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1.
Theor Appl Genet ; 137(7): 150, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38847846

ABSTRACT

Grain size is a crucial agronomic trait that determines grain weight and final yield. Although several genes have been reported to regulate grain size in rice (Oryza sativa), the function of Wall-Associated Kinase family genes affecting grain size is still largely unknown. In this study, we identified GRAIN WEIGHT AND NUMBER 1 (GWN1) using map-based cloning. GWN1 encodes the OsWAK74 protein kinase, which is conserved in plants. GWN1 negatively regulates grain length and weight by regulating cell proliferation in spikelet hulls. We also found that GWN1 negatively influenced grain number by influencing secondary branch numbers and finally increased plant grain yield. The GWN1 gene was highly expressed in inflorescences and its encoded protein is located at the cell membrane and cell wall. Moreover, we identified three haplotypes of GWN1 in the germplasm. GWN1hap1 showing longer grain, has not been widely utilized in modern rice varieties. In summary, GWN1 played a very important role in regulating grain length, weight and number, thereby exhibiting application potential in molecular breeding for longer grain and higher yield.


Subject(s)
Edible Grain , Oryza , Plant Proteins , Seeds , Oryza/genetics , Oryza/growth & development , Oryza/enzymology , Edible Grain/genetics , Edible Grain/growth & development , Plant Proteins/genetics , Plant Proteins/metabolism , Seeds/growth & development , Seeds/genetics , Phenotype , Gene Expression Regulation, Plant , Cloning, Molecular , Chromosome Mapping , Haplotypes , Cell Wall/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Genes, Plant
2.
Sci Adv ; 10(23): eadn7191, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38848361

ABSTRACT

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.


Subject(s)
Mitochondrial Membrane Transport Proteins , Mitochondrial Precursor Protein Import Complex Proteins , Protein Kinases , Saccharomyces cerevisiae , Protein Kinases/metabolism , Protein Kinases/genetics , Humans , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Mitochondria/metabolism , Protein Binding , Enzyme Activation , Models, Molecular , Protein Subunits/metabolism , Protein Subunits/genetics
3.
Cell Mol Biol (Noisy-le-grand) ; 70(6): 192-198, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38836662

ABSTRACT

Intervertebral disc degeneration (IDD) is characterized by the decreased function and number of nucleus pulposus cells (NPCs) caused by excessive intervertebral disc (IVD) pressure. This research aims to provide novel insights into IDD prevention and treatment by clarifying the effect of andrographolide (ANDR) on IDD cell autophagy and oxidative stress under mechanical stress. Human primary NPCs were extracted from the nucleus pulposus tissue of non-IDD trauma patients. An IDD cell model was established by posing mechanical traction on NPCs. Through the construction of an IDD rat model, the influence of ANDR on IDD pathological changes was explored in vivo. The proliferation and autophagy of NPCs were decreased while the apoptosis rate and oxidative stress reaction were increased by mechanical traction. ANDR intervention obviously alleviated this situation. MiR-9 showed upregulated expression in IDD cell model, while FoxO3 and PINK1/Parkin were downregulated. Decreased proliferation and autophagy as well as enhanced apoptosis and oxidative stress response of NPCs were observed following miR-9 mimics and H89 intervention, while the opposite trend was observed after FoxO3 overexpression. FoxO3 is a direct target downstream miR-9. The in vivo experiments revealed that after ANDR intervention, the number of apoptotic cells in rat IVD tissue decreased and the autophagy increased. In conclusion, ANDR improves NPC proliferation, and autophagy, inhibits apoptosis and oxidative stress, and alleviates the pathological changes of IDD via the miR-9/FoxO3/PINK1/Parkin axis, which may be a new and effective treatment for IDD in the future.


Subject(s)
Autophagy , Diterpenes , Forkhead Box Protein O3 , Intervertebral Disc Degeneration , MicroRNAs , Nucleus Pulposus , Oxidative Stress , Protein Kinases , Rats, Sprague-Dawley , Stress, Mechanical , Ubiquitin-Protein Ligases , MicroRNAs/metabolism , MicroRNAs/genetics , Autophagy/drug effects , Forkhead Box Protein O3/metabolism , Forkhead Box Protein O3/genetics , Oxidative Stress/drug effects , Animals , Intervertebral Disc Degeneration/metabolism , Intervertebral Disc Degeneration/pathology , Humans , Diterpenes/pharmacology , Nucleus Pulposus/metabolism , Nucleus Pulposus/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Protein Kinases/metabolism , Protein Kinases/genetics , Rats , Male , Apoptosis/drug effects , Cell Proliferation/drug effects , Signal Transduction/drug effects , Female , Adult , Disease Models, Animal
4.
Proc Natl Acad Sci U S A ; 121(25): e2312415121, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38875149

ABSTRACT

Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Immunity, Innate , Myosins , Plant Immunity , Protein Serine-Threonine Kinases , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Myosins/metabolism , Protein Kinases/metabolism , Cell Membrane/metabolism , Signal Transduction , Plant Diseases/immunology , Plant Diseases/microbiology
5.
Chem Res Toxicol ; 37(6): 1053-1061, 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38847154

ABSTRACT

Poisoning caused by the mushroom Amanita phalloides, due to the toxin α-amanitin, accounts for approximately 90% of food poisoning deaths in China with no specific antidotes. To investigate the role of salidroside (Sal) in α-amanitin (α-AMA)-induced mitophagy, mouse liver cells AML-12 were exposed to α-AMA in the presence of Sal or not. Intracellular reactive oxygen species (ROS) levels were measured using a ROS detection kit, mitochondrial activity was evaluated using a mitochondrial red fluorescent probe kit or JC-1 dye, and protein expression levels of PINK1, Parkin, LC3 II, P62, Bax, Bcl-2, Caspase 3, Cleaved-Caspase 3, PARP I, and Cleaved-PARP I were detected through Western blot. Results demonstrated that α-AMA led to increased intracellular ROS levels, cell apoptosis, and decreased mitochondrial membrane potential. Notably, expression levels of mitophagy-related proteins PINK1, Parkin, and LC3 increased significantly while the P62 protein expression decreased remarkably. Furthermore, Sal reversed the α-AMA-induced decrease in cell viability and mitochondrial membrane potential and increase in intracellular ROS level. In addition, Sal promoted expression levels of PINK1, Parkin, and LC3 II while suppressing the Bax/Bcl-2 ratio, Cleaved-Caspase 3, and Cleaved-PARP I as well as P62. The results above proved that salidroside alleviates α-AMA-induced mouse liver cells damage via promoting PINK1/Parkin-mediated mitophagy and reducing cell apoptosis.


Subject(s)
Apoptosis , Glucosides , Mitochondria , Mitophagy , Phenols , Protein Kinases , Reactive Oxygen Species , Ubiquitin-Protein Ligases , Animals , Apoptosis/drug effects , Ubiquitin-Protein Ligases/metabolism , Phenols/pharmacology , Phenols/chemistry , Glucosides/pharmacology , Glucosides/chemistry , Mice , Protein Kinases/metabolism , Mitophagy/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Membrane Potential, Mitochondrial/drug effects , Cell Line , Cell Survival/drug effects
6.
Med Oncol ; 41(7): 172, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38862702

ABSTRACT

Resistance to caspase-dependent apoptosis is often responsible for treatments failure in cancer. Necroptosis is a type of programmed necrosis that occurs under caspase-deficient conditions that could overcome apoptosis resistance. Our purpose was to investigate the interrelationship between apoptotic and necroptotic death pathways and their influence on the response of breast cancer cells to radiotherapy in vitro. Human BC cell lines MCF-7 and MDA-MB-231 were treated with ionizing radiation, and then several markers of apoptosis, necroptosis, and survival were assessed in the presence and absence of necroptosis inhibition. MLKL knockdown was achieved by siRNA transfection. Our main findings emphasize the role of necroptosis in cellular response to radiation represented in the dose- and time-dependent elevated expression of necroptotic markers RIPK1, RIPK3, and MLKL. Knockdown of necroptotic marker MLKL by siRNA led to a significant elevation in MDA-MB-231 and MCF-7 survival with a dose modifying factor (DMF) of 1.23 and 1.61, respectively. Apoptotic markers Caspase 8 and TRADD showed transitory or delayed upregulation, indicating that apoptosis was not the main mechanism by which cells respond to radiation exposure. Apoptotic markers also showed a significant elevation following MLKL knockdown, suggesting its role either as a secondary or death alternative pathway. The result of our study emphasizes the critical role of the necroptotic pathway in regulating breast cancer cells responses to radiotherapy and suggests a promising utilization of its key modulator, MLKL, as a treatment strategy to improve the response to radiotherapy.


Subject(s)
Apoptosis , Breast Neoplasms , Necroptosis , Protein Kinases , Humans , Apoptosis/radiation effects , Breast Neoplasms/radiotherapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Female , Protein Kinases/metabolism , Protein Kinases/genetics , Cell Line, Tumor , RNA, Small Interfering/genetics , Signal Transduction , MCF-7 Cells
7.
Cell Mol Biol Lett ; 29(1): 85, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38834954

ABSTRACT

The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given autophagy functions to reduce surplus to compensate for scarcity, it theoretically possesses the capability to selectively degrade specific substrates to meet distinct metabolic demands. However, direct evidence is still lacking that substantiates the idea that autophagy selectively targets specific substrates (known as selective autophagy) to address particular nutritional needs. Recently, Gross et al. found that during phosphate starvation (P-S), rather than nitrogen starvation (N-S), yeasts selectively eliminate peroxisomes by dynamically altering the composition of the Atg1/ULK kinase complex (AKC) to adapt to P-S. This study elucidates how the metabolite sensor Pho81 flexibly interacts with AKC and guides selective autophagic clearance of peroxisomes during P-S, providing novel insights into the metabolic contribution of autophagy to special nutritional needs.


Subject(s)
Autophagy , Phosphates , Saccharomyces cerevisiae Proteins , Phosphates/metabolism , Phosphates/deficiency , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Peroxisomes/metabolism , Saccharomyces cerevisiae/metabolism , Autophagy-Related Protein-1 Homolog/metabolism , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Proteins/metabolism , Autophagy-Related Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Kinases
8.
Nat Commun ; 15(1): 4796, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38839783

ABSTRACT

Powdery mildew is a devastating disease that affects wheat yield and quality. Wheat wild relatives represent valuable sources of disease resistance genes. Cloning and characterization of these genes will facilitate their incorporation into wheat breeding programs. Here, we report the cloning of Pm57, a wheat powdery mildew resistance gene from Aegilops searsii. It encodes a tandem kinase protein with putative kinase-pseudokinase domains followed by a von Willebrand factor A domain (WTK-vWA), being ortholog of Lr9 that mediates wheat leaf rust resistance. The resistance function of Pm57 is validated via independent mutants, gene silencing, and transgenic assays. Stable Pm57 transgenic wheat lines and introgression lines exhibit high levels of all-stage resistance to diverse isolates of the Bgt fungus, and no negative impacts on agronomic parameters are observed in our experimental set-up. Our findings highlight the emerging role of kinase fusion proteins in plant disease resistance and provide a valuable gene for wheat breeding.


Subject(s)
Aegilops , Ascomycota , Disease Resistance , Plant Diseases , Plant Proteins , Plants, Genetically Modified , Triticum , Triticum/microbiology , Triticum/genetics , Disease Resistance/genetics , Plant Diseases/microbiology , Plant Diseases/genetics , Plant Diseases/immunology , Ascomycota/genetics , Ascomycota/pathogenicity , Plant Proteins/genetics , Plant Proteins/metabolism , Aegilops/genetics , Aegilops/microbiology , Plant Breeding , Protein Kinases/genetics , Protein Kinases/metabolism , Cloning, Molecular , Gene Expression Regulation, Plant
9.
Commun Biol ; 7(1): 722, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38862688

ABSTRACT

The target of rapamycin complex 2 (TORC2) signaling is associated with plasma membrane (PM) integrity. In Saccharomyces cerevisiae, TORC2-Ypk1/2 signaling controls sphingolipid biosynthesis, and Ypk1/2 phosphorylation by TORC2 under PM stress conditions is increased in a Slm1/2-dependent manner, under which Slm1 is known to be released from an eisosome, a furrow-like invagination PM structure. However, it remains unsolved how the activation machinery of TORC2-Ypk1/2 signaling is regulated. Here we show that edelfosine, a synthetic lysophospholipid analog, inhibits the activation of TORC2-Ypk1/2 signaling, and the cell wall integrity (CWI) pathway is involved in this inhibitory effect. The activation of CWI pathway blocked the eisosome disassembly promoted by PM stress and the release of Slm1 from eisosomes. Constitutive activation of TORC2-Ypk1/2 signaling exhibited increased sensitivity to cell wall stress. We propose that the CWI pathway negatively regulates the TORC2-Ypk1/2 signaling, which is involved in the regulatory mechanism to ensure the proper stress response to cell wall damage.


Subject(s)
Cell Wall , Mechanistic Target of Rapamycin Complex 2 , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Signal Transduction , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/drug effects , Cell Wall/metabolism , Cell Wall/drug effects , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Mechanistic Target of Rapamycin Complex 2/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , Phosphorylation , Protein Kinases , Protein Serine-Threonine Kinases
10.
Nat Commun ; 15(1): 5111, 2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38877002

ABSTRACT

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.


Subject(s)
Carrier Proteins , Myocardium , Protein Phosphatase 1 , Protein Phosphatase 2 , Phosphorylation , Humans , Protein Phosphatase 1/metabolism , Carrier Proteins/metabolism , Animals , Protein Phosphatase 2/metabolism , Myocardium/metabolism , Protein Kinases/metabolism , Kinetics
11.
Int J Biol Macromol ; 270(Pt 2): 132370, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38763253

ABSTRACT

BACKGROUND: Polysaccharides from Grifola frondosa(GFP) have gained worldwide attention owing to their promising biological activities and potential health benefits. PURPOSE: This study aimed to investigate the effects of GFP on alleviation of osteoporosis in ovariectomized (OVX) mice and examine the underlying mechanism. METHOD: A mouse model of postmenopausal osteoporosis was established by OVX method, Forty eight C57BL/6 female mice were randomly divided into Normal group, OVX alone (Model group, n = 8), OVX + 10 mg/kg GFP (GFP-L group, n = 8), OVX + 20 mg/kg GFP (GFP-M group, n = 8), OVX + 40 mg/kg GFP (GFP-H group, n = 8), OVX + 10 mg/kg Estradiol valerate (Positive group, n = 8). RESULTS: The results showed that compared with Model group, the concentrations of interleukin (IL)-1ß, interleukin (IL)-6 and Tumor necrosis factor-α (TNF-α) were significantly reduced, the activity of superoxide dismutase (SOD) and glutathione (GSH) were significantly increased, the content of myeloperoxidase (MPO) and malondialdehyde (MDA) were significantly reduced, and the proteins levels of PINK1, Parkin, Beclin-1 and LC3-II were significantly decreased in the GFP groups. CONCLUSION: This study demonstrates that GFP alleviates ovariectomy-induced osteoporosis via reduced secretion of inflammatory cytokines, improvement in the oxidative stress status in the body, and inhibition of the PINK1/Parkin signaling pathway.


Subject(s)
Grifola , Inflammation , Osteoporosis , Ovariectomy , Oxidative Stress , Protein Kinases , Signal Transduction , Ubiquitin-Protein Ligases , Animals , Ovariectomy/adverse effects , Oxidative Stress/drug effects , Female , Mice , Signal Transduction/drug effects , Osteoporosis/drug therapy , Osteoporosis/etiology , Osteoporosis/prevention & control , Osteoporosis/metabolism , Protein Kinases/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Ubiquitin-Protein Ligases/metabolism , Grifola/chemistry , Mice, Inbred C57BL , Cytokines/metabolism , Fungal Polysaccharides/pharmacology , Fungal Polysaccharides/chemistry , Disease Models, Animal
12.
Discov Med ; 36(184): 992-1001, 2024 May.
Article in English | MEDLINE | ID: mdl-38798258

ABSTRACT

BACKGROUND: Diabetic neuropathic pain (DNP) is a complication of diabetes mellitus (DM). Hyperbaric lidocaine (HL), a local anesthetics drug, has neurotoxicity. The present study aims to study the effect and molecular mechanisms of HL on spinal nerve injury in DNP. METHODS: The DNP rat model was established through a high-fat-glucose diet in combination with Streptozotocin (STZ) administration. SB203580 and PD98059 were utilized to inhibit p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK). The mechanical paw withdrawal threshold (PWT) and the thermal paw withdrawal latency (PWL) were tested to evaluate rats' mechanical allodynia and thermal hyperalgesia. Hematoxylin-eosin (H&E) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end Labeling (TUNEL) staining were performed to evaluate the pathological changes and neuron apoptosis in spinal cord tissues of L4-5. Western blotting analysis and reverse transcription-polymerase chain reaction (RT-qPCR) assay were used to measure the levels of proteins and mRNAs, respectively. RESULTS: PWT and PWL were decreased in DNP rats with serious spinal nerve injury. HL administration downregulated the PWT and PWL and aggravated spinal nerve injury in DNP rats, but isobaric lidocaine had no effects on these changes. Meanwhile, p38 MAPK/ERK signaling and PTEN-induced kinase 1 (PINK1)-mediated mitophagy were activated in DNP, which was enhanced by HL but not isobaric lidocaine. Blocking p38 MAPK/ERK signaling could effectively attenuate HL-induced spinal nerve injury and inhibit mitophagy. CONCLUSION: In summary, HL can aggravate spinal cord tissue damage in DNP rats by inducing PINK1-mediated mitophagy via activating p38 MAPK/ERK signaling. Our data provide a novel insight that supports the potential role of p38 MAPK/ERK signaling in acting as a therapeutic target for HL-induced neurotoxicity.


Subject(s)
Diabetic Neuropathies , Lidocaine , Mitophagy , Protein Kinases , Rats, Sprague-Dawley , Ubiquitin-Protein Ligases , p38 Mitogen-Activated Protein Kinases , Animals , Lidocaine/pharmacology , Rats , Diabetic Neuropathies/pathology , Diabetic Neuropathies/metabolism , Diabetic Neuropathies/etiology , p38 Mitogen-Activated Protein Kinases/metabolism , Mitophagy/drug effects , Male , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/pathology , MAP Kinase Signaling System/drug effects , Signal Transduction/drug effects
13.
J Agric Food Chem ; 72(22): 12775-12787, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38776285

ABSTRACT

Excessive intake of fat and fructose in Western diets has been confirmed to induce renal lipotoxicity, thereby driving the progression of chronic kidney disease (CKD). This study was conducted to evaluate the efficacy of magnoflorine in a CKD mouse model subjected to high-fat and high-fructose diets. Our results demonstrated that magnoflorine treatment ameliorated abnormal renal function indices (serum creatinine, urea nitrogen, uric acid, and urine protein) in high-fat- and high-fructose-fed mice. Histologically, renal tubular cell steatosis, lipid deposition, tubular dilatation, and glomerular fibrosis were significantly reduced by the magnoflorine treatment in these mice. Mechanistically, magnoflorine promotes Parkin/PINK1-mediated mitophagy, thereby inhibiting NLRP3/Caspase-1-mediated pyroptosis. Consistent findings were observed in the palmitic acid-incubated HK-2 cell model. Notably, both silencing of Parkin and the use of a mitophagy inhibitor reversed the inhibitory effect of magnoflorine on NLRP3 inflammasome activation in vitro. Therefore, the present study provides compelling evidence that magnoflorine improves renal injury in high-fat- and high-fructose-fed mice by promoting Parkin/PINK1-dependent mitophagy to inhibit NLRP3 inflammasome activation and pyroptosis. Our findings suggest that dietary supplementation with magnoflorine and magnoflorine-rich foods (such as magnolia) might be an effective strategy for the prevention of CKD.


Subject(s)
Caspase 1 , Diet, High-Fat , Fructose , Mice, Inbred C57BL , Mitophagy , NLR Family, Pyrin Domain-Containing 3 Protein , Protein Kinases , Pyroptosis , Renal Insufficiency, Chronic , Ubiquitin-Protein Ligases , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Mice , Pyroptosis/drug effects , Fructose/adverse effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Male , Mitophagy/drug effects , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/prevention & control , Diet, High-Fat/adverse effects , Humans , Protein Kinases/metabolism , Protein Kinases/genetics , Caspase 1/metabolism , Caspase 1/genetics , Aporphines/pharmacology , Inflammasomes/metabolism
14.
Trends Cell Biol ; 34(6): 440-441, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38714421

ABSTRACT

Biomolecular condensation has gained considerable attention as a fundamental mechanism in cell signaling and various biological processes. A recent study by Egger et al. provides valuable insights into the constituents of topoisomerase IIß binding protein 1 (TopBP1) condensates and sheds light on the mechanism of Chk1 activation by ataxia telangiectasia-mutated and Rad3-related (ATR) at the interface of these condensates.


Subject(s)
Ataxia Telangiectasia Mutated Proteins , Carrier Proteins , Cell Cycle Proteins , Checkpoint Kinase 1 , DNA-Binding Proteins , Protein Kinases , Ataxia Telangiectasia Mutated Proteins/metabolism , Checkpoint Kinase 1/metabolism , Humans , DNA-Binding Proteins/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Protein Kinases/metabolism , Nuclear Proteins/metabolism , Animals , Protein Serine-Threonine Kinases/metabolism , Signal Transduction
15.
Physiol Res ; 73(2): 253-263, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38710055

ABSTRACT

Up to now, there's a limited number of studies on the relationship between PINK1/Park2 pathway and mitophagy in NAFLD. To investigate the effect of Park2-mediated mitophagy on non-alcoholic fatty liver disease (NAFLD). Oleic acid was used for the establishment of NAFLD model. Oil red-dyed lipid drops and mitochondrial alternations were observed by transmission electron microscopy. Enzymatic kit was used to test lipid content. The levels of IL-8 and TNF-alpha were determined by ELISA. Lenti-Park2 and Park2-siRNA were designed to upregulate and downregulate Park2 expression, respectively. The changing expression of PINK and Park2 was detected by RT-qPCR and Western blot. Immunofluorescence staining was applied to measure the amount of LC3. Successful NAFLD modeling was featured by enhanced lipid accumulation, as well as the elevated total cholesterol (TC), triglyceride (TG), TNF-alpha and IL-8 levels. Mitochondria in NAFLD model were morphologically and functionally damaged. Park2 expression was upregulated by lenti-Park2 and downregulated through Park2-siRNA. The PINK1 expression showed the same trend as Park2 expression. Immunofluorescence staining demonstrated that the when Park2 was overexpressed, more LC3 protein on mitochondrial autophagosome membrane was detected, whereas Park2 knockdown impeded LC3' locating on the membrane. The transmission electron microscopy image exhibited that the extent of damage to the mitochondrial in NAFLD model was revered by enhanced Park2 expression but further exacerbated by reduced Park2 expression. Park2-mediated mitophagy could relive NAFLD and may be a novel therapeutic target for NAFLD treatment. Keywords: Non-alcoholic Fatty Liver Disease (NAFLD), Mitophagy, PINK1/Park2, Park2, PINK1.


Subject(s)
Mitophagy , Non-alcoholic Fatty Liver Disease , Protein Kinases , Ubiquitin-Protein Ligases , Animals , Humans , Male , Mice , Mitophagy/physiology , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/genetics , Protein Kinases/metabolism , Protein Kinases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics
16.
PLoS Comput Biol ; 20(5): e1012100, 2024 May.
Article in English | MEDLINE | ID: mdl-38768223

ABSTRACT

The activities of most enzymes and drugs depend on interactions between proteins and small molecules. Accurate prediction of these interactions could greatly accelerate pharmaceutical and biotechnological research. Current machine learning models designed for this task have a limited ability to generalize beyond the proteins used for training. This limitation is likely due to a lack of information exchange between the protein and the small molecule during the generation of the required numerical representations. Here, we introduce ProSmith, a machine learning framework that employs a multimodal Transformer Network to simultaneously process protein amino acid sequences and small molecule strings in the same input. This approach facilitates the exchange of all relevant information between the two molecule types during the computation of their numerical representations, allowing the model to account for their structural and functional interactions. Our final model combines gradient boosting predictions based on the resulting multimodal Transformer Network with independent predictions based on separate deep learning representations of the proteins and small molecules. The resulting predictions outperform recently published state-of-the-art models for predicting protein-small molecule interactions across three diverse tasks: predicting kinase inhibitions; inferring potential substrates for enzymes; and predicting Michaelis constants KM. The Python code provided can be used to easily implement and improve machine learning predictions involving arbitrary protein-small molecule interactions.


Subject(s)
Computational Biology , Machine Learning , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Substrate Specificity , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Proteins/metabolism , Proteins/chemistry , Amino Acid Sequence , Deep Learning , Protein Binding , Protein Kinases/metabolism , Protein Kinases/chemistry , Humans
17.
Mol Med ; 30(1): 72, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822247

ABSTRACT

BACKGROUND: 8-Oxoguanine DNA glycosylase (OGG1), a well-known DNA repair enzyme, has been demonstrated to promote lung fibrosis, while the specific regulatory mechanism of OGG1 during pulmonary fibrosis remains unclarified. METHODS: A bleomycin (BLM)-induced mouse pulmonary fibrosis model was established, and TH5487 (the small molecule OGG1 inhibitor) and Mitochondrial division inhibitor 1 (Mdivi-1) were used for administration. Histopathological injury of the lung tissues was assessed. The profibrotic factors and oxidative stress-related factors were examined using the commercial kits. Western blot was used to examine protein expression and immunofluorescence analysis was conducted to assess macrophages polarization and autophagy. The conditional medium from M2 macrophages was harvested and added to HFL-1 cells for culture to simulate the immune microenvironment around fibroblasts during pulmonary fibrosis. Subsequently, the loss- and gain-of function experiments were conducted to further confirm the molecular mechanism of OGG1/PINK1. RESULTS: In BLM-induced pulmonary fibrosis, OGG1 was upregulated while PINK1/Parkin was downregulated. Macrophages were activated and polarized to M2 phenotype. TH5487 administration effectively mitigated pulmonary fibrosis, M2 macrophage polarization, oxidative stress and mitochondrial dysfunction while promoted PINK1/Parkin-mediated mitophagy in lung tissues of BLM-induced mice, which was partly hindered by Mdivi-1. PINK1 overexpression restricted M2 macrophages-induced oxidative stress, mitochondrial dysfunction and mitophagy inactivation in lung fibroblast cells, and OGG1 knockdown could promote PINK1/Parkin expression and alleviate M2 macrophages-induced mitochondrial dysfunction in HFL-1 cells. CONCLUSION: OGG1 inhibition protects against pulmonary fibrosis, which is partly via activating PINK1/Parkin-mediated mitophagy and retarding M2 macrophage polarization, providing a therapeutic target for pulmonary fibrosis.


Subject(s)
Bleomycin , DNA Glycosylases , Disease Models, Animal , Macrophages , Mitophagy , Protein Kinases , Pulmonary Fibrosis , Animals , Mitophagy/drug effects , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/pathology , DNA Glycosylases/metabolism , DNA Glycosylases/genetics , Mice , Macrophages/metabolism , Protein Kinases/metabolism , Bleomycin/adverse effects , Male , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Oxidative Stress/drug effects , Mice, Inbred C57BL , Macrophage Activation , Humans , Quinazolinones
18.
Cell Mol Biol Lett ; 29(1): 82, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822260

ABSTRACT

BACKGROUND: Hepatic stellate cells (HSCs) play a crucial role in the development of fibrosis in non-alcoholic fatty liver disease (NAFLD). Small extracellular vesicles (sEV) act as mediators for intercellular information transfer, delivering various fibrotic factors that impact the function of HSCs in liver fibrosis. In this study, we investigated the role of lipotoxic hepatocyte derived sEV (LTH-sEV) in HSCs activation and its intrinsic mechanisms. METHODS: High-fat diet (HFD) mice model was constructed to confirm the expression of LIMA1. The relationship between LIMA1-enriched LTH-sEV and LX2 activation was evaluated by measurement of fibrotic markers and related genes. Levels of mitophagy were detected using mt-keima lentivirus. The interaction between LIMA1 and PINK1 was discovered through database prediction and molecular docking. Finally, sEV was injected to investigate whether LIMA1 can accelerate HFD induced liver fibrosis in mice. RESULTS: LIMA1 expression was upregulated in lipotoxic hepatocytes and was found to be positively associated with the expression of the HSCs activation marker α-SMA. Lipotoxicity induced by OPA led to an increase in both the level of LIMA1 protein in LTH-sEV and the release of LTH-sEV. When HSCs were treated with LTH-sEV, LIMA1 was observed to hinder LX2 mitophagy while facilitating LX2 activation. Further investigation revealed that LIMA1 derived from LTH-sEV may inhibit PINK1-Parkin-mediated mitophagy, consequently promoting HSCs activation. Knocking down LIMA1 significantly attenuates the inhibitory effects of LTH-sEV on mitophagy and the promotion of HSCs activation. CONCLUSIONS: Lipotoxic hepatocyte-derived LIMA1-enriched sEVs play a crucial role in promoting HSCs activation in NAFLD-related liver fibrosis by negatively regulating PINK1 mediated mitophagy. These findings provide new insights into the pathological mechanisms involved in the development of fibrosis in NAFLD.


Subject(s)
Diet, High-Fat , Extracellular Vesicles , Hepatic Stellate Cells , Hepatocytes , Liver Cirrhosis , Mice, Inbred C57BL , Mitophagy , Animals , Humans , Male , Mice , Diet, High-Fat/adverse effects , Disease Models, Animal , Extracellular Vesicles/metabolism , Extracellular Vesicles/genetics , Hepatic Stellate Cells/metabolism , Hepatocytes/metabolism , Hepatocytes/pathology , Liver Cirrhosis/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/genetics , Mitophagy/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/genetics , Protein Kinases/metabolism , Protein Kinases/genetics
19.
Biochim Biophys Acta Mol Cell Res ; 1871(5): 119745, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38719029

ABSTRACT

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells.


Subject(s)
Necroptosis , Protein Kinases , Receptor-Interacting Protein Serine-Threonine Kinases , Rotavirus , Viral Nonstructural Proteins , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/genetics , Humans , Protein Kinases/metabolism , Protein Kinases/genetics , Rotavirus/metabolism , Animals , Host-Pathogen Interactions , Toxins, Biological/metabolism
20.
Infect Immun ; 92(6): e0013224, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38700334

ABSTRACT

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.


Subject(s)
Bacterial Adhesion , Disease Models, Animal , Epithelial Cells , Escherichia coli Infections , Escherichia coli , Fimbriae, Bacterial , Gene Expression Regulation, Bacterial , Animals , Mice , Fimbriae, Bacterial/metabolism , Fimbriae, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/pathogenicity , Epithelial Cells/microbiology , Escherichia coli Infections/microbiology , Bacterial Adhesion/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Virulence/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Intestines/microbiology , Female
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