

RESUMO

CRISPR/Cas9 has emerged as one of the most popular genome editing tools due to its simple design and high efficiency in multiple species. Myostatin (MSTN) negatively regulates skeletal muscle growth and mutations in myostatin cause double-muscled phenotype in various animals. Here, we generated myostatin mutation in Erhualian pigs using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. The protein level of myostatin precursor decreased dramatically in mutant cloned piglets. Unlike myostatin knockout Landrace, which often encountered health issues and died shortly after birth, Erhualian pigs harboring homozygous mutations were viable. Moreover, myostatin knockout Erhualian pigs exhibited partial double-muscled phenotype such as prominent muscular protrusion, wider back and hip compared with wild-type piglets. Genome editing in Chinese indigenous pig breeds thus holds great promise not only for improving growth performance, but also for protecting endangered genetic resources.


Assuntos
Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas , Mutação , Miostatina/genética , Suínos/genética , Animais , Feminino , Técnicas de Inativação de Genes , Homozigoto , Músculo Esquelético/fisiologia , Técnicas de Transferência Nuclear , Transfecção/métodos
14.
iScience ; 27(4): 109456, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38591005

RESUMO

Spermiogenesis defines the final phase of male germ cell differentiation. While multiple deubiquitinating enzymes have been linked to spermiogenesis, the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis. We selectively deleted Uaf1 in premeiotic germ cells using the Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. Further, UAF1 interacts and colocalizes with USP1 in the testes. Conditional knockout of Uaf1 in testes results in disturbed protein levels and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. Using tandem mass tag-based proteomics, we identified that conditional knockout of Uaf1 in the testes results in reduced levels of proteins that are essential for spermiogenesis. Thus, we conclude that the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.

15.
Cell Death Discov ; 10(1): 107, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429284

RESUMO

The cytoplasmic pattern recognition receptor, absent in melanoma 2 (AIM2), detects cytosolic DNA, activating the inflammasome and resulting in pro-inflammatory cytokine production and pyroptotic cell death. Recent research has illuminated AIM2's contributions to PANoptosis and host defense. However, the role of AIM2 in acetaminophen (APAP)-induced hepatoxicity remains enigmatic. In this study, we unveil AIM2's novel function as a negative regulator in the pathogenesis of APAP-induced liver damage in aged mice, independently of inflammasome activation. AIM2-deficient aged mice exhibited heightened lipid accumulation and hepatic triglycerides in comparison to their wild-type counterparts. Strikingly, AIM2 knockout mice subjected to APAP overdose demonstrated intensified liver injury, compromised mitochondrial stability, exacerbated glutathione depletion, diminished autophagy, and elevated levels of phosphorylated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, our investigation revealed AIM2's mitochondrial localization; its overexpression in mouse hepatocytes amplified autophagy while dampening JNK phosphorylation. Notably, induction of autophagy through rapamycin administration mitigated serum alanine aminotransferase levels and reduced the necrotic liver area in AIM2-deficient aged mice following APAP overdose. Mechanistically, AIM2 deficiency exacerbated APAP-induced acute liver damage and inflammation in aged mice by intensifying oxidative stress and augmenting the phosphorylation of JNK and ERK. Given its regulatory role in autophagy and lipid peroxidation, AIM2 emerges as a promising therapeutic target for age-related acute liver damage treatment.

16.
Cells ; 12(16)2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37626901

RESUMO

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm.


Assuntos
Sêmen , Motilidade dos Espermatozoides , Masculino , Animais , Camundongos , Espermatozoides , Flagelos , Cauda do Espermatozoide
17.
Research (Wash D C) ; 6: 0203, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37496633

RESUMO

Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.

19.
Elife ; 122023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36942942

RESUMO

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.


Assuntos
Acrossomo , Actinas , Masculino , Camundongos , Animais , Acrossomo/metabolismo , Actinas/metabolismo , Sêmen/metabolismo , Espermatogênese , Camundongos Knockout , Fatores de Despolimerização de Actina/metabolismo
20.
Biology (Basel) ; 11(1)2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35053139

RESUMO

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375-146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Bcl-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

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