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1.
Carbohydr Polym ; 332: 121889, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38431406

RESUMO

Metabolic alterations within mitochondria, encompassing processes such as autophagy and energy metabolism, play a pivotal role in facilitating the swift proliferation, invasion, and metastasis of cancer cells. Despite this, there is a scarcity of currently available medications with proven anticancer efficacy through the modulation of mitochondrial dysfunction in a clinical setting. Here, we introduce the structural characteristics of RN0D, a galactoglucan isolated and purified from Panax notoginseng flowers, mainly composed of ß-1,4-galactan and ß-1,3/1,6-glucan. RN0D demonstrates the capacity to induce mitochondrial impairment in cancer cells, leading to the accumulation of reactive oxygen species, initiation of mitophagy, and reduction in both mitochondrial number and size. This sequence of events ultimately results in the inhibition of mitochondrial and glycolytic bioenergetics, culminating in the demise of cancer cells due to adenosine triphosphate (ATP) deprivation. Notably, the observed bioactivity is attributed to RN0D's direct targeting of Galectin-3, as affirmed by surface plasmon resonance studies. Furthermore, RN0D is identified as an activator of the PTEN-induced kinase 1 (PINK1)/Parkin pathway, ultimately instigating cytotoxic mitophagy in tumor cells. This comprehensive study substantiates the rationale for advancing RN0D as a potentially efficacious anticancer therapeutic.


Assuntos
Neoplasias , Panax notoginseng , Polissacarídeos Bacterianos , Humanos , Mitofagia , Galactanos , Glucanos , Morte Celular , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases/metabolismo
2.
Nat Commun ; 15(1): 2210, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472229

RESUMO

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.


Assuntos
Proteínas Quinases , Transdução de Sinais , Proteínas Quinases/metabolismo , Fosforilação , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicação do DNA , Dano ao DNA , DNA de Cadeia Simples , Reparo do DNA
3.
Cells ; 13(5)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38474413

RESUMO

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.


Assuntos
Proteínas Ativadoras de GTPase , Mitofagia , Miócitos Cardíacos , Fosfatos de Fosfatidilinositol , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Actinas , Proteínas Ativadoras de GTPase/metabolismo , Mitofagia/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
4.
Immunity ; 57(3): 429-445, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38479360

RESUMO

Diverse inflammatory conditions, from infections to autoimmune disease, are often associated with cellular damage and death. Apoptotic cell death has evolved to minimize its inflammatory potential. By contrast, necrotic cell death via necroptosis and pyroptosis-driven by membrane-damaging MLKL and gasdermins, respectively-can both initiate and propagate inflammatory responses. In this review, we provide insights into the function and regulation of MLKL and gasdermin necrotic effector proteins and drivers of plasma membrane rupture. We evaluate genetic evidence that MLKL- and gasdermin-driven necrosis may either provide protection against, or contribute to, disease states in a context-dependent manner. These cumulative insights using gene-targeted mice underscore the necessity for future research examining pyroptotic and necroptotic cell death in human tissue, as a basis for developing specific necrotic inhibitors with the potential to benefit a spectrum of pathological conditions.


Assuntos
Apoptose , Gasderminas , Humanos , Animais , Camundongos , Necrose/metabolismo , Apoptose/fisiologia , Piroptose/fisiologia , Morte Celular , Inflamassomos/metabolismo , Proteínas Quinases/metabolismo
5.
Int J Mol Sci ; 25(5)2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38474242

RESUMO

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.


Assuntos
Ceramidas , Corantes Fluorescentes , Proteínas Quinases , Ceramidas/metabolismo , Transdução de Sinais/fisiologia , Fagocitose
6.
Nat Commun ; 15(1): 1962, 2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38438384

RESUMO

Myelinated axons form long-range connections that enable rapid communication between distant brain regions, but how genetics governs the strength and organization of these connections remains unclear. We perform genome-wide association studies of 206 structural connectivity measures derived from diffusion magnetic resonance imaging tractography of 26,333 UK Biobank participants, each representing the density of myelinated connections within or between a pair of cortical networks, subcortical structures or cortical hemispheres. We identify 30 independent genome-wide significant variants after Bonferroni correction for the number of measures studied (126 variants at nominal genome-wide significance) implicating genes involved in myelination (SEMA3A), neurite elongation and guidance (NUAK1, STRN, DPYSL2, EPHA3, SEMA3A, HGF, SHTN1), neural cell proliferation and differentiation (GMNC, CELF4, HGF), neuronal migration (CCDC88C), cytoskeletal organization (CTTNBP2, MAPT, DAAM1, MYO16, PLEC), and brain metal transport (SLC39A8). These variants have four broad patterns of spatial association with structural connectivity: some have disproportionately strong associations with corticothalamic connectivity, interhemispheric connectivity, or both, while others are more spatially diffuse. Structural connectivity measures are highly polygenic, with a median of 9.1 percent of common variants estimated to have non-zero effects on each measure, and exhibited signatures of negative selection. Structural connectivity measures have significant genetic correlations with a variety of neuropsychiatric and cognitive traits, indicating that connectivity-altering variants tend to influence brain health and cognitive function. Heritability is enriched in regions with increased chromatin accessibility in adult oligodendrocytes (as well as microglia, inhibitory neurons and astrocytes) and multiple fetal cell types, suggesting that genetic control of structural connectivity is partially mediated by effects on myelination and early brain development. Our results indicate pervasive, pleiotropic, and spatially structured genetic control of white-matter structural connectivity via diverse neurodevelopmental pathways, and support the relevance of this genetic control to healthy brain function.


Assuntos
Conectoma , Adulto , Humanos , Estudo de Associação Genômica Ampla , Semaforina-3A , Genes Reguladores , Encéfalo/diagnóstico por imagem , Proteínas Quinases , Proteínas Repressoras , Proteínas dos Microfilamentos , Peptídeos e Proteínas de Sinalização Intracelular
7.
Nat Commun ; 15(1): 2028, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459051

RESUMO

Copine proteins are highly conserved and ubiquitously found in eukaryotes, and their indispensable roles in different species were proposed. However, their exact function remains unclear. The phytohormone brassinosteroids (BRs) play vital roles in plant growth, development and environmental responses. A key event in effective BR signaling is the formation of functional BRI1-SERK receptor complex and subsequent transphosphorylation upon ligand binding. Here, we demonstrate that BONZAI (BON) proteins, which are plasma membrane-associated copine proteins, are critical components of BR signaling in both the monocot maize and the dicot Arabidopsis. Biochemical and molecular analyses reveal that BON proteins directly interact with SERK kinases, thereby ensuring effective BRI1-SERK interaction and transphosphorylation. This study advances the knowledge on BR signaling and provides an important target for optimizing valuable agronomic traits, it also opens a way to study steroid hormone signaling and copine proteins of eukaryotes in a broader perspective.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Transporte , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Zea mays/genética , Zea mays/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo
8.
Anal Chem ; 96(11): 4455-4462, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38458998

RESUMO

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.


Assuntos
Mitógenos , Proteínas Quinases , Espectrometria de Massas em Tandem/métodos , Processamento de Proteína Pós-Traducional , Peptídeos e Proteínas de Sinalização Intercelular , Íons
9.
Cell Biol Toxicol ; 40(1): 16, 2024 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-38472656

RESUMO

Intervertebral disc degeneration (IVDD) is an aging disease that results in a low quality of life and heavy socioeconomic burden. The mitochondrial unfolded protein response (UPRmt) take part in various aging-related diseases. Our research intents to explore the role and underlying mechanism of UPRmt in IVDD. Nucleus pulposus (NP) cells were exposed to IL-1ß and nicotinamide riboside (NR) served as UPRmt inducer to treat NP cells. Detection of ATP, NAD + and NADH were used to determine the function of mitochondria. MRI, Safranin O-fast green and Immunohistochemical examination were used to determine the degree of IVDD in vivo. In this study, we discovered that UPRmt was increased markedly in the NP cells of human IVDD tissues than in healthy controls. In vitro, UPRmt and mitophagy levels were promoted in NP cells treated with IL-1ß. Upregulation of UPRmt by NR and Atf5 overexpression inhibited NP cell apoptosis and further improved mitophagy. Silencing of Pink1 reversed the protective effects of NR and inhibited mitophagy induced by the UPRmt. In vivo, NR might attenuate the degree of IDD by activating the UPRmt in rats. In summary, the UPRmt was involved in IVDD by regulating Pink1-induced mitophagy. Mitophagy induced by the UPRmt might be a latent treated target for IVDD.


Assuntos
Degeneração do Disco Intervertebral , Mitofagia , Animais , Humanos , Ratos , Fatores Ativadores da Transcrição/metabolismo , Fatores Ativadores da Transcrição/farmacologia , Apoptose , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases/metabolismo , Qualidade de Vida , Ratos Sprague-Dawley
10.
J Cell Mol Med ; 28(7): e18172, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38494837

RESUMO

M1 macrophage polarization and synovitis play an important role in the pathogenesis of temporomandibular joint osteoarthritis (TMJOA). Reduced molecular weight of hyaluronic acid (HA) in synovial fluid of patients with TMJOA. In addition, high molecular weight hyaluronic acid (HMW-HA) is often used clinically to treat TMJ inflammation. As a pattern recognition receptor of the cytoplasm, ALPK1 was found to be pro-inflammatory in a variety of diseases. However, the relationship of ALPK1, HA and M1 macrophage polarization in TMJ synovitis remains unclear. We aimed to investigate the role of ALPK1 and HA in macrophage polarization and TMJ synovitis and the underlying mechanisms. The results demonstrated that ALPK1 was highly upregulated in the synovial macrophages in the inflamed TMJ synovium of patients. Low molecular weight hyaluronic acid (LMW-HA) promoted the expression of ALPK1 and M1 macrophage-associated genes. Besides, rhALPK1 promoted the expression of M1 macrophage-associated factors and the nuclear translocation of PKM2. Furthermore, ALPK1 knockout mice exhibited limited infiltration of macrophages and decreased expression levels of M1 macrophage-associated genes in CFA-induced TMJ synovitis. While HMW-HA inhibited the expression of ALPK1 and M1 macrophage polarization. Our results elucidated that ALPK1 promoted TMJ synovitis by promoting nuclear PKM2-mediated M1 macrophage polarization, whereas HMW-HA inhibited the expression of ALPK1 as well as M1 macrophage polarization.


Assuntos
Osteoartrite , Sinovite , Humanos , Animais , Camundongos , Ácido Hialurônico , Sinovite/patologia , Articulação Temporomandibular/patologia , Inflamação/patologia , Osteoartrite/metabolismo , Macrófagos/metabolismo , Proteínas Quinases
11.
Zhen Ci Yan Jiu ; 49(3): 221-230, 2024 Mar 25.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38500318

RESUMO

OBJECTIVES: To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on mitophagy mediated by silencing regulatory protein 3 (SIRT3)/ PTEN induced putative kinase 1 (PINK1)/PARK2 gene coding protein (Parkin) in the midbrain substantia nigra of Parkinson's disease (PD) mice, and to explore the potential mechanisms of EA in treating PD. METHODS: C57BL/6 mice were randomly divided into the control, model, EA, and sham EA groups, with 12 mice in each group. The PD mouse model was established by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). The EA group received EA stimulation at GV16, LR3 and ST36, while the sham EA group received shallow needling 1 mm away from the above acupoints without electrical stimulation. The motor ability of mice in each group was evaluated using an open field experiment. Immunohistochemistry was used to detect the expression of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the substantia nigra of mice. The ultrastructure of neurons in substantia nigra was observed by transmission electron microscope (TEM). Immunofluorescence was used to detect the expression of the autophagy marker autophagy-associated protein light chain 3 (LC3). The expression levels of TH, α-syn, SIRT3, PINK1, Parkin, P62, Beclin-1, LC3Ⅱ mRNA and protein were detected by PCR and Western blot. RESULTS: Compared with the control group, mice in the model group showed a decrease in the total exercise distance, time, movement speed and times of crossing central region (P<0.01);the positive expressions of TH and LC3 were decreased (P<0.01), while the positive expression of α-syn increased (P<0.01), accompanied by mitochondrial swelling, mitochondrial cristae fragmentation and decrease, and decreased lysosome count;the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1, and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were decreased (P<0.01), while the expression levels of α-syn and P62 mRNA and protein were increased (P<0.01, P<0.05). Compared with the model group, the mice in EA group showed a significant increase in the total exercise distance, time, movement speed and times of crossing central region (P<0.01, P<0.05);the positive expressions of TH and LC3 were increased (P<0.01, P<0.05), while the positive expression of α-syn was decreased (P<0.01), accompanied by an increase in mitochondrial count, appearance of autophagic va-cuoles, and a decrease in swelling, the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1 and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were increased (P<0.01, P<0.05), while the mRNA and protein expression levels of α-syn and P62 were decreased (P<0.01);the sham EA group showed an increase in the total exercise distance and time(P<0.05), with an increase in the positive expression of TH (P<0.05) and a decrease in the positive expression of α-syn (P<0.05);some mitochondria exhibited swelling, and no autophagic vacuoles were observed;the protein expression levels of TH, SIRT3, Parkin and LC3Ⅱ were increased (P<0.01, P<0.05), and the expression levels of P62 mRNA, α-syn mRNA and protein were decreased (P<0.01, P<0.05), and LC3Ⅱ mRNA expression was increased (P<0.05). In comparison to the sham EA group, the EA group showed an extension in the total exercise time (P<0.01), the positive expression and mRNA expression levels of α-syn were decreased (P<0.01, P<0.05), while the expression levels of TH, SIRT3, PINK1, Parkin mRNA and SIRT3 protein were increased (P<0.05). CONCLUSIONS: EA at GV16, LR3, and ST36 can exert neuroprotective function and improve the motor ability of PD mice by activating the SIRT3/PINK1/Parkin pathway to enhance the expression of TH and reduce α-syn aggregation in the substantia nigra of PD mice.


Assuntos
Eletroacupuntura , Doença de Parkinson , Sirtuína 3 , Camundongos , Animais , Doença de Parkinson/genética , Doença de Parkinson/terapia , Sirtuína 3/genética , Mitofagia/genética , Proteínas Quinases/genética , Proteína Beclina-1 , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , RNA Mensageiro
12.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474061

RESUMO

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl-/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl+/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl-/- and Mlkl+/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.


Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Frutose , Proteínas Quinases/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Modelos Animais de Doenças , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo/metabolismo , Inflamação , Colesterol , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
13.
Acta Cir Bras ; 39: e391424, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38511762

RESUMO

PURPOSE: XinJiaCongRongTuSiZiWan (XJCRTSZW) is a traditional Chinese medicine compound for invigorating the kidney, nourishing blood, and promoting blood circulation. This study aimed to explore the effect of XJCRTSZW on triptolide (TP)-induced oxidative stress injury. METHODS: Adult female Sprague-Dawley rats and human ovarian granulosa cell lines were treated with TP and XJCRTSZW. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, flow cytometry, CCK-8, JC-1 staining, transmission electron microscopy, reverse transcription-quantitative polymerase chain reaction, and Western blotting were performed in this study. RESULTS: XJCRTSZW treatment observably ameliorated the TP-induced pathological symptoms. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of estradiol, anti-Mullerian hormone, progesterone, superoxide dismutase, ATP content, mitochondrial membrane potential, p62, and Hsp60 mRNA, and protein levels in vivo and in vitro (p < 0.05). However, TP-induced elevation of follicle stimulating hormone and luteinizing hormone concentrations, malondialdehyde levels, reactive oxygen species levels, apoptosis rate, mitophagy, and the mRNA and protein expressions of LC3-II/LC3-I, PTEN-induced kinase 1 (PINK1), and Parkin were decreased (p < 0.05). In addition, XJCRTSZW treatment markedly increased cell viability in vitro (p < 0.05). CONCLUSIONS: XJCRTSZW protects TP-induced rats from oxidative stress injury via the mitophagy-mediated PINK1/Parkin pathway.


Assuntos
Diterpenos , Mitocôndrias , Mitofagia , Fenantrenos , Adulto , Ratos , Feminino , Humanos , Animais , Ratos Sprague-Dawley , Estresse Oxidativo , Ubiquitina-Proteína Ligases , Transdução de Sinais , Proteínas Quinases/metabolismo , Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Compostos de Epóxi
14.
Curr Protoc ; 4(3): e998, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38439594

RESUMO

Protein kinases catalyze the phosphorylation of proteins most commonly on Ser, Thr, and Tyr residues and regulate many cellular events in eukaryotic cells, such as cell cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site and one or more substrate-binding site(s) that exhibit recognition features for different protein substrates. By bringing ATP and a substrate into proximity, each protein kinase can transfer the γ phosphate of the ATP molecule to a hydroxyl group of the target residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated based on the phosphorylated versus dephosphorylated status of the substrate. Although there are a number of ways to assay the activity of protein kinases, most of them are technically cumbersome and/or are indirect or based on quenched reactions. This protocol describes an assay employing a fluorescent peptide substrate to detect phosphorylation by protein kinases in real time. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. We extend the application of this assay to an example of how to assess time-dependent covalent inhibition of kinases as well. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Measuring protein kinase activity using fluorescent peptides Alternate Protocol: Measuring protein kinase activity using a fluorescence plate reader Support Protocol: Labeling peptides with sox fluorophore Basic Protocol 2: Measuring time-dependent ATP-competitive inhibition of protein kinases using fluorescent peptides.


Assuntos
Peptídeos , Proteínas Quinases , Fosforilação , Corantes Fluorescentes , Trifosfato de Adenosina
15.
Nat Commun ; 15(1): 2449, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503771

RESUMO

Wheat powdery mildew is one of the most destructive diseases threatening global wheat production. The wild relatives of wheat constitute rich sources of diversity for powdery mildew resistance. Here, we report the map-based cloning of the powdery mildew resistance gene Pm13 from the wild wheat species Aegilops longissima. Pm13 encodes a mixed lineage kinase domain-like (MLKL) protein that contains an N-terminal-domain of MLKL (MLKL_NTD) domain in its N-terminus and a C-terminal serine/threonine kinase (STK) domain. The resistance function of Pm13 is validated by mutagenesis, gene silencing, transgenic assay, and allelic association analyses. The development of introgression lines with significantly reduced chromosome segments of Ae. longissima encompassing Pm13 enables widespread deployment of this gene into wheat cultivars. The cloning of Pm13 may provide valuable insights into the molecular mechanisms underlying Pm13-mediated powdery mildew resistance and highlight the important roles of kinase fusion proteins (KFPs) in wheat immunity.


Assuntos
Aegilops , Ascomicetos , Triticum/genética , Genes de Plantas , Resistência à Doença/genética , Ascomicetos/genética , Aegilops/genética , Proteínas Quinases/genética , Doenças das Plantas/genética
16.
Sci Rep ; 14(1): 6574, 2024 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503865

RESUMO

Cell cycle-dependent protein kinase 12 (CDK12) plays a key role in a variety of carcinogenesis processes and represents a promising therapeutic target for cancer treatment. However, to date, there have been no systematic studies addressing its diagnostic, prognostic and immunological value across cancers. Here, we found that CDK12 was significantly upregulated in various types of cancers, and it expression increased with progression in ten cancer types, including breast cancer, cholangiocarcinoma and colon adenocarcinoma. Moreover, the ROC curves indicated that CDK12 showed diagnostic value in eight cancer types. High CDK12 expression was associated with poor prognosis in eight types of cancer, including low-grade glioma, mesothelioma, melanoma and pancreatic cancer. Furthermore, we conducted immunoassays to explore the exact mechanisms underlying CDK12-induced carcinogenesis, which revealed that increased expression of CDK12 allowed tumours to evade immune surveillance and upregulate immune checkpoint genes. Additionally, mutational studies have shown that amplification and missense mutations are the predominant mutational events affecting CDK12 across cancers. These findings establish CDK12 as a significant biological indicator of cancer diagnosis, prognosis, and immunotherapeutic targeting. Early surveillance and employment of CDK12 inhibitors, along with concomitant immunotherapy interventions, may enhance the clinical outcomes of cancer patients.


Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Proteínas Quinases , Quinases Ciclina-Dependentes/metabolismo , Prognóstico , Carcinogênese , Biomarcadores Tumorais/metabolismo , Imunomodulação/genética
17.
Eur J Pharmacol ; 969: 176459, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38438063

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fatal and insidious interstitial lung disease. So far, there are no effective drugs for preventing the disease process. Cellular senescence plays a critical role in the development of IPF, with the senescence and insufficient mitophagy of alveolar epithelial cells being implicated in its pathogenesis. Tetrandrine is a natural alkaloid which is now produced synthetically. It was known that the tetrandrine has anti-fibrotic effects, but the efficacy and mechanisms are still not well evaluated. Here, we reveal the roles of tetrandrine on AECs senescence and the antifibrotic effects by using a bleomycin challenged mouse model of pulmonary fibrosis and a bleomycin-stimulated mouse alveolar epithelial cell line (MLE-12). We performed the ß-galactosidase staining, immunohistochemistry and fluorescence to assess senescence in MLE-12 cells. The mitophagy levels were detected by co-localization of LC3 and COVIX. Our findings indicate that tetrandrine suppressed bleomycin-induced fibroblast activation and ultimately blocked the increase of collagen deposition in mouse model lung tissue. It has significantly inhibited the bleomycin-induced senescence and senescence-associated secretory phenotype (SASP) in alveolar epithelial cells (AECs). Mechanistically, tetrandrine suppressed the decrease of mitochondrial autophagy-related protein expression to rescue the bleomycin-stimulated impaired mitophagy in MLE-12 cells. We revealed that knockdown the putative kinase 1 (PINK1) gene by a short interfering RNA (siRNA) could abolish the ability of tetrandrine and reverse the MLE-12 cells senescence, which indicated the mitophagy of MLE-12 cells is PINK1 dependent. Our data suggest the tetrandrine could be a novel and effective drug candidate for lung fibrosis and senescence-related fibrotic diseases.


Assuntos
Células Epiteliais Alveolares , Benzilisoquinolinas , Fibrose Pulmonar Idiopática , Camundongos , Animais , Mitofagia , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Senescência Celular , Fibrose , Proteínas Quinases/metabolismo , Bleomicina/toxicidade , Ubiquitina-Proteína Ligases/metabolismo
18.
Bioorg Med Chem ; 102: 117659, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38442525

RESUMO

Necroptosis is an important form of programmed cell death (PCD), which is mediated by a death receptor and independent of the caspase proteolytic enzyme. Mixed lineage kinase domain-like (MLKL) is the final effector of necroptosis, playing an irreplaceable role in the execution of necroptosis. However, the studies on MLKL inhibitors are in their infancy. Necrosulfonamide (NSA) is an early-discovered covalent MLKL inhibitor, possessing medium anti-necroptosis activity and a structure-activity relationship (SAR) not widely disclosed. In this study, with the covalent motif maintained, we aim to improve the activity by introducing the terminal fused heterocycles and meanwhile revealing the SAR on the part. As a result, compounds 9 and 14 showed the best activity (EC50 = 148.4 and 595.9 nM) against necroptosis among the analogues by covalently binding to MLKL. The SAR was also concluded to guide further structural optimization in this field.


Assuntos
Necroptose , Proteínas Quinases , Proteínas Quinases/metabolismo , Apoptose , Fosforilação
19.
Protein Sci ; 33(4): e4918, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501429

RESUMO

Protein kinases are key actors of signaling networks and important drug targets. They cycle between active and inactive conformations, distinguished by a few elements within the catalytic domain. One is the activation loop, whose conserved DFG motif can occupy DFG-in, DFG-out, and some rarer conformations. Annotation and classification of the structural kinome are important, as different conformations can be targeted by different inhibitors and activators. Valuable resources exist; however, large-scale applications will benefit from increased automation and interpretability of structural annotation. Interpretable machine learning models are described for this purpose, based on ensembles of decision trees. To train them, a set of catalytic domain sequences and structures was collected, somewhat larger and more diverse than existing resources. The structures were clustered based on the DFG conformation and manually annotated. They were then used as training input. Two main models were constructed, which distinguished active/inactive and in/out/other DFG conformations. They considered initially 1692 structural variables, spanning the whole catalytic domain, then identified ("learned") a small subset that sufficed for accurate classification. The first model correctly labeled all but 3 of 3289 structures as active or inactive, while the second assigned the correct DFG label to all but 17 of 8826 structures. The most potent classifying variables were all related to well-known structural elements in or near the activation loop and their ranking gives insights into the conformational preferences. The models were used to automatically annotate 3850 kinase structures predicted recently with the Alphafold2 tool, showing that Alphafold2 reproduced the active/inactive but not the DFG-in proportions seen in the Protein Data Bank. We expect the models will be useful for understanding and engineering kinases.


Assuntos
Inibidores de Proteínas Quinases , Proteínas Quinases , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Conformação Proteica , Proteínas Quinases/química , Aprendizado de Máquina
20.
Hum Genomics ; 18(1): 24, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38475971

RESUMO

BACKGROUND: Protein Phosphatase Enzymes (PPE) and protein kinases simultaneously control phosphorylation mechanisms that tightly regulate intracellular signalling pathways and stimulate cellular responses. In human malignancies, PPE and protein kinases are frequently mutated resulting in uncontrolled kinase activity and PPE suppression, leading to cell proliferation, migration and resistance to anti-cancer therapies. Cancer associated DNA hypermethylation at PPE promoters gives rise to transcriptional silencing (epimutations) and is a hallmark of cancer. Despite recent advances in sequencing technologies, data availability and computational capabilities, only a fraction of PPE have been reported as transcriptionally inactive as a consequence of epimutations. METHODS: In this study, we examined promoter-associated DNA methylation profiles in Protein Phosphatase Enzymes and their Interacting Proteins (PPEIP) in a cohort of 705 cancer patients in five tissues (Large intestine, Oesophagus, Lung, Pancreas and Stomach) in three cell models (primary tumours, cancer cell lines and 3D embedded cancer cell cultures). As a subset of PPEIP are known tumour suppressor genes, we analysed the impact of PPEIP promoter hypermethylation marks on gene expression, cellular networks and in a clinical setting. RESULTS: Here, we report epimutations in PPEIP are a frequent occurrence in the cancer genome and manifest independent of transcriptional activity. We observed that different tumours have varying susceptibility to epimutations and identify specific cellular signalling networks that are primarily affected by epimutations. Additionally, RNA-seq analysis showed the negative impact of epimutations on most (not all) Protein Tyrosine Phosphatase transcription. Finally, we detected novel clinical biomarkers that inform on patient mortality and anti-cancer treatment sensitivity. CONCLUSIONS: We propose that DNA hypermethylation marks at PPEIP frequently contribute to the pathogenesis of malignancies and within the precision medicine space, hold promise as biomarkers to inform on clinical features such as patient survival and therapeutic response.


Assuntos
Epigênese Genética , Neoplasias , Humanos , Metilação de DNA , Fosfoproteínas Fosfatases , Proteínas Quinases , Biomarcadores , DNA , Regulação Neoplásica da Expressão Gênica
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