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1.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500630

RESUMO

Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL2-154 into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL2-154 becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL2-154 is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.


Assuntos
Membrana Celular/metabolismo , Necroptose/fisiologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Ácido Glutâmico/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/fisiologia
2.
Biomolecules ; 11(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34356632

RESUMO

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Relacionadas à Autofagia/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Proteínas Quinases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/metabolismo , Termodinâmica , Ubiquitina/metabolismo
3.
BMC Plant Biol ; 21(1): 382, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34412592

RESUMO

BACKGROUND: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development. RESULTS: CaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane. CONCLUSIONS: Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.


Assuntos
Capsicum/genética , Capsicum/microbiologia , Cisteína/genética , Cisteína/metabolismo , Resistência à Doença/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ralstonia solanacearum/patogenicidade , Capsicum/fisiologia , China , Resistência à Doença/fisiologia , Regulação da Expressão Gênica de Plantas
4.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360883

RESUMO

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.


Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Suplementos Nutricionais , Mitofagia/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 3/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Valeratos/farmacologia , Adenocarcinoma/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445680

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is the most common degenerative motor neuron disease in adults. About 97% of ALS patients present TDP-43 aggregates with post-translational modifications, such as hyperphosphorylation, in the cytoplasm of affected cells. GSK-3ß is one of the protein kinases involved in TDP-43 phosphorylation. Up-regulation of its expression and activity is reported on spinal cord and cortex tissues of ALS patients. Here, we propose the repurposing of Tideglusib, an in-house non-ATP competitive GSK-3ß inhibitor that is currently in clinical trials for autism and myotonic dystrophy, as a promising therapeutic strategy for ALS. With this aim we have evaluated the efficacy of Tideglusib in different experimental ALS models both in vitro and in vivo. Moreover, we observed that GSK-3ß activity is increased in lymphoblasts from sporadic ALS patients, with a simultaneous increase in TDP-43 phosphorylation and cytosolic TDP-43 accumulation. Treatment with Tideglusib decreased not only phospho-TDP-43 levels but also recovered its nuclear localization in ALS lymphoblasts and in a human TDP-43 neuroblastoma model. Additionally, we found that chronic oral treatment with Tideglusib is able to reduce the increased TDP-43 phosphorylation in the spinal cord of Prp-hTDP-43A315T mouse model. Therefore, we consider Tideglusib as a promising drug candidate for ALS, being proposed to start a clinical trial phase II by the end of the year.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Tiadiazóis/farmacologia , Idoso , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Glicogênio Sintase Quinase 3 beta/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Preparações Farmacêuticas/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Medula Espinal/metabolismo
6.
Theriogenology ; 174: 160-168, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34455243

RESUMO

Vitrification is an effective technique for fertility preservation, but is known to lead to mitochondrial dysfunction in porcine oocytes. Mitophagy is induced to rebalance mitochondrial function, a process in which reactive oxygen species (ROS) plays a role. In this study, vitrified-warmed porcine oocytes were incubated for 4 h with the oxidant AAPH or antioxidant α-tocopherol to alter ROS levels. A series of tests suggested that vitrification damaged mitochondrial structure and caused dysfunction, including blurred mitochondrial cristae, decreased mitochondrial membrane potential, decreased mtDNA copy number and increased ROS generation. This dysfunction resulted in mitophagy and the loss of embryonic developmental potential. Incubation with AAPH or α-tocopherol altered mitochondrial function and mitophagy flux status in vitrified oocytes. The PINK1/Parkin pathway was involved in oxidative stress regulation in vitrified oocytes. Under AAPH-induced oxidative stress, increased fluorescence intensity of Parkin, increased expression of PINK1, Parkin, and LC3B-II, and decreased expression of MFN2 and p62 were observed, whereas the opposite effects were induced under α-tocopherol treatment. The inhibition of ROS by α-tocopherol benefitted mitochondrial homeostasis and alleviated PINK1/Parkin-mediated mitophagy, resulting in the recovery of embryonic developmental potential in vitrified porcine oocytes. Therefore, this study provides a new mechanism for the application of antioxidants to aid the cryopreservation of porcine oocytes.


Assuntos
Mitocôndrias , Mitofagia , Animais , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Oócitos/metabolismo , Estresse Oxidativo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Suínos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
7.
Zool Res ; 42(4): 469-477, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34213093

RESUMO

Mutations of PTEN-induced kinase I (PINK1) cause early-onset Parkinson's disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients. This suggests that generating PINK1 disease models in non-human primates (NHPs) that are close to humans is essential to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both of which can reduce off-target effects without compromising on-target editing, are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA. However, western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts. We further reprogramed mutant fibroblasts into induced pluripotent stem cells (iPSCs), which showed similar ability to differentiate into dopamine (DA) neurons. Taken together, our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.


Assuntos
Modelos Animais de Doenças , Macaca fascicularis/genética , Doença de Parkinson/veterinária , Proteínas Quinases/metabolismo , Animais , Animais Recém-Nascidos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Cultura Embrionária , Transferência Embrionária , Fibroblastos/fisiologia , Mutação da Fase de Leitura , Regulação da Expressão Gênica , Macaca fascicularis/embriologia , Doenças dos Macacos/genética , Mutação , Doença de Parkinson/genética , Proteínas Quinases/genética , RNA Guia
8.
J Physiol Biochem ; 77(3): 405-417, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34212313

RESUMO

Cholesterol efflux from macrophages is the first step of reverse cholesterol transport (RCT), whose increase inhibits cholesterol accumulation and foam cell formation to suppress atherogenesis. Hesperetin has been reported to exert several protective effects on cardiovascular diseases, while little is known about the role of hesperetin and its underlying mechanism in macrophage foam cell formation. In this study, we sought to investigate the potential effects of hesperetin on foam cell formation and cholesterol efflux by using human macrophages, focusing on liver X receptor alpha (LXRα) and AMPK. We found that hesperetin treatment reduced foam cell formation, intracellular cholesterol levels and the cholesterol esterification rate, and increased cholesterol efflux in THP-1 macrophages. Hesperetin increased the levels of LXRα protein and its targets, including ABCA1, ABCG1, SR-BI, and phosphorylated-AMPK. Meanwhile, the hesperetin-induced increase in LXRα expression was further increased by the AMPK agonist and inhibited by an AMPK inhibitor. Meanwhile, hesperetin increased the levels of LXRα mRNA and its target genes, all of which were decreased in cells transfected with the AMPKα1/α2 small interfering RNA (siRNA). Furthermore, the hesperetin-induced inhibition of foam cell formation and promotion of cholesterol efflux were decreased by transfection of AMPKα1/α2 siRNA. In conclusions, We are the first to report that hesperetin activate AMPK in THP-1-derived macrophages. This activation upregulats LXRα and its targets, including ABCA1, ABCG1 and SR-BI, which significantly inhibits foam cell formation and promotes cholesterol efflux. Our results highlight the therapeutic potential of hesperetin to possibly reduce foam cell formation. This new mechanism might contribute the anti-atherogenic effects of hesperetin.


Assuntos
Colesterol/metabolismo , Células Espumosas/efeitos dos fármacos , Hesperidina/farmacologia , Aterosclerose/metabolismo , Células Espumosas/patologia , Humanos , Receptores X do Fígado/metabolismo , Proteínas Quinases/metabolismo , Células THP-1
9.
Chem Commun (Camb) ; 57(66): 8154-8157, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34313270

RESUMO

Herein, we demonstrate that the active surface of nanoceria can be fine-tuned by phosphorylated peptides. Accordingly, a colorimetric and fluorometric dual-readout strategy is rationally developed for assaying protein kinase activity. This feature not only enables the versatile monitoring of peptide phosphorylation but also broadens the application scope of nanoceria.


Assuntos
Cério/metabolismo , Colorimetria , Fluorometria , Proteínas Quinases/metabolismo , Cério/química , Humanos , Células MCF-7 , Proteínas Quinases/química , Propriedades de Superfície
10.
Biomolecules ; 11(7)2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209255

RESUMO

Various environmental stimuli, including oxidative stress, could lead to granulosa cell (GC) death through mitophagy. Recently, it was reported that melatonin (MEL) has a significant effect on GC survival during oxidative damage. Here, we found that MEL inhibited oxidative stress-induced mitophagy to promote GC survival. The loss of cell viability upon H2O2 exposure was significantly restored after MEL treatment. Concomitantly, MEL inhibited the activation of mitophagy during oxidative stress. Notably, blocking mitophagy repressed GC death caused by oxidative stress. However, MEL cannot further restore viability of cells treated with mitophagy inhibitor. Moreover, PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase, was inhibited by MEL during oxidative stress. As a result, the E3 ligase Parkin failed to translocate to mitochondria, leading to impaired mitochondria clearance. Using RNAi to knock down PINK1 expression, we further verified the role of the MEL-PINK1-Parkin (MPP) pathway in maintaining GC survival by suppressing mitophagy. Our findings not only clarify the protective mechanisms of MEL against oxidative damage in GCs, but also extend the understanding about how circadian rhythms might influence follicles development in the ovary. These findings reveal a new mechanism of melatonin in defense against oxidative damage to GCs by repressing mitophagy, which may be a potential therapeutic target for anovulatory disorders.


Assuntos
Células da Granulosa/metabolismo , Melatonina/farmacologia , Mitofagia/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células da Granulosa/fisiologia , Peróxido de Hidrogênio/farmacologia , Masculino , Melatonina/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Substâncias Protetoras/farmacologia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
11.
Cell Death Dis ; 12(7): 661, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210956

RESUMO

Bladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Neoplasias da Bexiga Urinária/enzimologia , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Bases de Dados Genéticas , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas Quinases/genética , Transdução de Sinais , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
12.
Nat Cell Biol ; 23(7): 796-807, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239062

RESUMO

Inflammatory bowel diseases present with elevated levels of intestinal epithelial cell (IEC) death, which compromises the gut barrier, activating immune cells and triggering more IEC death. The endogenous signals that prevent IEC death and break this vicious cycle, allowing resolution of intestinal inflammation, remain largely unknown. Here we show that prostaglandin E2 signalling via the E-type prostanoid receptor 4 (EP4) on IECs represses epithelial necroptosis and induces resolution of colitis. We found that EP4 expression correlates with an improved IBD outcome and that EP4 activation induces a transcriptional signature consistent with resolution of intestinal inflammation. We further show that dysregulated necroptosis prevents resolution, and EP4 agonism suppresses necroptosis in human and mouse IECs. Mechanistically, EP4 signalling on IECs converges on receptor-interacting protein kinase 1 to suppress tumour necrosis factor-induced activation and membrane translocation of the necroptosis effector mixed-lineage kinase domain-like pseudokinase. In summary, our study indicates that EP4 promotes the resolution of colitis by suppressing IEC necroptosis.


Assuntos
Colite/metabolismo , Colo/metabolismo , Dinoprostona/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Necroptose , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Colite/induzido quimicamente , Colite/patologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose/efeitos dos fármacos , Organoides , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de Sinais
14.
Mol Genet Genomics ; 296(5): 1135-1145, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196769

RESUMO

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.


Assuntos
Proteínas Fúngicas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Magnaporthe/enzimologia , Dioxóis/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 12(1): 4472, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294691

RESUMO

Alzheimer's disease (AD) is influenced by both genetic and environmental factors; thus, brain epigenomic alterations may provide insights into AD pathogenesis. Multiple array-based Epigenome-Wide Association Studies (EWASs) have identified robust brain methylation changes in AD; however, array-based assays only test about 2% of all CpG sites in the genome. Here, we develop EWASplus, a computational method that uses a supervised machine learning strategy to extend EWAS coverage to the entire genome. Application to six AD-related traits predicts hundreds of new significant brain CpGs associated with AD, some of which are further validated experimentally. EWASplus also performs well on data collected from independent cohorts and different brain regions. Genes found near top EWASplus loci are enriched for kinases and for genes with evidence for physical interactions with known AD genes. In this work, we show that EWASplus implicates additional epigenetic loci for AD that are not found using array-based AD EWASs.


Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Doença de Alzheimer/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Coortes , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Aprendizado de Máquina Supervisionado
16.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
17.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205396

RESUMO

Members of the lectin receptor-like kinase (LecRLKs) family play a vital role in innate plant immunity. Few members of the LecRLKs family have been characterized in rice and Arabidopsis, respectively. However, little literature is available about LecRLKs and their role against fungal infection in cucumber. In this study, 60 putative cucumber LecRLK (CsLecRLK) proteins were identified using genome-wide analysis and further characterized into L-type LecRLKs (24) and G-type LecRLKs (36) based on domain composition and phylogenetic analysis. These proteins were allocated to seven cucumber chromosomes and found to be involved in the expansion of the CsLecRLK gene family. Subcellular localization of CsaLecRLK9 and CsaLecRLK12 showed green fluorescence signals in the plasma membrane of leaves. The transcriptional profiling of CsLecRLK genes showed that L-type LecRLKs exhibited functional redundancy as compared to G-type LecRLKs. The qRT-PCR results indicated that both L- and G-type LecRLKs showed significant response against plant growth-promoting fungi (PGPF-Trichoderma harzianum Rifai), powdery mildew pathogen (PPM-Golovinomyces orontii (Castagne) V.P. Heluta), and combined (PGPF+PPM) treatments. The findings of this study contribute to a better understanding of the role of cucumber CsLecRLK genes in response to PGPF, PPM, and PGPF+PPM treatments and lay the basis for the characterization of this important functional gene family.


Assuntos
Cucumis sativus/enzimologia , Erysiphe/imunologia , Imunidade Vegetal , Proteínas Quinases/genética , Estresse Fisiológico , Cromossomos de Plantas , Cucumis sativus/genética , Cucumis sativus/imunologia , Perfilação da Expressão Gênica , Genes de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo
18.
FASEB J ; 35(8): e21771, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34275172

RESUMO

Impaired mitochondrial fusion, due in part to decreased mitofusin 2 (Mfn2) expression, contributes to unrestricted cell proliferation and apoptosis-resistance in hyperproliferative diseases like pulmonary arterial hypertension (PAH) and non-small cell lung cancer (NSCLC). We hypothesized that Mfn2 levels are reduced due to increased proteasomal degradation of Mfn2 triggered by its phosphorylation at serine 442 (S442) and investigated the potential kinase mediators. Mfn2 expression was decreased and Mfn2 S442 phosphorylation was increased in pulmonary artery smooth muscle cells from PAH patients and in NSCLC cells. Mfn2 phosphorylation was mediated by PINK1 and protein kinase A (PKA), although only PINK1 expression was increased in these diseases. We designed a S442 phosphorylation deficient Mfn2 construct (PD-Mfn2) and a S442 constitutively phosphorylated Mfn2 construct (CP-Mfn2). The effects of these modified Mfn2 constructs on Mfn2 expression and biological function were compared with those of the wildtype Mfn2 construct (WT-Mfn2). WT-Mfn2 increased Mfn2 expression and mitochondrial fusion in both PAH and NSCLC cells resulting in increased apoptosis and decreased cell proliferation. Compared to WT-Mfn2, PD-Mfn2 caused greater Mfn2 expression, suppression of proliferation, apoptosis induction, and cell cycle arrest. Conversely, CP-Mfn2 caused only a small increase in Mfn2 expression and did not restore mitochondrial fusion, inhibit cell proliferation, or induce apoptosis. Silencing PINK1 or PKA, or proteasome blockade using MG132, increased Mfn2 expression, enhanced mitochondrial fusion and induced apoptosis. In a xenotransplantation NSCLC model, PD-Mfn2 gene therapy caused greater tumor regression than did therapy with WT-Mfn2. Mfn2 deficiency in PAH and NSCLC reflects proteasomal degradation triggered by Mfn2-S442 phosphorylation by PINK1 and/or PKA. Inhibiting Mfn2 phosphorylation has potential therapeutic benefit in PAH and lung cancer.


Assuntos
Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Hipertensão Pulmonar/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Proteólise , Células A549 , Animais , GTP Fosfo-Hidrolases/genética , Humanos , Hipertensão Pulmonar/genética , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Quinases/genética
19.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299248

RESUMO

Parkinson's disease (PD) is a complex and progressive neurodegenerative disorder with a prevalence of approximately 0.5-1% among those aged 65-70 years. Although most of its clinical manifestations are due to a loss of dopaminergic neurons, the PD etiology is largely unknown. PD is caused by a combination of genetic and environmental factors, and the exact interplay between genes and the environment is still debated. Several biological processes have been implicated in PD, including mitochondrial or lysosomal dysfunctions, alteration in protein clearance, and neuroinflammation, but a common molecular mechanism connecting the different cellular alterations remains incompletely understood. Accumulating evidence underlines a significant role of lipids in the pathological pathways leading to PD. Beside the well-described lipid alteration in idiopathic PD, this review summarizes the several lipid alterations observed in experimental models expressing PD-related genes and suggests a possible scenario in relationship to the molecular mechanisms of neuronal toxicity. PD could be considered a lipid-induced proteinopathy, where alteration in lipid composition or metabolism could induce protein alteration-for instance, alpha-synuclein accumulation-and finally neuronal death.


Assuntos
Metabolismo dos Lipídeos/genética , Lipídeos/fisiologia , Doença de Parkinson/genética , Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
20.
Int J Mol Sci ; 22(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299293

RESUMO

Brassinosteroids (BRs) are growth-promoting phytohormones that can efficiently function by exogenous application at micromolar concentrations or by endogenous fine-tuning of BR-related gene expression, thus, precisely controlling BR signal strength is a key factor in exploring the agricultural potential of BRs. BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor, is the rate-limiting enzyme in BR signal transduction, and the phosphorylation of each phosphorylation site of SlBRI1 has a distinct effect on BR signal strength and botanic characteristics. We recently demonstrated that modifying the phosphorylation sites of tomato SlBRI1 could improve the agronomic traits of tomato to different extents; however, the associated agronomic potential of SlBRI1 phosphorylation sites in tomato has not been fully exploited. In this research, the biological functions of the phosphorylation site threonine-825 (Thr-825) of SlBRI1 in tomato were investigated. Phenotypic analysis showed that, compared with a tomato line harboring SlBRI1, transgenic tomato lines expressing SlBRI1 with a nonphosphorylated Thr-825 (T825A) exhibited a larger plant size due to a larger cell size and higher yield, including a greater plant height, thicker stems, longer internodal lengths, greater plant expansion, a heavier fruit weight, and larger fruits. Molecular analyses further indicated that the autophosphorylation level of SlBRI1, BR signaling, and gibberellic acid (GA) signaling were elevated when SlBRI1 was dephosphorylated at Thr-825. Taken together, the results demonstrated that dephosphorylation of Thr-825 can enhance the functions of SlBRI1 in BR signaling, which subsequently activates and cooperates with GA signaling to stimulate cell elongation and then leads to larger plants and higher yields per plant. These results also highlight the agricultural potential of SlBRI1 phosphorylation sites for breeding high-yielding tomato varieties through precise control of BR signaling.


Assuntos
Brassinosteroides/metabolismo , Lycopersicon esculentum/genética , Proteínas Serina-Treonina Quinases/genética , Tamanho Celular , Frutas/metabolismo , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Treonina/metabolismo
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