Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis. / æ ‘çªçŠ¶ç»†èƒžäºšç¾¤ä»‹å¯¼ç³»ç»Ÿæ€§çº¢æ–‘ç‹¼ç–®å’Œç‹¼ç–®è‚¾ç‚Žçš„ç¨‹åºæ€§ç»†èƒžæ­»äº¡æ¨¡å¼å¤±è¡¡ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. METHODS: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 µmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 µmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. RESULTS: Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. CONCLUSIONS: This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Canagliflozin Mitigates Diabetic Cardiomyopathy through Enhanced PINK1-Parkin Mitophagy.

Diabetic cardiomyopathy (DCM) is a major determinant of mortality in diabetic populations, and the potential strategies are insufficient. Canagliflozin has emerged as a potential cardioprotective agent in diabetes, yet its underlying molecular mechanisms remain unclear. We employed a high-glucose challenge (60 mM for 48 h) in vitro to rat cardiomyocytes (H9C2), with or without canagliflozin treatment (20 µM). In vivo, male C57BL/6J mice were subjected to streptozotocin and a high-fat diet to induce diabetes, followed by canagliflozin administration (10, 30 mg·kg-1·d-1) for 12 weeks. Proteomics and echocardiography were used to assess the heart. Histopathological alterations were assessed by the use of Oil Red O and Masson's trichrome staining. Additionally, mitochondrial morphology and mitophagy were analyzed through biochemical and imaging techniques. A proteomic analysis highlighted alterations in mitochondrial and autophagy-related proteins after the treatment with canagliflozin. Diabetic conditions impaired mitochondrial respiration and ATP production, alongside decreasing the related expression of the PINK1-Parkin pathway. High-glucose conditions also reduced PGC-1α-TFAM signaling, which is responsible for mitochondrial biogenesis. Canagliflozin significantly alleviated cardiac dysfunction and improved mitochondrial function both in vitro and in vivo. Specifically, canagliflozin suppressed mitochondrial oxidative stress, enhancing ATP levels and sustaining mitochondrial respiratory capacity. It activated PINK1-Parkin-dependent mitophagy and improved mitochondrial function via increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Notably, PINK1 knockdown negated the beneficial effects of canagliflozin on mitochondrial integrity, underscoring the critical role of PINK1 in mediating these protective effects. Canagliflozin fosters PINK1-Parkin mitophagy and mitochondrial function, highlighting its potential as an effective treatment for DCM.

Populus euphratica CPK21 Interacts with NF-YC3 to Enhance Cadmium Tolerance in Arabidopsis.

The toxic metal cadmium (Cd) poses a serious threat to plant growth and human health. Populus euphratica calcium-dependent protein kinase 21 (CPK21) has previously been shown to attenuate Cd toxicity by reducing Cd accumulation, enhancing antioxidant defense and improving water balance in transgenic Arabidopsis. Here, we confirmed a protein-protein interaction between PeCPK21 and Arabidopsis nuclear transcription factor YC3 (AtNF-YC3) by yeast two-hybrid and bimolecular fluorescence complementation assays. AtNF-YC3 was induced by Cd and strongly expressed in PeCPK21-overexpressed plants. Overexpression of AtNF-YC3 in Arabidopsis reduced the Cd inhibition of root length, fresh weight and membrane stability under Cd stress conditions (100 µM, 7 d), suggesting that AtNF-YC3 appears to contribute to the improvement of Cd stress tolerance. AtNF-YC3 improved Cd tolerance by limiting Cd uptake and accumulation, activating antioxidant enzymes and reducing hydrogen peroxide (H2O2) production under Cd stress. We conclude that PeCPK21 interacts with AtNF-YC3 to limit Cd accumulation and enhance the reactive oxygen species (ROS) scavenging system and thereby positively regulate plant adaptation to Cd environments. This study highlights the interaction between PeCPK21 and AtNF-YC3 under Cd stress conditions, which can be utilized to improve Cd tolerance in higher plants.

Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

Andrographolide influences IDD cell autophagy and oxidative stress under mechanical pressure via miR-9/FoxO3/PINK1/Parkin molecular axis.

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.

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system.

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.

Pm57 from Aegilops searsii encodes a tandem kinase protein and confers wheat powdery mildew resistance.

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.

An acidic pH environment converts necroptosis to apoptosis.

Cardiac ischemia results in anaerobic metabolism and lactic acid accumulation and with time, intracellular and extracellular acidosis. Ischemia and subsequent reperfusion injury (IRI) lead to various forms of programmed cell death. Necroptosis is a major form of programmed necrosis that worsens cardiac function directly and also promotes inflammation by the release of cellular contents. Potential effects of increasing acidosis on programmed cell death and their specific components have not been well studied. While apoptosis is caspase-dependent, in contrast, necroptosis is mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1/3). In our study, we observed that at physiological pH = 7.4, caspase-8 inhibition did not prevent TNFα-induced cell death in mouse cardiac vascular endothelial cells (MVECs) but promoted necroptotic cell death. As expected, necroptosis was blocked by RIPK1 inhibition. However, at pH = 6.5, TNFα induced an apoptosis-like pattern which was inhibited by caspase-8 inhibition. Interestingly phosphorylation of necroptotic molecules RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) was enhanced in an acidic pH environment. However, RIPK3 and MLKL phosphorylation was self-limited which may have limited their participation in necroptosis. In addition, an acidic pH promoted apoptosis-inducing factor (AIF) cleavage and nuclear translocation. AIF RNA silencing inhibited cell death, supporting the role of AIF in this cell death. In summary, our study demonstrated that the pH of the micro-environment during inflammation can bias cell death pathways by altering the function of necroptosis-related molecules and promoting AIF-mediated cell death. Further insights into the mechanisms by which an acidic cellular micro-environment influences these and perhaps other forms of regulated cell death, may lead to therapeutic strategies to attenuate IRI.

Brassinosteroid-signaling kinase ZmBSK7 enhances salt stress tolerance in maize.

Salinity has become a crucial environmental factor that restricts plant growth, development, and productivity. Nevertheless, the mechanisms by which plants react to salt stress remain inadequately comprehended. In this study, we identified maize brassinosteroid-signaling kinase gene ZmBSK7 which is homologous to AtBSK1. Our results showed that ZmBSK7 is induced by salt stress and ZmBSK7 localizes in the plasma membrane. ZmBSK7 overexpression increases salt tolerance, while its knockdown decreases salt tolerance in maize. ZmBSK7 reduces the malondialdehyde (MDA) content and the percentage of electrolyte leakage, and also elevates the activities of antioxidant enzymes. Furthermore, ZmBSK7 promotes K+ content accumulation and reduces Na+/K+ ratio. Further found that ZmBSK7 physically interacts with K+ efflux antiporter 2 (ZmKEA2) in vivo and in vitro. Salt stress also increased the expression of ZmKEA2. Thus, ZmBSK7 improves salt tolerance in maize by affecting ZmKEA2 expression to promote K+ content accumulation and reduce Na+/K+ ratio. This study enhances the comprehension of BSK proteins and establishes a theoretical foundation for investigating salt stress tolerance in plants.

MLKL regulates radiation-induced death in breast cancer cells: an interplay between apoptotic and necroptotic signals.

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.

A multifaceted kinase axis regulates plant organ abscission through conserved signaling mechanisms.

Plants have evolved mechanisms to abscise organs as they develop or when exposed to unfavorable conditions.1 Uncontrolled abscission of petals, fruits, or leaves can impair agricultural productivity.2,3,4,5 Despite its importance for abscission progression, our understanding of the IDA signaling pathway and its regulation remains incomplete. IDA is secreted to the apoplast, where it is perceived by the receptors HAESA (HAE) and HAESA-LIKE2 (HSL2) and somatic embryogenesis receptor kinase (SERK) co-receptors.6,7,8,9 These plasma membrane receptors activate an intracellular cascade of mitogen-activated protein kinases (MAPKs) by an unknown mechanism.10,11,12 Here, we characterize brassinosteroid signaling kinases (BSKs) as regulators of floral organ abscission in Arabidopsis. BSK1 localizes to the plasma membrane of abscission zone cells, where it interacts with HAESA receptors to regulate abscission. Furthermore, we demonstrate that YODA (YDA) has a leading role among other MAPKKKs in controlling abscission downstream of the HAESA/BSK complex. This kinase axis, comprising a leucine-rich repeat receptor kinase, a BSK, and an MAPKKK, is known to regulate stomatal patterning, early embryo development, and immunity.10,13,14,15,16 How specific cellular responses are obtained despite signaling through common effectors is not well understood. We show that the identified abscission-promoting allele of BSK1 also enhances receptor signaling in other BSK-mediated pathways, suggesting conservation of signaling mechanisms. Furthermore, we provide genetic evidence supporting independence of BSK1 function from its kinase activity in several developmental processes. Together, our findings suggest that BSK1 facilitates signaling between plasma membrane receptor kinases and MAPKKKs via conserved mechanisms across multiple facets of plant development.

The wall-associated kinase GWN1 controls grain weight and grain number in rice.

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.

Kinematic analysis of kinases and their oncogenic mutations - Kinases and their mutation kinematic analysis.

Protein kinases are crucial cellular enzymes that facilitate the transfer of phosphates from adenosine triphosphate (ATP) to their substrates, thereby regulating numerous cellular activities. Dysfunctional kinase activity often leads to oncogenic conditions. Chosen by using structural similarity to 5UG9, we selected 79 crystal structures from the PDB and based on the position of the phenylalanine side chain in the DFG motif, we classified these 79 crystal structures into 5 group clusters. Our approach applies our kinematic flexibility analysis (KFA) to explore the flexibility of kinases in various activity states and examine the impact of the activation loop on kinase structure. KFA enables the rapid decomposition of macromolecules into different flexibility regions, allowing comprehensive analysis of conformational structures. The results reveal that the activation loop of kinases acts as a "lock" that stabilizes the active conformation of kinases by rigidifying the adjacent α-helices. Furthermore, we investigate specific kinase mutations, such as the L858R mutation commonly associated with non-small cell lung cancer, which induces increased flexibility in active-state kinases. In addition, through analyzing the hydrogen bond pattern, we examine the substructure of kinases in different states. Notably, active-state kinases exhibit a higher occurrence of α-helices compared to inactive-state kinases. This study contributes to the understanding of biomolecular conformation at a level relevant to drug development.

Pneumococcal hydrogen peroxide regulates host cell kinase activity.

Introduction: Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. Methods: We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Results: Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation.

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.

Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5 kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42 kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

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.

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.

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.

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.