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.

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.

BACKGROUND: Airway epithelial cell (AEC) necroptosis contributes to airway allergic inflammation and asthma exacerbation. Targeting the tumor necrosis factor-like ligand 1 A (TL1A)/death receptor 3 (DR3) axis has a therapeutic effect on asthmatic airway inflammation. The role of TL1A in mediating necroptosis of AECs challenged with ovalbumin (OVA) and its contribution to airway inflammation remains unclear.
METHODS: We evaluated the expression of the receptor-interacting serine/threonine-protein kinase 3(RIPK3) and the mixed lineage kinase domain-like protein (MLKL) in human serum and lung, and histologically verified the level of MLKL phosphorylation in lung tissue from asthmatics and OVA-induced mice. Next, using MLKL knockout mice and the RIPK3 inhibitor GSK872, we investigated the effects of TL1A on airway inflammation and airway barrier function through the activation of necroptosis in experimental asthma.
RESULTS: High expression of necroptosis marker proteins was observed in the serum of asthmatics, and necroptosis was activated in the airway epithelium of both asthmatics and OVA-induced mice. Blocking necroptosis through MLKL knockout or RIPK3 inhibition effectively attenuated parabronchial inflammation, mucus hypersecretion, and airway collagen fiber accumulation, while also suppressing type 2 inflammatory factors secretion. In addition, TL1A/ DR3 was shown to act as a death trigger for necroptosis in the absence of caspases by silencing or overexpressing TL1A in HBE cells. Furthermore, the recombinant TL1A protein was found to induce necroptosis in vivo, and knockout of MLKL partially reversed the pathological changes induced by TL1A. The necroptosis induced by TL1A disrupted the airway barrier function by decreasing the expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin, possibly through the activation of the NF-κB signaling pathway.
CONCLUSIONS: TL1A-induced airway epithelial necroptosis plays a significant role in promoting airway inflammation and barrier dysfunction in asthma. Inhibition of the TL1A-induced necroptosis pathway could be a promising therapeutic strategy.

A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)-1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes.

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.

Loss-of-function mutants are fundamental resources for gene function studies. However, it is difficult to generate viable and heritable knockout mutants for essential genes. Here, we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1 (OsMPK1) results in weak mutants. This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations. Using the same C-terminal editing approach, we also obtained viable mutants for a wall-associated protein kinase (Os07g0493200) and a leucine-rich repeat receptor-like protein kinase (Os01g0239700), while the null mutations of these genes were lethal. These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus, which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00165-5.

OBJECTIVE: 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.
目的: 免疫细胞的异常程序性死亡与自身免疫性疾病相关，但系统性红斑狼疮(systemic lupus erythematosus，SLE)，尤其是狼疮肾炎(lupus nephritis，LN)的程序性死亡模式尚不清楚。本研究旨在探究SLE和LN与免疫细胞死亡模式的关联。方法: 在高通量基因表达(Gene Expression Omnibus，GEO)数据库中下载批量RNA测序(bulk RNA sequencing，bulk RNA-seq)和单细胞RNA测序(single-cell RNA sequencing，scRNA-seq)数据，通过生物信息学分析探究SLE患者外周血单个核细胞中3种细胞死亡模式相关基因的表达水平；通过scRNA-seq确定参与细胞死亡模式失衡的关键细胞亚群；采用免疫荧光法检测树突状细胞(dendritic cell，DC)中受体相互作用丝氨酸/苏氨酸激酶3(receptor interacting serine/threonine kinase 3，RIPK3)、混合谱系激酶结构域样蛋白(mixed-lineage kinase domain-like protein、MLKL)、磷酸化MLKL(phosphorylated MLKL，pMLKL)、含半胱氨酸的天冬氨酸蛋白水解酶1(caspase 1、CASP1)、CD1c分子(CD1c molecule、CD1C)、含C型凝集素结构域9A(C-type lectin domain containing 9A、CLEC9A)和X-C基序趋化因子受体1(X-C motif chemokine receptor 1，XCR1)的表达水平；对中南大学湘雅三医院收集的LN患者和健康对照者(healthy control，HC)的肾组织进行scRNA-seq，并通过生物信息学分析参与细胞死亡模式失衡的关键细胞亚群；采用拟时序分析和配受体分析探究不同DC亚群的分化方向和细胞通信。采用瞬时转染技术分别在RAW264.7细胞中转染空质粒、空质粒+dsDNA(HSV-DNA)、空质粒+200 μmol/L叔丁基过氧化氢(tert-butyl-hydroperoxide，TBHP)、STING shRNA质粒、STING shRNA质粒+dsDNA(HSV-DNA)、STING shRNA质粒+200 μmol/L TBHP；采用Annexin V-mCherry和SYTOX Green染色检测各组细胞死亡情况；蛋白质印迹法检测各组CASP1、消皮素D(gasdermin D，GSDMD)、RIPK3和MLKL的活化情况。结果: 生物信息学分析显示SLE和LN患者存在3种细胞死亡模式的失衡：促炎型细胞焦亡和坏死性凋亡被激活，抗炎型细胞凋亡被抑制。其中的关键细胞亚群为DC亚群，并聚焦于CLEC9A+cDC1。免疫荧光法结果显示在外周血中SLE组DC中RIPK3、MLKL和CASP1表达水平较HC组DC升高；通过CLEC9A和XCR1标记肾组织中的cDC1，结果显示LN组cDC1的pMLKL和CASP1表达水平高于HC组。拟时序分析和配受体分析提示LN肾组织中CLEC9A+cDC1亚群存在外周循环起源。Annexin V-mCherry和SYTOX Green染色结果显示，在RAW264.7细胞中，与转染空质粒组相比，转染STING shRNA质粒组的死亡细胞数目减少；蛋白质印迹法结果显示与转染空质粒组相比，转染STING shRNA质粒组CASP1、GSDMD、RIPK3和MLKL的活化减少。结论: 本研究为CLEC9A+cDC1在SLE和LN的细胞死亡模式失衡中的作用提供了新的思路。.

Damage to renal tubular epithelial cells (RTECs) signaled the onset and progression of sepsis-associated acute kidney injury (SA-AKI). Recent research on mitochondria has revealed that mitophagy plays a crucial physiological role in alleviating injury to RTECs and it is suppressed progressively by the inflammation response in SA-AKI. However, the mechanism by which inflammation influences mitophagy remains poorly understood. We examined how macrophage migration inhibitory factor (MIF), a pro-inflammatory protein, influences the PINK1-Parkin pathway of mitophagy by studying protein-protein interactions when MIF was inhibited or overexpressed. Surprisingly, elevated levels of MIF were found to directly bind to PINK1, disrupting its interaction with Parkin. This interference hindered the recruitment of Parkin to mitochondria and impeded the initiation of mitophagy. Furthermore, this outcome led to significant apoptosis of RTECs, which could, however, be reversed by an MIF inhibitor ISO-1 and/or a new mitophagy activator T0467. These findings highlight the detrimental impact of MIF on renal damage through its disruption of the interaction between PINK1 and Parkin, and the therapeutic potential of ISO-1 and T0467 in mitigating SA-AKI. This study offers a fresh perspective on treating SA-AKI by targeting MIF and mitophagy.

The prevalence of diabetes is increasing worldwide. Massive death of pancreatic beta-cells causes type 1 diabetes. Progressive loss of beta-cell function and mass characterizes type 2 diabetes. To date, none of the available antidiabetic drugs promotes the maintenance of a functional mass of endogenous beta-cells, revealing an unmet medical need. Dysfunction and apoptotic death of beta-cells occur, in particular, through the activation of intracellular protein kinases. In recent years, protein kinases have become highly studied targets of the pharmaceutical industry for drug development. A number of drugs that inhibit protein kinases have been approved for the treatment of cancers. The question of whether safe drugs that inhibit protein kinase activity can be developed and used to protect the function and survival of beta-cells in diabetes is still unresolved. This review presents arguments suggesting that several protein kinases in beta-cells may represent targets of interest for the development of drugs to treat diabetes.

NUAK family kinase 2 (NUAK2) is a promising target for cancer therapeutics due to its reported role in protein phosphorylation, a critical process in cancer cell survival, proliferation, invasion, and senescence. This study aimed to identify novel inhibitors that disrupt NUAK2 activity. We have already identified two KRICT Hippo kinase inhibitor (KHKI) compounds, such as KHKI-01128 and KHKI-01215. Our aim was to evaluate the impact of KHKI-01128 and KHKI-01215 on NUAK2 activity and elucidate its mechanism in colorectal cancer cells.
To evaluate anticancer properties of these inhibitors, four in vitro assays in the SW480 cell line (time-resolved fluorescence resonance energy transfer assay, KINOMEscan kinase profiling, viability, and apoptosis assays) and two pharmacological mechanism analyses (Gene Set Enrichment Analysis and western blotting) were performed.
KHKI-01128 and KHKI-01215 exhibited potent inhibitory activity against NUAK2 (half-maximal inhibitory concentration=0.024±0.015 μM and 0.052±0.011 μM, respectively). These inhibitors suppressed cell proliferation, with half-maximal inhibitory concentrations of 1.26±0.17 μM and 3.16±0.30 μM, respectively, and induced apoptosis of SW480 cells. Gene Set Enrichment Analysis revealed negative enrichment scores of -0.84 for KHKI-01128 (false-discovery rate=0.70) and 1.37 for KHKI-01215 (false-discovery rate=0.18), indicating that both effectively suppressed the expression of YES1-associated transcriptional regulator (YAP) target genes.
These results suggest that KHKI-01128 and KHKI-01215 are potent NUAK2 inhibitors with promising potential for pharmaceutical applications.