Acupotomy alleviates knee osteoarthritis in rabbit by regulating chondrocyte mitophagy Pink1-Parkin pathway.

OBJECTIVE: To investigate the effect of acupotomy, on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis (KOA). METHODS: A KOA model was established via the modified Videman method. Rabbits were randomly divided into a control group (CON), KOA group and KOA + acupotomy group (Acu). Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment. The behavior of the rabbits before and after intervention was recorded. Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy. The level of mitophagy was evaluated by transmission electron microscopy, immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The expression of phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence, Western blotting and real-time polymerase chain reaction. RESULTS: In rabbits with KOA, joint pain, mobility disorders and cartilage degeneration were observed, the Mankin score was increased, collagen type Ⅱ (Col-Ⅱ) expression was significantly decreased, mitophagy was inhibited, mitochondrial function was impaired, and factors associated with the Pink1-Parkin pathway were inhibited. Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins, the mitophagy-related protein microtubule-associated protein-1 light chain-3, the translocase of the outer membrane, and the inner mitochondrial membrane 23; increased the colocalization of mitochondria and autophagosomes; promoted the removal of damaged mitochondria; restored mitochondrial adenosine-triphosphate (ATP) production; and alleviated cartilage degeneration in rabbits with KOA. CONCLUSIONS: Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.

Electropositive Citric Acid-Polyethyleneimine Carbon Dots Carrying the PINK1 Gene Regulate ATP-Related Metabolic Dysfunction in APP/PS1-N2a Cells.

(1) Background: Alzheimer's disease (AD) is characterized by ß-amyloid (Aß) peptide accumulation and mitochondrial dysfunction during the early stage of disease. PINK1 regulates the balance between mitochondrial homeostasis and bioenergy supply and demand via the PINK1/Parkin pathway, Na+/Ca2+ exchange, and other pathways. (2) Methods: In this study, we synthesized positively charged carbon dots (CA-PEI CDs) using citric acid (CA) and polyethyleneimine (PEI) and used them as vectors to express PINK1 genes in the APP/PS1-N2a cell line to determine mitochondrial function, electron transport chain (ETC) activity, and ATP-related metabolomics. (3) Results: Our findings showed that the CA-PEI CDs exhibit the characteristics of photoluminescence, low toxicity, and concentrated DNA. They are ideal biological carriers for gene delivery. PINK1 overexpression significantly increased the mitochondrial membrane potential in APP/PS1-N2a cells and reduced reactive-oxygen-species generation and Aß1-40 and Aß1-42 levels. An increase in the activity of NADH ubiquinone oxidoreductase (complex I, CI) and cytochrome C oxidase (complex IV, CIV) induces the oxidative phosphorylation of mitochondria, increasing ATP generation. (4) Conclusions: These findings indicate that the PINK gene can alleviate AD by increasing bioenergetic metabolism, reducing Aß1-40 and Aß1-42, and increasing ATP production.

Identification of cysteine-rich receptor-like kinase gene family in potato: revealed StCRLK9 in response to heat, salt and drought stresses.

The investigation into cysteine-rich receptor-like kinases (CRLKs) holds pivotal significance as these conserved, upstream signalling molecules intricately regulate fundamental biological processes such as plant growth, development and stress adaptation. This study undertakes a comprehensive characterisation of CRLKs in Solanum tuberosum (potato), a staple food crop of immense economic importance. Employing comparative genomics and evolutionary analyses, we identified 10 distinct CRLK genes in potato. Further categorisation into three major groups based on sequence similarity was performed. Each CRLK member in potato was systematically named according to its chromosomal position. Multiple sequence alignment and phylogenetic analyses unveiled conserved gene structures and motifs within the same groups. The genomic distribution of CRLKs was observed across Chromosomes 2-5, 8 and 12. Gene duplication analysis highlighted a noteworthy trend, with most gene pairs exhibiting a Ka/Ks ratio greater than one, indicating positive selection of StCRLKs in potato. Salt and drought stresses significantly impacted peroxidase and catalase activities in potato seedlings. The presence of diverse cis -regulatory elements, including hormone-responsive elements, underscored their involvement in myriad biotic and abiotic stress responses. Interestingly, interactions between the phytohormone auxin and CRLK proteins unveiled a potential auxin-mediated regulatory mechanism. A holistic approach combining transcriptomics and quantitative PCR validation identified StCRLK9 as a potential candidate involved in plant response to heat, salt and drought stresses. This study lays a robust foundation for future research on the functional roles of the CRLK gene family in potatoes, offering valuable insights into their diverse regulatory mechanisms and potential applications in stress management.

PINK1/Park2-Mediated Mitophagy Relieve Non-Alcoholic Fatty Liver Disease.

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.

Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Previous studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae. RESULT: This study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress. CONCLUSION: Our results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.

Liquid-liquid phase separation of TZP promotes PPK-mediated phosphorylation of the phytochrome A photoreceptor.

Phytochrome A (phyA) is the plant far-red (FR) light photoreceptor and plays an essential role in regulating photomorphogenic development in FR-rich conditions, such as canopy shade. It has long been observed that phyA is a phosphoprotein in vivo; however, the protein kinases that could phosphorylate phyA remain largely unknown. Here we show that a small protein kinase family, consisting of four members named PHOTOREGULATORY PROTEIN KINASES (PPKs) (also known as MUT9-LIKE KINASES), directly phosphorylate phyA in vitro and in vivo. In addition, TANDEM ZINC-FINGER/PLUS3 (TZP), a recently characterized phyA-interacting protein required for in vivo phosphorylation of phyA, is also directly phosphorylated by PPKs. We reveal that TZP contains two intrinsically disordered regions in its amino-terminal domain that undergo liquid-liquid phase separation (LLPS) upon light exposure. The LLPS of TZP promotes colocalization and interaction between PPKs and phyA, thus facilitating PPK-mediated phosphorylation of phyA in FR light. Our study identifies PPKs as a class of protein kinases mediating the phosphorylation of phyA and demonstrates that the LLPS of TZP contributes significantly to more production of the phosphorylated phyA form in FR light.

KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy.

It has recently been shown that KAT8, a genome-wide association study candidate risk gene for Parkinson's Disease, is involved in PINK1/Parkin-dependant mitophagy. The KAT8 gene encodes a lysine acetyltransferase and represents the catalytically active subunit of the non-specific lethal epigenetic remodelling complex. In the current study, we show that contrary to KAT5 inhibition, dual inhibition of KAT5 and KAT8 via the MG149 compound inhibits the initial steps of the PINK1-dependant mitophagy process. More specifically, our study shows that following mitochondrial depolarisation induced by mitochondrial toxins, MG149 treatment inhibits PINK1-dependant mitophagy initiation by impairing PINK1 activation, and subsequent phosphorylation of Parkin and ubiquitin. While this inhibitory effect of MG149 on PINK1-activation is potent, MG149 treatment in the absence of mitochondrial toxins is sufficient to depolarise the mitochondrial membrane, recruit PINK1 and promote partial downstream recruitment of the autophagy receptor p62, leading to an increase in mitochondrial delivery to the lysosomes. Altogether, our study provides additional support for KAT8 as a regulator of mitophagy and autophagy processes.

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb.

Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.

Comprehensive analysis of stress-activated protein kinase genes (OsSAPKs) in rice flowering time.

MAIN CONCLUSION: The rice SnRK2 members SAPK4, SAPK5, SAPK7 and SAPK10 are positive regulators involved in the regulation of rice flowering, while other single mutants exhibited no effect on rice flowering. The rice SnRK2 family, comprising 10 members known as SAPK (SnRK2-Associated Protein Kinase), is pivotal in the abscisic acid (ABA) pathway and crucial for various biological processes, such as drought resistance and salt tolerance. Additionally, these members have been implicated in the regulation of rice heading date, a key trait influencing planting area and yield. In this study, we utilized gene editing technology to create mutants in the Songjing 2 (SJ2) background, enabling a comprehensive analyze the role of each SAPK member in rice flowering. We found that SAPK1, SAPK2, and SAPK3 may not directly participate in the regulatory network of rice heading date, while SAPK4, SAPK5, and SAPK7 play positive roles in rice flowering regulation. Notably, polygene deletion resulted in an additive effect on delaying flowering. Our findings corroborate the previous studies indicating the positive regulatory role of SAPK10 in rice flowering, as evidenced by delayed flowering observed in sapk9/10 double mutants. Moving forward, our future research will focus on analyzing the molecular mechanisms underlying SAPKs involvement in rice flowering regulation, aiming to enhance our understanding of the rice heading date relationship network and lay a theoretical foundation for breeding efforts to alter rice ripening dates.

Bioinformatics analysis of PANoptosis regulators in the diagnosis and subtyping of steroid-induced osteonecrosis of the femoral head.

In this study, we aimed to investigate the involvement of PANoptosis, a form of regulated cell death, in the development of steroid-induced osteonecrosis of the femoral head (SONFH). The underlying pathogenesis of PANoptosis in SONFH remains unclear. To address this, we employed bioinformatics approaches to analyze the key genes associated with PANoptosis. Our analysis was based on the GSE123568 dataset, allowing us to investigate both the expression profiles of PANoptosis-related genes (PRGs) and the immune profiles in SONFHallowing us to investigate the expression profiles of PRGs as well as the immune profiles in SONFH. We conducted cluster classification based on PRGs and assessed immune cell infiltration. Additionally, we used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific hub genes. Furthermore, we developed an optimal machine learning model to identify the key predictive genes responsible for SONFH progression. We also constructed a nomogram model with high predictive accuracy for assessing risk factors in SONFH patients, and validated the model using external data (area under the curve; AUC = 1.000). Furthermore, we identified potential drug targets for SONFH through the Coremine medical database. Using the optimal machine learning model, we found that 2 PRGs, CASP1 and MLKL, were significantly correlated with the key predictive genes and exhibited higher expression levels in SONFH. Our analysis revealed the existence of 2 distinct PANoptosis molecular subtypes (C1 and C2) within SONFH. Importantly, we observed significant variations in the distribution of immune cells across these subtypes, with C2 displaying higher levels of immune cell infiltration. Gene set variation analysis indicated that C2 was closely associated with multiple immune responses. In conclusion, our study sheds light on the intricate relationship between PANoptosis and SONFH. We successfully developed a risk predictive model for SONFH patients and different SONFH subtypes. These findings enhance our understanding of the pathogenesis of SONFH and offer potential insights into therapeutic strategies.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

VDR restores the expression of PINK1 and BNIP3 in TECs of streptozotocin-induced diabetic mice.

Defective mitophagy in renal tubular epithelial cells is one of the main drivers of renal fibrosis in diabetic kidney disease. Our gene sequencing data showed the expression of PINK1 and BNIP3, two key molecules of mitophagy, was decreased in renal tissues of VDR-knockout mice. Herein, streptozotocin (STZ) was used to induce renal interstitial fibrosis in mice. VDR deficiency exacerbated STZ-induced renal impairment and defective mitophagy. Paricalcitol (pari, a VDR agonist) and the tubular epithelial cell-specific overexpression of VDR restored the expression of PINK1 and BNIP3 in the renal cortex and attenuated STZ-induced kidney fibrosis and mitochondrial dysfunction. In HK-2 cells under high glucose conditions, an increased level of α-SMA, COL1, and FN and a decreased expression of PINK1 and BNIP3 with severe mitochondrial damage were observed, and these alterations could be largely reversed by pari treatment. ChIP-qPCR and luciferase reporter assays showed VDR could positively regulate the transcription of Pink1 and Bnip3 genes. These findings reveal that VDR could restore mitophagy defects and attenuate STZ-induced fibrosis in diabetic mice through regulation of PINK1 and BNIP3.

Aging accelerates locomotor decline in PINK1 knockout rats in association with decreased nigral, but not striatal, dopamine and tyrosine hydroxylase expression.

Parkinson's disease (PD) rodent models provide insight into the relationship between nigrostriatal dopamine (DA) signaling and locomotor function. Although toxin-based rat models produce frank nigrostriatal neuron loss and eventual motor decline characteristic of PD, the rapid nature of neuronal loss may not adequately translate premotor traits, such as cognitive decline. Unfortunately, rodent genetic PD models, like the Pink1 knockout (KO) rat, often fail to replicate the differential severity of striatal DA and tyrosine hydroxylase (TH) loss, and a bradykinetic phenotype, reminiscent of human PD. To elucidate this inconsistency, we evaluated aging as a progression factor in the timing of motor and non-motor cognitive impairments. Male PINK1 KO and age-matched wild type (WT) rats were evaluated in a longitudinal study from 3 to 16 months old in one cohort, and in a cross-sectional study of young adult (6-7 months) and aged (18-19 months) in another cohort. Young adult PINK1 KO rats exhibited hyperkinetic behavior associated with elevated DA and TH in the substantia nigra (SN), which decreased therein, but not striatum, in the aged KO rats. Additionally, norepinephrine levels decreased in aged KO rats in the prefrontal cortex (PFC), paired with a higher DA levels in young and aged KO. Although a younger age of onset characterizes familial forms of PD, our results underscore the critical need to consider age-related factors. Moreover, the results indicate that compensatory mechanisms may exist to preserve locomotor function, evidenced by increased DA in the SN early in the lifespan, in response to deficient PINK1 function, which declines with aging and the onset of motor decline.

TROP2 promotes PINK1-mediated mitophagy and apoptosis to accelerate the progression of senile chronic obstructive pulmonary disease by up-regulating DRP1 expression.

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterised by irreversible airflow limitation. The elderly are a vulnerable population for developing COPD. With the growth of age, physiological degenerative changes occur in the thorax, bronchus, lung and vascular wall, which can lead to age-related physiological attenuation of lung function in the elderly, so the prevalence of COPD increases with age. Its pathogenesis has not yet been truly clarified. Mitophagy plays an important role in maintaining the stability of mitochondrial function and intracellular environment by scavenging damaged mitochondria. Currently, studies have shown that trophoblast antigen 2 (TROP2) expression is up-regulated in airway basal cells of patients with COPD, suggesting that TROP2 is involved in the progression of COPD. However, whether it is involved in disease progression by regulating mitochondrial function remains unclear. In this study, compared with non-smoking non-COPD patients, the expression of TROP2 in lung tissues of smoking non-COPD patients and patients with COPD increased, and TROP2 expression in patients with COPD was higher than that in smoking non-COPD patients. To further explore the role of TROP2, we stimulated BEAS-2B with cigarette smoke to construct an in vitro model. We found that TROP2 expression increased, whereas TROP2 silencing reversed the cigarette smoke extract-induced decrease in mitochondrial membrane potential, increased reactive oxygen species content, decreased adenosine triphosphate (ATP) production, increased inflammatory factor secretion and increased apoptosis. In addition, we searched online bioinformatics and screened the gene dynamin-related protein 1 (DRP1) related to mitophagy as the research object. Co-IP assay verified the binding relationship between DRP1 and TROP2. Further study found that TROP2 promoted mitophagy and apoptosis of BEAS-2B cells by up-regulating the expression of DRP1. In addition, PTEN-induced putative kinase 1 (PINK1) is a potential binding protein of DRP1, and DRP1 accelerated mitophagy and apoptosis of BEAS-2B cells by promoting the expression of PINK1. We established a COPD SD rat model by cigarette smoke exposure and LPS instillation and treated it by intraperitoneal injection of si-TROP2. The results showed that TROP2 silencing restored lung function and reduced the secretion of inflammatory factors in bronchoalveolar lavage fluid. In conclusion, TROP2 can be used as a new reference for COPD treatment.

PINK1 deficiency alters muscle stem cell fate decision and muscle regenerative capacity.

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions.

Integrated genome-wide association and transcriptomic analysis to identify receptor kinase genes to stripe rust resistance in wheat germplasm from southwestern China.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Identification of new and elite Pst-resistance loci or genes has the potential to enhance overall resistance to this pathogen. Here, we conducted an integrated genome-wide association study (GWAS) and transcriptomic analysis to screen for loci associated with resistance to stripe rust in 335 accessions from Yunnan, including 311 landraces and 24 cultivars. Based on the environmental phenotype, we identified 113 protein kinases significantly associated with Pst resistance using mixed linear model (MLM) and generalized linear model (GLM) models. Transcriptomic analysis revealed that 52 of 113 protein kinases identified by GWAS were up and down regulated in response to Pst infection. Among these genes, a total of 15 receptor kinase genes were identified associated with Pst resistance. 11 candidate genes were newly discovered in Yunnan wheat germplasm. Our results revealed that resistance alleles to stripe rust were accumulated in Yunnan wheat germplasm, implying direct or indirect selection for improving stripe rust resistance in elite wheat breeding programs.

Inhibiting Pink1/Parkin-mediated mitophagy enhances the anticancer effects of quercetin in hepatocellular carcinomaf.

Quercetin, a naturally occurring flavonoid, has been investigated for its potential anti-cancer effects in various types of cancer, including hepatocellular carcinoma (HCC). However, its suppressing effect on reactive oxygen species (ROS) production might limited its anti-cancer effects. In this study, we aimed to explore the interplay among quercetin, mitochondrial dynamics and mitophagy and whether mitophagy-inhibition synergistically enhances the anti-tumor effects of quercetin. Huh7 and Hep3B cells were utilized for in vitro and in vivo studies. Results showed that quercetin treatment significantly increased the expression of mitochondrial fusion genes (MFN1 and MFN2) and decreased the expression of fission genes (DRP1 and FIS1) in Huh7 and Hep3B cells, leading to a more fused and elongated mitochondrial network. Quercetin upregulated the expression of key mitophagy regulators, PINK1 and PARK2, and enhanced the colocalization of mitochondria with lysosomes, indicating increased mitophagy. Knockdown of PINK1, PARK2, or SIRT1 attenuated quercetin-induced mitophagy and reduction of intracellular ROS levels. Quercetin treatment upregulates SIRT1 expression, which subsequently enhances PINK1 and PARK2 expression in Huh7 and Hep3B cells. In vivo experiments using Hep3B xenograft models revealed that the combination of quercetin with the mitophagy inhibitor hydroxychloroquine or SIRT1 knockdown significantly enhanced the anticancer effects of quercetin, as evidenced by reduced tumor size and weight, increased necrosis and apoptosis, and decreased proliferation in tumor tissues. These findings suggest that quercetin-induced mitochondrial fusion and Pink1/Parkin-dependent mitophagy may negatively influence its anti-cancer effects in HCC. Targeting mitophagy may enhance the therapeutic potential of quercetin in HCC treatment.

KdpD is a tandem serine histidine kinase that controls K+ pump KdpFABC transcriptionally and post-translationally.

Two-component systems, consisting of a histidine kinase and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a histidine kinase of the KdpDE two-component system, which controls production of the potassium pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high potassium levels, using not its C-terminal histidine kinase domain but an N-terminal atypical serine kinase domain. Sequence analysis of KdpDs from different species highlights that some KdpDs are much shorter than others. We show that, while Escherichia coli KdpD's atypical serine kinase domain responds directly to potassium levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial potassium homeostasis.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

The SCF-FBW7ß E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1.

Understanding the mechanisms that govern the stability of functionally crucial proteins is essential for various cellular processes, development, and overall cell viability. Disturbances in protein homeostasis are linked to the pathogenesis of neurodegenerative diseases. PTEN-induced kinase 1 (PINK1), a protein kinase, plays a significant role in mitochondrial quality control and cellular stress response, and its mutated forms lead to early-onset Parkinson's disease. Despite its importance, the specific mechanisms regulating PINK1 protein stability have remained unclear. This study reveals a cytoplasmic interaction between PINK1 and F-box and WD repeat domain-containing 7ß (FBW7ß) in mammalian cells. FBW7ß, a component of the Skp1-Cullin-1-F-box protein complex-type ubiquitin ligase, is instrumental in recognizing substrates. Our findings demonstrate that FBW7ß regulates PINK1 stability through the Skp1-Cullin-1-F-box protein complex and the proteasome pathway. It facilitates the K48-linked polyubiquitination of PINK1, marking it for degradation. When FBW7 is absent, PINK1 accumulates, leading to heightened mitophagy triggered by carbonyl cyanide 3-chlorophenylhydrazone treatment. Moreover, exposure to the toxic compound staurosporine accelerates PINK1 degradation via FBW7ß, correlating with increased cell death. This study unravels the intricate mechanisms controlling PINK1 protein stability and sheds light on the novel role of FBW7ß. These findings deepen our understanding of PINK1-related pathologies and potentially pave the way for therapeutic interventions.