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OBJECTIVE: Health-promoting behaviors carry substantial significance for miners' overall health and well-being. This study aimed to examine the association between cumulative risk (CR) and miners' health-promoting behaviors and test the mediating role of health beliefs in this relationship. METHODS: Data were collected from a sequential survey conducted among 712 frontline miners (Mage=41.7 ± 10.1 years) in China. The survey entailed online questionnaire measurements at three distinct time points, each spaced two weeks apart. This study utilized the conceptual model of health-promoting behaviors, the CR model, and structural equation modeling in the analysis of relationships. RESULTS: CR was negatively related to health-promoting behaviors, with a negative acceleration effect. CR was positively associated with perceived threat in a gradient effect, while negatively associated with perceived benefits in a gradient effect. Furthermore, CR was negatively related to self-efficacy, following a negative acceleration effect. Perceived threat, perceived benefits, and self-efficacy emerged as significant mediators in the relationship between CR and health-promoting behaviors. CONCLUSION: This study highlights the critical role of considering both CR and health beliefs in shaping miners' health-promoting behaviors. Understanding these dynamics is pivotal for developing interventions to enhance miners' health and well-being.
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A multichannel high-dimensional data encoding/decoding scheme based on composite elliptic optical vortex (EOV) arrays is proposed. By exploiting the rotation angle of the EOV, a 4 × 4 composite EOV array is used for high-dimensional data encoding. The conjugate symmetric extension Fourier computer-generated holography algorithm with controllable reconstruction focus is used to assign different reconstruction focus to the data of the three channels (R, G, and B) of the color image. Then, the data of the three channels is transmitted simultaneously by a single hologram to further improve the transmission efficiency. At the receiver, the initial information sequence is decoded by directly identifying the captured intensity patterns with a deep learning-based convolutional neural network. In the experiment, a 128 × 128-pixel color image is successfully transmitted, which confirms the feasibility of our proposed encoding/decoding scheme. This method has great potential for future high-capacity optical communications.
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Nonalcoholic fatty liver disease (NAFLD) is substantiated by the reprogramming of liver metabolic pathways that disrupts the homeostasis of lipid and glucose metabolism and thus promotes the progression of the disease. The metabolic pathways associated with NAFLD are regulated at different levels from gene transcription to various post-translational modifications including ubiquitination. Here, we used a novel orthogonal ubiquitin transfer platform to identify pyruvate dehydrogenase A1 (PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two important enzymes that regulate glycolysis and ketogenesis, as substrates of E3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3A accelerated the degradation of PDHA1 and promoted glycolytic activities in HEK293 cells. Furthermore, a high-fat diet suppressed the expression of UBE3A in the mouse liver, which was associated with increased ACAT1 protein levels, while forced expression of UBE3A in the mouse liver resulted in decreased ACAT1 protein contents. As a result, the mice with forced expression of UBE3A in the liver exhibited enhanced accumulation of triglycerides, cholesterol, and ketone bodies. These results reveal the role of UBE3A in NAFLD development by inducing the degradation of ACAT1 in the liver and promoting lipid storage. Overall, our work uncovers an important mechanism underlying the regulation of glycolysis and lipid metabolism through UBE3A-mediated ubiquitination of PDHA1 and ACAT1 to regulate their stabilities and enzymatic activities in the cell.
Assuntos
Acetiltransferases , Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Acetiltransferases/genética , Células HEK293 , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Oxirredutases/metabolismo , Lipídeos , Acetil-CoA C-Acetiltransferase/genéticaRESUMO
Lung transplantation is a life-saving treatment for both chronic end-stage lung diseases and acute respiratory distress syndrome, including those caused by infectious agents like COVID-19. Despite its increasing utilization, outcomes post-lung transplantation are worse than other solid organ transplants. Primary graft dysfunction (PGD)-a condition affecting more than half of the recipients post-transplantation-is the chief risk factor for post-operative mortality, transplant-associated multi-organ dysfunction, and long-term graft loss due to chronic rejection. While donor-specific antibodies targeting allogenic human leukocyte antigens have been linked to transplant rejection, the role of recipient's pre-existing immunoglobulin G autoantibodies against lung-restricted self-antigens (LRA), like collagen type V and k-alpha1 tubulin, is less understood in the context of lung transplantation. Recent studies have found an increased risk of PGD development in lung transplant recipients with LRA. This review will synthesize past and ongoing research-utilizing both mouse models and human subjects-aimed at unraveling the mechanisms by which LRA heightens the risk of PGD. Furthermore, it will explore prospective approaches designed to mitigate the impact of LRA on lung transplant patients.
RESUMO
Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1ß-mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.
Assuntos
Interleucina-1beta/metabolismo , Transplante de Pulmão , Disfunção Primária do Enxerto , Traumatismo por Reperfusão , Animais , Autoanticorpos , Proteínas do Sistema Complemento , Humanos , Imunoglobulina G , Pulmão/patologia , Transplante de Pulmão/efeitos adversos , Lectinas de Ligação a Manose , Camundongos , Disfunção Primária do Enxerto/genética , Disfunção Primária do Enxerto/metabolismo , Receptores Fc , Traumatismo por Reperfusão/patologiaRESUMO
Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1ß production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1ß production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R-dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1ß-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1ß, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.
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Transplante de Pulmão , Macrófagos Alveolares/imunologia , Monócitos/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Disfunção Primária do EnxertoRESUMO
Recently, a new form of laser beam, called the "centrosymmetric optical vortex" (CSOV), has been proposed. We employ the modified calculation method for studying the propagation of the CSOV beam, which is constructed via four canonical optical vortices with different topological charges. The speed of calculation is more convenient and faster than the usual means by using the diffraction integral directly. With a modified calculation method, the propagation properties of the CSOV in free space are illustrated and analyzed using numerical examples. Several parameters influencing the CSOV beams and the ABCD optical system are discussed in detail. Our experimental results are consistent with theoretical predictions. Furthermore, the orbital angular momentum states and density are also studied. The research results are expected to provide a basis for the application of CSOV beams in beam reshaping, optical trapping, and rotating microparticle manipulation, particularly in separating cells.
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Lung transplant is a definitive treatment for several end-stage lung diseases. However, the high incidence of allograft rejection limits the overall survival following lung transplantation. Traditionally, alloimmunity directed against human leukocyte antigens (HLA) has been implicated in transplant rejection. Recently, the clinical impact of non-HLA lung-restricted antibodies (LRA) has been recognized and extensive research has demonstrated that they may play a dominant role in the development of lung allograft rejection. The immunogenic lung-restricted antigens that have been identified include amongst others, collagen type I, collagen type V, and k-alpha 1 tubulin. Pre-existing antibodies against these lung-restricted antigens are prevalent in patients undergoing lung transplantation and have emerged as one of the predominant risk factors for primary graft dysfunction which limits short-term survival following lung transplantation. Additionally, LRA have been shown to predispose to chronic lung allograft rejection, the predominant cause of poor long-term survival. This review will discuss ongoing research into the mechanisms of development of LRA as well as the pathogenesis of associated lung allograft injury.
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Autoanticorpos/metabolismo , Bronquiolite Obliterante/imunologia , Rejeição de Enxerto/imunologia , Transplante de Pulmão , Pulmão/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Colágeno Tipo I/imunologia , Colágeno Tipo V/imunologia , Humanos , Especificidade de Órgãos , Tubulina (Proteína)/imunologiaRESUMO
Hybridly polarized Laguerre-Gaussian vector beams (HPLGVBs) with zero radial index are obtained based on the third type of Laguerre-Gaussian vector beams. Polarization distributions of HPLGVBs are controlled by the phase retardation of a wave plate. The ellipticity angle and polarization orientation angle are used to describe the polarization distributions of the HPLGVBs. The electric field intensity distributions of tightly focused HPLGVBs are analyzed in the focal plane by the Richards-Wolf vectorial diffraction method. It is found that the tightly focused HPLGVBs have a focal shift phenomenon. The dependence of the focal shift of the HPLGVBs on the different parameters is discussed in detail. The simulation results show that the magnitude of the focal shift is related to the polarization distribution, and the focal shift is quite obvious for large azimuthal mode index, long focal length, large numerical aperture, and narrow beam waist. The effective control of the focal shift will have great potential applications in optical micromanipulation.
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E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and ß-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.
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Proteínas Supressoras de Tumor/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Bacteriófagos , Biocatálise , Quinase 4 Dependente de Ciclina/metabolismo , Estresse do Retículo Endoplasmático , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Proteólise , Reprodutibilidade dos Testes , Transdução de Sinais , Especificidade por Substrato , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , UbiquitinaçãoRESUMO
E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, ß-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.
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Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Sanguíneas/metabolismo , Proteína Quinase CDC2/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteômica , Saccharomyces cerevisiae , Enzimas Ativadoras de Ubiquitina , beta Catenina/metabolismoRESUMO
Ubiquitination plays critical roles in the regulation of oncoproteins and tumor suppressors during carcinogenesis. The two ubiquitin activating enzymes (E1) in human genome, UBA1 and UBA6, initiate ubiquitination by ATP-dependent activation of ubiquitin. Recent evidence suggests that UBA1 and UBA6 play partially overlapped yet distinct roles in controlling the proteome. Here we demonstrate that ubiquitination pathways initiated specifically by UBA6 set a suppressive barrier against critical steps of mammary carcinogenesis such as loss of polarity, anoikis resistance and epithelial-mesenchymal transition (EMT). Mammary epithelial MCF-10A cells expressing shRNA against UBA6 fail in establishing cell cycle arrest in response to detachment from extracellular matrix, confluency with fully engaged cell-cell contact or growth factor deprivation. Moreover, UBA6-deficient MCF-10A cells undergo spontaneous EMT under growth factor deprivation and exhibit accelerated kinetics of TGF-ß-induced EMT. The Rho-GTPase CDC42 is one of the specific targets of UBA6-initiated ubiquitination and plays a key role in the function of UBA6 in controlling epithelial homeostasis, since a CDC42 inhibitor, ML141, rescues UBA6-deficient cells from the EMT phenotype. Immunohistochemical analysis of human breast cancer tissues demonstrates that 38% of invasive carcinomas express low or undetectable expression of UBA6, suggesting that downregulation of this non-canonical E1 plays a role in breast cancer development.
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Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.
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Diferenciação Celular/fisiologia , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Proteínas CELF1/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Proteínas do Citoesqueleto/metabolismo , Epitélio/crescimento & desenvolvimento , Células HEK293 , Humanos , Morfogênese/fisiologia , Mutagênese Insercional , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Especificidade por Substrato/fisiologia , Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
BACKGROUND: The HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells. METHODS: Human breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines. RESULTS: Immunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs. CONCLUSIONS: This study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.
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Carcinoma Ductal de Mama/enzimologia , Carcinoma Intraductal não Infiltrante/enzimologia , MicroRNAs/metabolismo , Proteína do Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Mutação , Fenótipo , Interferência de RNA , Proteína do Retinoblastoma/genética , Transdução de Sinais , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The Ku70-Ku80 ring complex encloses DNA ends to facilitate telomere maintenance and DNA break repair. Many studies focus on the ring-forming regions of subunits Ku70 and Ku80. Less is known about the Ku70 C-terminal tail, which lies outside the ring. Our results suggest that this region is responsible for dynamic sumoylation of Yku70 upon DNA association in budding yeast. Mutating a cluster of five lysines in this region largely eliminates Yku70 sumoylation. Chromatin immunoprecipitation analyses show that yku70 mutants with these lysines replaced by arginines exhibit reduced Ku-DNA association at both telomeres and internal DNA breaks. Consistent with this physical evidence, Yku70 sumoylation deficiency is associated with impaired ability to block DNA end resection and suppression of multiple defects caused by inefficient resection. Correlating with these, yku70 mutants with reduced sumoylation levels exhibit shorter telomeres, increased G overhang levels, and altered levels of non-homologous end joining. We also show that diminution of sumoylation does not affect Yku70 protein levels or its interactions with protein and RNA partners. These results suggest a model whereby Yku70 sumoylation upon DNA association strengthens Ku-DNA interaction to promote multiple functions of Ku.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Regulação Fúngica da Expressão Gênica , Lisina/química , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Sumoilação , Telômero/ultraestruturaRESUMO
The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.
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Apoptose , Proteínas Culina/biossíntese , Hipóxia/enzimologia , Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Animais , Proteínas Reguladoras de Apoptose/biossíntese , Hipóxia Celular , Proteínas Culina/genética , Dano ao DNA , Humanos , Hipóxia/genética , Hipóxia/patologia , Células PC12 , Ratos , Transcrição Gênica , Ubiquitina-Proteína Ligases/genéticaRESUMO
Telomere length homeostasis is an important aspect of telomere biology. Here, we show that SUMOylation limits telomere length and targets multiple telomere proteins in Saccharomyces cerevisiae. A main target is Cdc13, which both positively and negatively regulates telomerase and confers end protection. We demonstrate that Cdc13 SUMOylation restrains telomerase functions by promoting Cdc13 interaction with the telomerase inhibitor Stn1 without affecting end protection. Mutation of the Cdc13 SUMOylation site (cdc13-snm) lengthens telomeres and reduces the Stn1 interaction, whereas Cdc13-SUMO fusion has the opposite effects. cdc13-snm's effect on telomere length is epistatic with stn1, but not with yku70, tel1 or est1 alleles, and is suppressed by Stn1 overexpression. Cdc13 SUMOylation peaks in early-mid S phase, prior to its known Cdk1-mediated phosphorylation, and the two modifications act antagonistically, suggesting that the opposite roles of Cdc13 in telomerase regulation can be separated temporally and regulated by distinct modifications.
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Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Epistasia Genética , Homeostase , Fosforilação , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Sumoilação , Proteínas de Ligação a Telômeros/químicaRESUMO
Hydroxyurea (HU) inhibits ribonucleotide reductase (RNR), which catalyzes the rate-limiting synthesis of deoxyribonucleotides for DNA replication. HU is used to treat HIV, sickle-cell anemia and some cancers. We found that, compared with vector control cells, low levels of alpha-synuclein (α-syn) protect S. cerevisiae cells from the growth inhibition and reactive oxygen species (ROS) accumulation induced by HU. Analysis of this effect using different α-syn mutants revealed that the α-syn protein functions in the nucleus and not the cytoplasm to modulate S-phase checkpoint responses: α-syn up-regulates histone acetylation and RNR levels, maintains helicase minichromosome maintenance protein complexes (Mcm2-7) on chromatin and inhibits HU-induced ROS accumulation. Strikingly, when residues 2-10 or 96-140 are deleted, this protective function of α-syn in the nucleus is abolished. Understanding the mechanism by which α-syn protects against HU could expand our knowledge of the normal function of this neuronal protein.
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Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Hidroxiureia/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo , Núcleo Celular/genética , Ribonucleotídeo Redutases/metabolismo , Saccharomyces cerevisiae/genética , alfa-Sinucleína/genéticaRESUMO
The Smc5/6 complex is an evolutionarily conserved chromosomal ATPase required for cell growth and DNA repair. Its Mms21 subunit supports both functions by docking to the arm region of Smc5 and providing SUMO ligase activity. Here, we report the crystal structure of Mms21 in complex with the Smc5 arm. Our structure revealed two distinct structural and functional domains of the Smc5-bound Mms21: its N-terminal half is dedicated to Smc5 binding by forming a helix bundle with a coiled-coil structure of Smc5; its C-terminal half includes the SUMO ligase domain, which adopts a new type of RING E3 structure. Mutagenesis and structural analyses showed that the Mms21-Smc5 interface is required for cell growth and resistance to DNA damage, while the unique Mms21 RING domain confers specificity to the SUMO E2-E3 interaction. Through structure-based dissection of Mms21 functions, our studies establish a framework for understanding its roles in the Smc5/6 complex.