The Na+-Cl- cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.

Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.

Alfalfa (Medicago L.) is a high-quality perennial leguminous forage with the advantages of salt tolerance, mowing tolerance, high protein content, and other economically valuable characteristics. As the sixth class of plant hormones, brassinosteroids (BRs) play indispensable roles in modulating a variety of plant growth, maturation, and environmental adaptation processes, thereby influencing vegetal expansion and development. Brassinosteroid signal kinases (BSKs) are key cytoplasmic receptor kinases downstream of the BR signaling transduction pathway, participating in plant growth, development, and stress regulation. However, the phylogenetic and expression pattern analyses of the BSK gene family among the five alfalfa species have rarely been reported; in this study, 52 BSK family members were found in the genomes of the five subspecies, and phylogenetic trees were constructed according to protein sequences, allowing us to categorize all BSKs into seven distinct groups. Domain, conserved motif, and exon-intron structural analyses showed that most BSK members were relatively conserved, except for MtBSK3-2, MtBSK7-1, and MtBSK7-2, which may be truncated members. Intra-species collinearity and Ka/Ks analyses showed that purifying selection influenced BSK genes during evolution; most of the cis-acting elements in the promoter region were associated with responses, such as light, defense, and stress, anaerobic induction, MeJA, and abscisic acid. Expression pattern analysis indicated that the majority of alfalfa genes exhibited downregulation after reaching a peak at 0.5 h after treatment with 250 mM NaCl, especially for MsBSK14, MsBSK15, MsBSK17, MsBSK19, and MsBSK21; meanwhile, MsBSK4, MsBSK7, and MsBSK9 increased and were highly expressed at 12 h, demonstrating significantly altered expression patterns under salt stress; furthermore, MsBSK4, MsBSK7, and MsBSK9 exhibited expression specifically in the leaves. qRT-PCR analysis confirmed the expression trends for MsBSK4, MsBSK7, MsBSK9, MsBSK14, MsBSK15, and MsBSK16 matched the transcriptome data. However, the trends for MsBSK17, MsBSK19, and MsBSK21 diverged from the transcriptome data. Our study may provide a foundation for further functional analyses of BSK genes in growth, development, and salt stress tolerance in alfalfa.

Cotton is essential for the textile industry as a primary source of natural fibers. However, environmental factors like drought present significant challenges to its cultivation, adversely affecting both production levels and fiber quality. Enhancing cotton\'s drought resilience has the potential to reduce yield losses and support the growth of cotton farming. In this study, the cotton calcium-dependent protein kinase GhCDPK16 was characterized, and the transcription level of GhCDPK16 was significantly upregulated under drought and various stress-related hormone treatments. Physiological analyses revealed that the overexpression of GhCDPK16 improved drought stress resistance in Arabidopsis by enhancing osmotic adjustment capacity and boosting antioxidant enzyme activities. In contrast, silencing GhCDPK16 in cotton resulted in increased dehydration compared with the control. Furthermore, reduced antioxidant enzyme activities and downregulation of ABA-related genes were observed in GhCDPK16-silenced plants. These findings not only enhanced our understanding of the biological functions of GhCDPK16 and the mechanisms underlying drought stress resistance but also underscored the considerable potential of GhCDPK16 in improving drought resilience in cotton.

Brassinosteroids (BRs) are an essential group of plant hormones regulating numerous aspects of plant growth, development, and stress responses. BRI1, along with its co-receptor BAK1, are involved in brassinosteroid sensing and early events in the BR signal transduction cascade. Mutational analysis of a particular gene is a powerful strategy for investigating its biochemical role. Molecular genetic studies, predominantly in Arabidopsis thaliana, but progressively in numerous other plants, have identified many mutants of the BRI1 gene and its orthologs to gain insight into its structure and function. So far, the plant kingdom has identified up to 40 bri1 alleles in Arabidopsis and up to 30 bri1 orthologs in different plants. These alleles exhibit phenotypes that are identical in terms of development and growth. Here, we have summarized bri1 alleles in Arabidopsis and its orthologs present in various plants including monocots and dicots. We have discussed the possible mechanism responsible for the specific allele. Finally, we have briefly debated the importance of these alleles in the research field and the agronomically valuable traits they offer to improve plant varieties.

This study aimed to investigate whether the beneficial effects of PCA on chondrocyte senescence are mediated through the regulation of mitophagy. Chondrocyte senescence plays a significant role in the development and progression of knee osteoarthritis (OA). The compound protocatechuic aldehyde (PCA), which is abundant in the roots of Salvia miltiorrhiza, has been reported to have antioxidant properties and the ability to protect against cellular senescence. To achieve this goal, a destabilization of the medial meniscus (DMM)-induced mouse OA model and a lipopolysaccharide (LPS)-induced chondrocyte senescence model were used, in combination with PINK1 gene knockdown or overexpression. After treatment with PCA, cellular senescence was assessed using Senescence-Associated β-Galactosidase (SA-β-Gal) staining, DNA damage was evaluated using Hosphorylation of the Ser-139 (γH2AX) staining, reactive oxygen species (ROS) levels were measured using Dichlorodihydrofluorescein diacetate (DCFH-DA) staining, mitochondrial membrane potential was determined using a 5,5\',6,6\'-TETRACHLORO-1,1\',3,3\'-*. TETRAETHYBENZIMIDA (JC-1) kit, and mitochondrial autophagy was examined using Mitophagy staining. Western blot analysis was also performed to detect changes in senescence-related proteins, PINK1/Parkin pathway proteins, and mitophagy-related proteins. Our results demonstrated that PCA effectively reduced chondrocyte senescence, increased the mitochondrial membrane potential, facilitated mitochondrial autophagy, and upregulated the PINK1/Parkin pathway. Furthermore, silencing PINK1 weakened the protective effects of PCA, whereas PINK1 overexpression enhanced the effects of PCA on LPS-induced chondrocytes. PCA attenuates chondrocyte senescence by regulating PINK1/Parkin-mediated mitochondrial autophagy, ultimately reducing cartilage degeneration.

Hexavalent chromium (Cr(VI)) is a hazardous metallic compound commonly used in industrial processes. The liver, responsible for metabolism and detoxification, is the main target organ of Cr(VI). Toxicity experiments were performed to investigate the impacts of low-dose exposure to Cr(VI) on rat livers. It was revealed that exposure of 0.05 mg/kg potassium dichromate (K2Cr2O7) and 0.25 mg/kg K2Cr2O7 notably increased malondialdehyde (MDA) levels and the expressions of P-AMPK, P-ULK, PINK1, P-Parkin, and LC3II/LC3I, and significantly reduced SOD activity and P-mTOR and P62 expression levels in liver. Electron microscopy showed that CR(VI) exposure significantly increased mitophagy and the destruction of mitochondrial structure. This study simulates the respiratory exposure mode of CR(VI) workers through intratracheal instillation of CR(VI) in rats. It confirms that autophagy in hepatocytes is induced by low concentrations of CR(VI) and suggest that the liver damage caused by CR(VI) may be associated with the AMPK-related PINK/Parkin signaling pathway.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy.
METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis.
RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them.
CONCLUSIONS: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

Many disease resistance genes have been introgressed into wheat from its wild relatives. However, reduced recombination within the introgressed segments hinders the cloning of the introgressed genes. Here, we have cloned the powdery mildew resistance gene Pm13, which is introgressed into wheat from Aegilops longissima, using a method that combines physical mapping with radiation-induced chromosomal aberrations and transcriptome sequencing analysis of ethyl methanesulfonate (EMS)-induced loss-of-function mutants. Pm13 encodes a kinase fusion protein, designated MLKL-K, with an N-terminal domain of mixed lineage kinase domain-like protein (MLKL_NTD domain) and a C-terminal serine/threonine kinase domain bridged by a brace. The resistance function of Pm13 is validated through transient and stable transgenic complementation assays. Transient over-expression analyses in Nicotiana benthamiana leaves and wheat protoplasts reveal that the fragment Brace-Kinase122-476 of MLKL-K is capable of inducing cell death, which is dependent on a functional kinase domain and the three α-helices in the brace region close to the N-terminus of the kinase domain.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.