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1.
Int J Mol Sci ; 25(19)2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39408915

RESUMO

Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions.


Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-raf , Humanos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Membrana Celular/metabolismo , MAP Quinase Quinase 2/metabolismo , Quinases raf/metabolismo , Proteína Quinase C/metabolismo , Células HeLa , Fosforilação , Animais , Transporte Proteico , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas Quinases
2.
Nucleic Acids Res ; 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39291733

RESUMO

Replication stresses are the major source of break-induced replication (BIR). Here, we show that in alternative lengthening of telomeres (ALT) cells, replication stress-induced polyubiquitinated proliferating cell nuclear antigen (PCNA) (polyUb-PCNA) triggers BIR at telomeres and the common fragile site (CFS). Consistently, depleting RAD18, a PCNA ubiquitinating enzyme, reduces the occurrence of ALT-associated promyelocytic leukemia (PML) bodies (APBs) and mitotic DNA synthesis at telomeres and CFS, both of which are mediated by BIR. In contrast, inhibiting ubiquitin-specific protease 1 (USP1), an Ub-PCNA deubiquitinating enzyme, results in an increase in the above phenotypes in a RAD18- and UBE2N (the PCNA polyubiquitinating enzyme)-dependent manner. Furthermore, deficiency of ATAD5, which facilitates USP1 activity and unloads PCNAs, augments recombination-associated phenotypes. Mechanistically, telomeric polyUb-PCNA accumulates SLX4, a nuclease scaffold, at telomeres through its ubiquitin-binding domain and increases telomere damage. Consistently, APB increase induced by Ub-PCNA depends on SLX4 and structure-specific endonucleases. Taken together, our results identified the polyUb-PCNA-SLX4 axis as a trigger for directing BIR.

3.
Am J Hum Genet ; 111(9): 1970-1993, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39106866

RESUMO

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.


Assuntos
Proteína de Replicação C , Humanos , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Masculino , Células HeLa , Feminino , Fenótipo , Replicação do DNA/genética , Adulto , Mutação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Alelos
4.
Proc Natl Acad Sci U S A ; 121(34): e2315759121, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39145935

RESUMO

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , Ubiquitinação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Ubiquitina/metabolismo , Dano ao DNA , Ligação Proteica , Proteases Específicas de Ubiquitina
5.
Mutat Res ; 829: 111878, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39151334

RESUMO

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

6.
Anim Cells Syst (Seoul) ; 28(1): 401-416, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39176289

RESUMO

Lamin A/C, a core component of the nuclear lamina, forms a mesh-like structure beneath the inner nuclear membrane. While its structural role is well-studied, its involvement in DNA metabolism remains unclear. We conducted sequential protein fractionation to determine the subcellular localization of early DNA damage response (DDR) proteins. Our findings indicate that most DDR proteins, including ATM and the MRE11-RAD50-NBS1 (MRN) complex, are present in the nuclease - and high salt-resistant pellet fraction. Notably, ATM and MRN remain stably associated with these structures throughout the cell cycle, independent of ionizing radiation (IR)-induced DNA damage. Although Lamin A/C interacts with ATM and MRN, its depletion does not disrupt their association with nuclease-resistant structures. However, it impairs the IR-enhanced association of ATM with the nuclear matrix and ATM-mediated DDR signaling, as well as the interaction between ATM and MRN. This disruption impedes the recruitment of MRE11 to damaged DNA and the association of damaged DNA with the nuclear matrix. Additionally, Lamin A/C depletion results in reduced protein levels of CtIP and RAD51, which is mediated by transcriptional regulation. This, in turn, impairs the efficiency of homologous recombination (HR). Our findings indicate that Lamin A/C plays a pivotal role in DNA damage repair (DDR) by orchestrating ATM-mediated signaling, maintaining HR protein levels, and ensuring efficient DNA repair processes.

7.
Res Sq ; 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39070640

RESUMO

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

8.
Exp Mol Med ; 56(8): 1736-1749, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39085352

RESUMO

The SARS-CoV-2 pandemic has had an unprecedented impact on global public health and the economy. Although vaccines and antivirals have provided effective protection and treatment, the development of new small molecule-based antiviral candidates is imperative to improve clinical outcomes against SARS-CoV-2. In this study, we identified UNI418, a dual PIKfyve and PIP5K1C inhibitor, as a new chemical agent that inhibits SARS-CoV-2 entry into host cells. UNI418 inhibited the proteolytic activation of cathepsins, which is regulated by PIKfyve, resulting in the inhibition of cathepsin L-dependent proteolytic cleavage of the SARS-CoV-2 spike protein into its mature form, a critical step for viral endosomal escape. We also demonstrated that UNI418 prevented ACE2-mediated endocytosis of the virus via PIP5K1C inhibition. Our results identified PIKfyve and PIP5K1C as potential antiviral targets and UNI418 as a putative therapeutic compound against SARS-CoV-2.


Assuntos
Antivirais , COVID-19 , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool) , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Antivirais/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/virologia , COVID-19/metabolismo , Tratamento Farmacológico da COVID-19 , Animais , Catepsina L/metabolismo , Catepsina L/antagonistas & inibidores , Chlorocebus aethiops , Endocitose/efeitos dos fármacos , Células Vero , Enzima de Conversão de Angiotensina 2/metabolismo , Células HEK293
9.
DNA Repair (Amst) ; 140: 103696, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38820807

RESUMO

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Animais , Reparo do DNA , DNA Polimerase teta , Proteína 9 Associada à CRISPR/metabolismo , DNA/metabolismo
10.
iScience ; 27(4): 109524, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38577109

RESUMO

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

11.
Trends Genet ; 40(6): 526-539, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38485608

RESUMO

Proliferating cell nuclear antigen (PCNA) is a eukaryotic replicative DNA clamp. Furthermore, DNA-loaded PCNA functions as a molecular hub during DNA replication and repair. PCNA forms a closed homotrimeric ring that encircles the DNA, and association and dissociation of PCNA from DNA are mediated by clamp-loader complexes. PCNA must be actively released from DNA after completion of its function. If it is not released, abnormal accumulation of PCNA on chromatin will interfere with DNA metabolism. ATAD5 containing replication factor C-like complex (RLC) is a PCNA-unloading clamp-loader complex. ATAD5 deficiency causes various DNA replication and repair problems, leading to genome instability. Here, we review recent progress regarding the understanding of the action mechanisms of PCNA unloading complex in DNA replication/repair pathways.


Assuntos
Reparo do DNA , Replicação do DNA , Mamíferos , Antígeno Nuclear de Célula em Proliferação , Replicação do DNA/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Reparo do DNA/genética , Animais , Humanos , Mamíferos/genética , Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo
12.
Biomedicines ; 11(11)2023 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-38001935

RESUMO

Patient-derived xenograft (PDX) models, which can retain the characteristics of original tumors in an in vivo-mimicking environment, have been developed to identify better treatment options. However, although original tumors and xenograft tissues mostly share oncogenic mutations and global gene expression patterns, their detailed mutation profiles occasionally do not overlap, indicating that selection occurs in the xenograft environment. To understand this mutational alteration in xenografts, we established 13 PDX models derived from 11 brain tumor patients and confirmed their histopathological similarity. Surprisingly, only a limited number of somatic mutations were shared between the original tumor and xenograft tissue. By analyzing deleteriously mutated genes in tumors and xenografts, we found that previously reported brain tumor-related genes were enriched in PDX samples, demonstrating that xenografts are a valuable platform for studying brain tumors. Furthermore, mutated genes involved in cilium movement, microtubule depolymerization, and histone methylation were enriched in PDX samples compared with the original tumors. Even with the limitations of the heterogeneity of clinical lesions with a heterotropic model, our study demonstrates that PDX models can provide more information in genetic analysis using samples with high heterogeneity, such as brain tumors.

13.
Exp Mol Med ; 55(11): 2357-2375, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37907739

RESUMO

Dopamine neurons are essential for voluntary movement, reward learning, and motivation, and their dysfunction is closely linked to various psychological and neurodegenerative diseases. Hence, understanding the detailed signaling mechanisms that functionally modulate dopamine neurons is crucial for the development of better therapeutic strategies against dopamine-related disorders. Phospholipase Cγ1 (PLCγ1) is a key enzyme in intracellular signaling that regulates diverse neuronal functions in the brain. It was proposed that PLCγ1 is implicated in the development of dopaminergic neurons, while the physiological function of PLCγ1 remains to be determined. In this study, we investigated the physiological role of PLCγ1, one of the key effector enzymes in intracellular signaling, in regulating dopaminergic function in vivo. We found that cell type-specific deletion of PLCγ1 does not adversely affect the development and cellular morphology of midbrain dopamine neurons but does facilitate dopamine release from dopaminergic axon terminals in the striatum. The enhancement of dopamine release was accompanied by increased colocalization of vesicular monoamine transporter 2 (VMAT2) at dopaminergic axon terminals. Notably, dopamine neuron-specific knockout of PLCγ1 also led to heightened expression and colocalization of synapsin III, which controls the trafficking of synaptic vesicles. Furthermore, the knockdown of VMAT2 and synapsin III in dopamine neurons resulted in a significant attenuation of dopamine release, while this attenuation was less severe in PLCγ1 cKO mice. Our findings suggest that PLCγ1 in dopamine neurons could critically modulate dopamine release at axon terminals by directly or indirectly interacting with synaptic machinery, including VMAT2 and synapsin III.


Assuntos
Dopamina , Proteínas Vesiculares de Transporte de Monoamina , Animais , Camundongos , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genética , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
14.
Cell Rep ; 42(10): 113239, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37819757

RESUMO

Dopamine synapses play a crucial role in volitional movement and reward-related behaviors, while dysfunction of dopamine synapses causes various psychiatric and neurological disorders. Despite this significance, the true biological nature of dopamine synapses remains poorly understood. Here, we show that dopamine transmission is strongly correlated with GABA co-transmission across the brain and dopamine synapses are structured and function like GABAergic synapses with marked regional heterogeneity. In addition, GABAergic-like dopamine synapses are clustered on the dendrites, and GABA transmission at dopamine synapses has distinct physiological properties. Interestingly, the knockdown of neuroligin-2, a key postsynaptic protein at GABAergic synapses, unexpectedly does not weaken GABA co-transmission but instead facilitates it at dopamine synapses in the striatal neurons. More importantly, the attenuation of GABA co-transmission precedes deficits in dopaminergic transmission in animal models of Parkinson's disease. Our findings reveal the spatial and functional nature of GABAergic-like dopamine synapses in health and disease.


Assuntos
Encéfalo , Dopamina , Animais , Dopamina/metabolismo , Encéfalo/metabolismo , Sinapses/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Receptores de GABA-A/metabolismo
15.
Nucleic Acids Res ; 51(19): 10519-10535, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37739427

RESUMO

Homologous recombination (HR) requires bidirectional end resection initiated by a nick formed close to a DNA double-strand break (DSB), dysregulation favoring error-prone DNA end-joining pathways. Here we investigate the role of the ATAD5, a PCNA unloading protein, in short-range end resection, long-range resection not being affected by ATAD5 deficiency. Rapid PCNA loading onto DNA at DSB sites depends on the RFC PCNA loader complex and MRE11-RAD50-NBS1 nuclease complexes bound to CtIP. Based on our cytological analyses and on an in vitro system for short-range end resection, we propose that PCNA unloading by ATAD5 is required for the completion of short-range resection. Hampering PCNA unloading also leads to failure to remove the KU70/80 complex from the termini of DSBs hindering DNA repair synthesis and the completion of HR. In line with this model, ATAD5-depleted cells are defective for HR, show increased sensitivity to camptothecin, a drug forming protein-DNA adducts, and an augmented dependency on end-joining pathways. Our study highlights the importance of PCNA regulation at DSB for proper end resection and HR.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Endodesoxirribonucleases/metabolismo , Recombinação Homóloga/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Humanos
16.
NAR Cancer ; 5(3): zcad042, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37554969

RESUMO

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

17.
Exp Mol Med ; 55(8): 1720-1733, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37524868

RESUMO

Autophagy functions in cellular quality control and metabolic regulation. Dysregulation of autophagy is one of the major pathogenic factors contributing to the progression of nonalcoholic fatty liver disease (NAFLD). Autophagy is involved in the breakdown of intracellular lipids and the maintenance of healthy mitochondria in NAFLD. However, the mechanisms underlying autophagy dysregulation in NAFLD remain unclear. Here, we demonstrate that the hepatic expression level of Thrap3 was significantly increased in NAFLD conditions. Liver-specific Thrap3 knockout improved lipid accumulation and metabolic properties in a high-fat diet (HFD)-induced NAFLD model. Furthermore, Thrap3 deficiency enhanced autophagy and mitochondrial function. Interestingly, Thrap3 knockout increased the cytosolic translocation of AMPK from the nucleus and enhanced its activation through physical interaction. The translocation of AMPK was regulated by direct binding with AMPK and the C-terminal domain of Thrap3. Our results indicate a role for Thrap3 in NAFLD progression and suggest that Thrap3 is a potential target for NAFLD treatment.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/genética , Dieta Hiperlipídica/efeitos adversos , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Células Hep G2
18.
Nucleic Acids Res ; 51(11): 5584-5602, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37140056

RESUMO

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.


Assuntos
Reparo do DNA , Exodesoxirribonucleases , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Exodesoxirribonucleases/metabolismo , Recombinação Homóloga , Proteína 2 Homóloga a MutS/metabolismo , Humanos , Linhagem Celular , DNA Helicases/metabolismo , Proteína 3 Homóloga a MutS/metabolismo
19.
PLoS One ; 18(5): e0285337, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37205694

RESUMO

Proliferating cell nuclear antigen (PCNA) is a maestro of DNA replication. PCNA forms a homotrimer and interacts with various proteins, such as DNA polymerases, DNA ligase I (LIG1), and flap endonuclease 1 (FEN1) for faithful DNA replication. Here, we identify the crucial role of Ser46-Leu47 residues of PCNA in maintaining genomic integrity using in vitro, and cell-based assays and structural prediction. The predicted PCNAΔSL47 structure shows the potential distortion of the central loop and reduced hydrophobicity. PCNAΔSL47 shows a defective interaction with PCNAWT leading to defects in homo-trimerization in vitro. PCNAΔSL47 is defective in the FEN1 and LIG1 interaction. PCNA ubiquitination and DNA-RNA hybrid processing are defective in PCNAΔSL47-expressing cells. Accordingly, PCNAΔSL47-expressing cells exhibit an increased number of single-stranded DNA gaps and higher levels of γH2AX, and sensitivity to DNA-damaging agents, highlighting the importance of PCNA Ser46-Leu47 residues in maintaining genomic integrity.


Assuntos
Replicação do DNA , Endonucleases Flap , Antígeno Nuclear de Célula em Proliferação/metabolismo , Endonucleases Flap/química , DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Genômica
20.
Hum Genomics ; 17(1): 44, 2023 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-37208785

RESUMO

BACKGROUND: Ubiquitin-related rare diseases are generally characterized by developmental delays and mental retardation, but the exact incidence or prevalence is not yet fully understood. The clinical application of next-generation sequencing for pediatric seizures and developmental delay of unknown causes has become common in studies aimed at identification of a causal gene in patients with ubiquitin-related rare diseases that cannot be diagnosed using conventional fluorescence in situ hybridization or chromosome microarray tests. Our study aimed to investigate the effects of ubiquitin-proteasome system on ultra-rare neurodevelopmental diseases, through functional identification of candidate genes and variants. METHODS: In our present work, we carried out genome analysis of a patient with clinical phenotypes of developmental delay and intractable convulsion, to identify causal mutations. Further characterization of the candidate gene was performed using zebrafish, through gene knockdown approaches. Transcriptomic analysis using whole embryos of zebrafish knockdown morphants and additional functional studies identified downstream pathways of the candidate gene affecting neurogenesis. RESULTS: Through trio-based whole-genome sequencing analysis, we identified a de novo missense variant of the ubiquitin system-related gene UBE2H (c.449C>T; p.Thr150Met) in the proband. Using zebrafish, we found that Ube2h is required for normal brain development. Differential gene expression analysis revealed activation of the ATM-p53 signaling pathway in the absence of Ube2h. Moreover, depletion of ube2h led to induction of apoptosis, specifically in the differentiated neural cells. Finally, we found that a missense mutation in zebrafish, ube2h (c.449C>T; p.Thr150Met), which mimics a variant identified in a patient with neurodevelopmental defects, causes aberrant Ube2h function in zebrafish embryos. CONCLUSION: A de novo heterozygous variant in the UBE2H c.449C>T (p.Thr150Met) has been identified in a pediatric patient with global developmental delay and UBE2H is essential for normal neurogenesis in the brain.


Assuntos
Doenças Raras , Enzimas de Conjugação de Ubiquitina , Peixe-Zebra , Animais , Humanos , Encéfalo/metabolismo , Deficiências do Desenvolvimento , Hibridização in Situ Fluorescente , Mutação , Mutação de Sentido Incorreto/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
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