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Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

CDKL5 Deficiency Disorder (CDD) is a debilitating epileptic encephalopathy disorder affecting young children with no effective treatments. CDD is caused by pathogenic variants in Cyclin-Dependent Kinase-Like 5 (CDKL5), a protein kinase that regulates key phosphorylation events in neurons. For therapeutic intervention, it is essential to understand molecular pathways and phosphorylation targets of CDKL5. Using an unbiased phosphoproteomic approach we identified novel targets of CDKL5, including GTF2I, PPP1R35, GATAD2A and ZNF219 in human iPSC-derived neuronal cells. The phosphoserine residue in the target proteins lies in the CDKL5 consensus motif. We validated direct phosphorylation of GTF2I and PPP1R35 by CDKL5 using complementary approaches. GTF2I controls axon guidance, cell cycle and neurodevelopment by regulating expression of neuronal genes. PPP1R35 is critical for centriole elongation and cilia morphology, processes that are impaired in CDD. PPP1R35 interacts with CEP131, a known CDKL5 phospho-target. GATAD2A and ZNF219 belong to the Nucleosome Remodelling Deacetylase (NuRD) complex, which regulates neuronal activity-dependent genes and synaptic connectivity. In-depth knowledge of molecular pathways regulated by CDKL5 will allow a better understanding of druggable disease pathways to fast-track therapeutic development. [ABSTRACT FROM AUTHOR]

Background: Cellular retinoic acid binding protein 1 (CRABP1) mediates rapid, non-canonical activity of retinoic acid (RA) by forming signalosomes via protein-protein interactions. Two signalosomes have been identified previously: CRABP1-MAPK and CRABP1-CaMKII. Crabp1 knockout (CKO) mice exhibited altered exosome profiles, but the mechanism of CRABP1 action was unclear. This study aimed to screen for and identify novel CRABP1 signalosomes that could modulate exosome secretion by using a combinatorial approach involving biochemical, bioinformatic and molecular studies. Methods: Immunoprecipitation coupled with mass spectrometry (IP-MS) identified candidate CRABP1-interacting proteins which were subsequently analyzed using GO Term Enrichment, Functional Annotation Clustering; and Pathway Analysis. Gene expression analysis of CKO samples revealed altered expression of genes related to exosome biogenesis and secretion. The effect of CRABP1 on exosome secretion was then experimentally validated using CKO mice and a Crabp1 knockdown P19 cell line. Results: IP-MS identified CRABP1-interacting targets. Bioinformatic analyses revealed significant association with actin cytoskeletal dynamics, kinases, and exosome secretion. The effect of CRABP1 on exosome secretion was experimentally validated by comparing circulating exosome numbers of CKO and wild type (WT) mice, and secreted exosomes from WT and siCRABP1-P19 cells. Pathway analysis identified kinase signaling and Arp2/3 complex as the major pathways where CRABP1-signalosomes modulate exosome secretion, which was validated in the P19 system. Conclusion: The combinatorial approach allowed efficient screening for and identification of novel CRABP1-signalosomes. The results uncovered a novel function of CRABP1 in modulating exosome secretion, and suggested that CRABP1 could play roles in modulating intercellular communication and signal propagation. [ABSTRACT FROM AUTHOR]

SUMMARY: Phytohormones are essential signaling molecules regulating various processes in growth, development, and stress responses. Genetic and molecular studies, especially using Arabidopsis thaliana (Arabidopsis), have discovered many important players involved in hormone perception, signal transduction, transport, and metabolism. Phytohormone signaling pathways are extensively interconnected with other endogenous and environmental stimuli. However, our knowledge of the huge and complex molecular network governed by a hormone remains limited. Here we report a global overview of downstream events of an abscisic acid (ABA) receptor, REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) 6 (also known as PYRABACTIN RESISTANCE 1 [PYR1]‐LIKE [PYL] 12), by integrating phosphoproteomic, proteomic and metabolite profiles. Our data suggest that the RCAR6 overexpression constitutively decreases the protein levels of its coreceptors, namely clade A protein phosphatases of type 2C, and activates sucrose non‐fermenting‐1 (SNF1)‐related protein kinase 1 (SnRK1) and SnRK2, the central regulators of energy and ABA signaling pathways. Furthermore, several enzymes in sugar metabolism were differentially phosphorylated and expressed in the RCAR6 line, and the metabolite profile revealed altered accumulations of several organic acids and amino acids. These results indicate that energy‐ and water‐saving mechanisms mediated by the SnRK1 and SnRK2 kinases, respectively, are under the control of the ABA receptor‐coreceptor complexes. Significance Statement: Phosphoproteomic, proteomic, and metabolite profiles of the Arabidopsis overexpressing an abscisic acid (ABA) receptor were integrated, providing a global overview of the complex network governed by a hormone. The overproduction of an ABA receptor caused the decreased protein levels of its coreceptor phosphatases, activating the central regulatory kinases of energy and stress signaling. [ABSTRACT FROM AUTHOR]

Remarkable progress has been made in the development of cysteine‐targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K–E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D‐targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide‐based small‐molecule inhibitors capable of inducing intramolecular cross‐linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide‐facilitated, ligand‐induced mechanism leading to intramolecular kinase cross‐linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative. [ABSTRACT FROM AUTHOR]

Receptor-interacting protein kinase 1 (RIPK1) plays a crucial role in controlling inflammation and cell death. Its function is tightly controlled through post-translational modifications, enabling its dynamic switch between promoting cell survival and triggering cell death. Phosphorylation of RIPK1 at various sites serves as a critical mechanism for regulating its activity, exerting either activating or inhibitory effects. Perturbations in RIPK1 phosphorylation status have profound implications for the development of severe inflammatory diseases in humans. This review explores the intricate regulation of RIPK1 phosphorylation and dephosphorylation and highlights the potential of targeting RIPK1 phosphorylation as a promising therapeutic strategy for mitigating human diseases. [ABSTRACT FROM AUTHOR]

Mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti‐cancer therapies. The limitations of ATP‐competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC50 = 2.3 nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor‐promoting macrophage M2 phenotype in vitro and in vivo. In macrophage‐rich tumor model, compound 11 notably slowed growth in a dose‐dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions. [ABSTRACT FROM AUTHOR]

Due to the similarity and diversity among kinases, small molecule kinase inhibitors (SMKIs) often display multi-target effects or selectivity, which have a strong correlation with the efficacy and safety of these inhibitors. However, due to the limited number of well-known popular databases and their restricted data mining capabilities, along with the significant scarcity of databases focusing on the pharmacological similarity and diversity of SMIKIs, researchers find it challenging to quickly access relevant information. The KLIFS database is representative of specialized application databases in the field, focusing on kinase structure and co-crystallised kinase-ligand interactions, whereas the KLSD database in this paper emphasizes the analysis of SMKIs among all reported kinase targets. To solve the current problem of the lack of professional application databases in kinase research and to provide centralized, standardized, reliable and efficient data resources for kinase researchers, this paper proposes a research program based on the ChEMBL database. It focuses on kinase ligands activities comparisons. This scheme extracts kinase data and standardizes and normalizes them, then performs kinase target difference analysis to achieve kinase activity threshold judgement. It then constructs a specialized and personalized kinase database platform, adopts the front-end and back-end separation technology of SpringBoot architecture, constructs an extensible WEB application, handles the storage, retrieval and analysis of the data, ultimately realizing data visualization and interaction. This study aims to develop a kinase database platform to collect, organize, and provide standardized data related to kinases. By offering essential resources and tools, it supports kinase research and drug development, thereby advancing scientific research and innovation in kinaserelated fields. [ABSTRACT FROM AUTHOR]

Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5–SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5S10A exhibit centriole assembly failure, while those expressing SAS-5S331/338/340A possess extra centrioles. We show that in the absence of serine 10 phosphorylation, the SAS-5–SAS-6 complex is recruited to centrioles, but is not stably incorporated, possibly due to a failure to coordinately recruit the microtubule-binding protein SAS-4. Our work defines the critical role of phosphorylation during centriole assembly and reveals that ZYG-1 might play a role in preventing the formation of excess centrioles. Synopsis: In C. elegans, a critical step in centriole assembly involves phosphorylation of the centriole scaffold protein SAS-5 by ZYG-1, which is required for recruitment of SAS-4 and the stable incorporation of the SAS-5-SAS-6 complex into the nascent centriole. In C. elegans, the kinase ZYG-1 phosphorylates the central scaffold protein SAS-5 to drive centriole assembly. Phosphorylation of SAS-5 on serine 10 is specifically required for stable incorporation of the SAS-5-SAS-6 complex into nascent centrioles. SAS-4, a stabilizer of nascent centrioles, is not recruited to the assembling centriole when phosphorylation of serine 10 is blocked. Serine-to-alanine mutations within the C-terminus of SAS-5 results in elevated levels of the protein and centriole amplification, suggesting that SAS-5 might be regulated by phosphorylation. In C. elegans, a critical step in centriole assembly involves phosphorylation of the centriole scaffold protein SAS-5 by ZYG-1, which is required for recruitment of SAS-4 and the stable incorporation of the SAS-5-SAS-6 complex into the nascent centriole. [ABSTRACT FROM AUTHOR]

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases. [ABSTRACT FROM AUTHOR]

Mitochondria are organelles involved in cellular energetics in all eukaryotes, and changes in their dynamics, fission, fusion, or localization can lead to cell defects and disease in humans. Budding yeast, Saccharomyces cerevisiae, has been shown to be an effective model organism in elucidating mechanisms underpinning these mitochondrial processes. In the work presented here, a genetic screen was performed to identify overexpressing kinases, phosphatases, and ubiquitin ligases, which resulted in mitochondrial defects. A total of 33 overexpressed genes showed mitochondrial phenotypes but without severe growth defects. These included a subset that affected the timing of mitochondrial inheritance and were the focus of further study. Using cell and biochemical approaches, the roles of the PAK-family kinase Cla4 and the E3-ubiquitin ligases Dma1 and Dma2 were investigated. Previous studies have indicated the roles of kinase Cla4 and ligases Dma1 and Dma2 in triggering the degradation of trafficking adaptors in the bud, which leads to disruption of the interaction with the transporting class V myosin, Myo2. Here, we map a key interface between Cla4 and the mitochondrial adaptor Mmr1 necessary for phosphorylation and identify a region of Mmr1 required for its degradation via Dma1 and Dma2. Together, our data provide insights into key regulatory regions of Mmr1 responsible for its function in mitochondrial inheritance. [ABSTRACT FROM AUTHOR]

Kinase is an enzyme that helps in the phosphorylation of the targeted molecules and can affect their ability to react with other molecules. So, kinase influences metabolic reactions like cell signaling, secretory processes, transport of molecules, etc. The increased activity of certain kinases may cause various types of cancer, i.e. leukemia, glioblastoma, and neuroblastomas. So, the growth of particular cancer cells can be prevented by the inhibition of the kinase responsible for those cancers. Natural products are the key resources for the development of new drugs where approximately 60% of anti-tumor drugs are being developed with the same including specific kinase dwellers. This study comprised molecular interactions of various molecules (obtained from natural sources) as kinase inhibitors for the treatment of cancer. It is expected that by analyzing the skeleton behavior, the process of action, and the body-related activity of these organic products, new cancer-avoiding molecules can be developed. [ABSTRACT FROM AUTHOR]

Purpose: Joint pain is one of the most commonly reported pain types in the United States. In the case of patients suffering from inflammatory diseases such as osteoarthritis (OA) and gout, persistent inflammation due to long-term overexpression of several key cytokines has been linked to neuronal hypersensitivity and damage within the joints. Ultimately, a subset of patients develop chronic pain. Pharmacologic treatment of joint pain involves the use of analgesics such as acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), opioids, antidepressants, as well as intra-articular injections of corticosteroids and hyaluronic acid. However, NSAIDs are short-acting and fail to alleviate severe pain, opioids are generally ineffective at managing chronic pain, and all therapeutic options involve increased risks of serious side effects. Methods: We explored the therapeutic and analgesic effects of transforming growth factor-β-activated kinase 1 (TAK1) inhibition in both the monoiodoacetate (MIA) and monosodium urate (MSU) models of joint pain as an innovative strategy for alleviating chronic inflammatory pain. Mechanical allodynia (Von Frey), weight-bearing and histological changes were measured in separate groups of rats receiving either the selective TAK1 inhibitor, HS-276, gabapentin or vehicle. Results: Our data support that TAK1 inhibition effectively prevented the development of mechanical allodynia and differential weight-bearing in the MIA model. In the MSU model of gouty arthritis, treatment with HS-276 significantly reduced mechanical allodynia and knee edema in female rats, but not male rats. Histological evaluation of effected joints in both models showed that HS-276 treatment significantly reduced disease-induced degradation of the joint. Conclusion: Our results support that TAK1 is a critical signaling node in inflammatory joint diseases such as OA and gouty arthritis. Selective pharmacological inhibition significantly attenuated several aspects of the disease, including joint degeneration and mechanical pain. Thus, TAK1 is a novel therapeutic target for the treatment of painful inflammatory joint diseases. Perspective: This article reports on the therapeutic potential of TAK1 in the treatment of chronic inflammatory joint diseases such as OA and gout. Using the selective TAK1 inhibitor, HS-276, we show the therapeutic and analgesic effects of TAK1 inhibition in two preclinical murine models of inflammatory joint pain. [ABSTRACT FROM AUTHOR]

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Bifidobacterium species are one of the most important probiotic microorganisms which are present in both, infants and adults. Nowadays, growing data describing their healthy properties arise, indicating they could act at the cellular and molecular level. However, still little is known about the specific mechanisms promoting their beneficial effects. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is involved in the protective mechanisms in the gastrointestinal tract, where it can be provided by epithelial cells, macrophages, or bacteria. The present study explored whether induction of iNOS-dependent NO synthesis in macrophages stems from the cellular action of Bifidobacterium species. The ability of ten Bifidobacterium strains belonging to 3 different species (Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium animalis) to activate MAP kinases, NF-κB factor, and iNOS expression in a murine bone-marrow-derived macrophages cell line was determined by Western blotting. Changes in NO production were determined by the Griess reaction. It was performed that the Bifidobacterium strains were able to induce NF-қB-dependent iNOS expression and NO production; however, the efficacy depends on the strain. The highest stimulatory activity was observed for Bifidobacterium animalis subsp. animals CCDM 366, whereas the lowest was noted for strains Bifidobacterium adolescentis CCDM 371 and Bifidobacterium longum subsp. longum CCDM 372. Both TLR2 and TLR4 receptors are involved in Bifidobacterium-induced macrophage activation and NO production. We showed that the impact of Bifidobacterium on the regulation of iNOS expression is determined by MAPK kinase activity. Using pharmaceutical inhibitors of ERK 1/2 and JNK, we confirmed that Bifidobacterium strains can activate these kinases to control iNOS mRNA expression. Concluding, the induction of iNOS and NO production may be involved in the protective mechanism of action observed for Bifidobacterium in the intestine, and the efficacy is strain-dependent. [ABSTRACT FROM AUTHOR]

Abstract Background Cellular retinoic acid binding protein 1 (CRABP1) mediates rapid, non-canonical activity of retinoic acid (RA) by forming signalosomes via protein-protein interactions. Two signalosomes have been identified previously: CRABP1-MAPK and CRABP1-CaMKII. Crabp1 knockout (CKO) mice exhibited altered exosome profiles, but the mechanism of CRABP1 action was unclear. This study aimed to screen for and identify novel CRABP1 signalosomes that could modulate exosome secretion by using a combinatorial approach involving biochemical, bioinformatic and molecular studies. Methods Immunoprecipitation coupled with mass spectrometry (IP-MS) identified candidate CRABP1-interacting proteins which were subsequently analyzed using GO Term Enrichment, Functional Annotation Clustering; and Pathway Analysis. Gene expression analysis of CKO samples revealed altered expression of genes related to exosome biogenesis and secretion. The effect of CRABP1 on exosome secretion was then experimentally validated using CKO mice and a Crabp1 knockdown P19 cell line. Results IP-MS identified CRABP1-interacting targets. Bioinformatic analyses revealed significant association with actin cytoskeletal dynamics, kinases, and exosome secretion. The effect of CRABP1 on exosome secretion was experimentally validated by comparing circulating exosome numbers of CKO and wild type (WT) mice, and secreted exosomes from WT and siCRABP1-P19 cells. Pathway analysis identified kinase signaling and Arp2/3 complex as the major pathways where CRABP1-signalosomes modulate exosome secretion, which was validated in the P19 system. Conclusion The combinatorial approach allowed efficient screening for and identification of novel CRABP1-signalosomes. The results uncovered a novel function of CRABP1 in modulating exosome secretion, and suggested that CRABP1 could play roles in modulating intercellular communication and signal propagation.

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.

Abstract Mutations in PINK1 and Parkin cause early-onset Parkinson’s Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5–3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

The study of a receptor-ligand system generally relies on the analysis of its doseresponse (or concentration-effect) curve, which quantifies the relation between ligand concentration and the biological effect (or cellular response) induced when binding its specific cell surface receptor. Mathematical models of receptor-ligand systems have been developed to compute a dose-response curve under the assumption that the biological effect is proportional to the number of ligand-bound receptors. Given a dose-response curve, two quantities (or metrics) have been defined to characterize the properties of the ligand-receptor system under consideration: amplitude and potency (or halfmaximal effective concentration, and denoted by EC50). Both the amplitude and the EC50 are key quantities commonly used in pharmaco-dynamic modeling, yet a comprehensive mathematical investigation of the behavior of these two metrics is still outstanding; for a large (and important) family of receptors, called cytokine receptors, we still do not know how amplitude and EC50 depend on receptor copy numbers. Here we make use of algebraic approaches (Gr\"obner basis) to study these metrics for a large class of receptor-ligand models, with a focus on cytokine receptors. In particular, we introduce a method, making use of two motivating examples based on the interleukin-7 (IL-7) receptor, to compute analytic expressions for the amplitude and the EC50. We then extend the method to a wider class of receptor-ligand systems, sequential receptor-ligand systems with extrinsic kinase, and provide some examples. The algebraic methods developed in this paper not only reduce computational costs and numerical errors, but allow us to explicitly identify key molecular parameters and rates which determine the behavior of the dose-response curve. Thus, the proposed methods provide a novel and useful approach to perform model validation, assay design and parameter exploration of receptor-ligand systems. [ABSTRACT FROM AUTHOR]

Imidazole peptides possess multiple functions, including antioxidant effects, although their biological activities are largely unclear. Their production in humans and animals suggests their physiological roles and validates their safety for pharmaceutical or supplemental use. This study investigated the in vitro anti-anaphylactic potential of two histidine-containing dipeptides, carnosine and anserine in mast cells and basophils. Carnosine and anserine reduced mast cell degranulation elicited by anti-ovalbumin monoclonal IgE and ovalbumin or ionomycin in rat basophilic leukemia RBL2H3 cells without affecting cell viability. In contrast, interleukin-4 production following stimulation was enhanced in the presence of carnosine. Carnosine and anserine strongly inhibited Akt phosphorylation and moderately inhibited ERK phosphorylation. However, these peptides enhanced the increase in phosphorylated JNK levels upon IgE stimulation. The phosphorylation levels of p38 were not affected by carnosine or anserine. To determine the effect of carnosine on basophils, we established a method for detecting IgE-dependent activation in primary cultured mouse splenic basophils via CD63 expression for the first time. Carnosine treatment significantly reduced the increase in CD63 surface expression, a marker of degranulation, in mouse splenic basophils stimulated with anti-IgE. Flow cytometory analysis revealed that mean fluorescence intensities for non-stimulated, anti-IgE-stimulated, and carnosine-pre-treated/anti-IgE-stimulated basophils were 3853 ± 320, 5548 ± 282, and 3853 ± 203, respectively. The findings indicate carnosine and anserine suppress mast cell and basophil IgE-dependent degranulation. The proposed mechanism of the inhibitory effect is the suppression of the activation of phosphoinositide 3-kinase–Akt. Carnosine and anserine may exert anti-anaphylactic effects under physiological and pathological conditions and serve as safe potential drug candidates. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer, a life‐threatening disease, is the seventh most commonly detected cancer among women throughout the world. The present study investigated the effect of tretinoin on cervical cancer growth and metastasis in vitro and in vivo in the mice model. Materials and Methods: Cell Counting Kit‐8, clonogenic survival, and transwell chamber assays were used for determination cells proliferation, colony formation, and invasiveness. Western blotting assay was used for assessment of protein expression whereas AutoDock Vina and Discovery studio software for in silico studies. Results: Tretinoin treatment significantly (p <.05) reduced the proliferation of HT‐3 and Caski cells in concentration‐based manner. Incubation with tretinoin caused a significant decrease in clonogenic survival of HT‐3 and Caski cells compared with the control cultures. The invasive potential of HT‐3 cells was decreased to 18%, whereas that of Caski cells to 21% on treatment with 8 μM concentration of tretinoin. In HT‐3 cells, tretinoin treatment led to a prominent reduction in p‐focal adhesion kinase (FAK), matrix metalloproteinases (MMP)‐2, and MMP‐9 expression in HT‐3 cells. Treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. The metastasis of tumor in model cervical cancer mice group was effectively inhibited in spleen, intestines, and peritoneal cavity. In silico studies showed that tretinoin interacts with alanine, proline, isoleucine, and glycine amino acid residues of FAK protein to block its activation. The 2‐dimensional diagram of interaction of tretinoin with FAK protein revealed that tretinoin binds to alanine and glycine amino acids through conventional hydrogen bonding. Conclusion: In summary, tretinoin suppressed the proliferation, colony formation, and invasiveness of cervical cancer cells in vitro. It decreased the expression of activated focal adhesion kinase, MMP‐2, and MMP‐9 in HT‐3 cells in dose‐dependent manner. In silico studies showed that tretinoin interacts with alanine and glycine amino acids through conventional hydrogen bonding. In vivo data demonstrated that treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. Therefore, tretinoin can be developed as an effective therapeutic agent for cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Peripheral nerve injury disease is a prevalent traumatic condition in current medical practice. Despite the present treatment approaches, encompassing surgical sutures, autologous nerve or allograft nerve transplantation, tissue engineering techniques, and others, an eective clinical treatment method still needs to be discovered. Exploring novel treatment methods to improve peripheral nerve regeneration requires more eort in investigating the cellular and molecular mechanisms involved. Many factors are associated with the regeneration of injured peripheral nerves, including the cross-sectional area of the injured nerve, the length of the nerve gap defect, and various cellular and molecular factors such as Schwann cells, inflammation factors, kinases, and growth factors. As crucial mediators of cellular communication, kinases exert regulatory control over numerous signaling cascades, thereby participating in various vital biological processes, including peripheral nerve regeneration after nerve injury. In this review, we examined diverse kinase classifications, distinct nerve injury types, and the intricate mechanisms involved in peripheral nerve regeneration. Then we stressed the significance of kinases in regulating autophagy, inflammatory response, apoptosis, cell cycle, oxidative processes, and other aspects in establishing conductive microenvironments for nerve tissue regeneration. Finally, we briefly discussed the functional roles of kinases in dierent types of cells involved in peripheral nerve regeneratio [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK/MPK) cascade is an important intercellular signaling module that regulates plant growth, development, reproduction, and responses to biotic and abiotic stresses. A MAPK cascade usually consists of a MAPK kinase kinase (MAPKKK/MEKK), a MAPK kinase (MAPKK/MKK/MEK), and a MAPK. The well-characterized MAPK cascades in plant immunity to date are the MEKK1-MKK1/2-MPK4 cascade and the MAPKKK3/4/5-MKK4/5-MPK3/6 cascade. Recently, major breakthroughs have been made in understanding the molecular mechanisms associated with the regulation of immune signaling by both of these MAPK cascades. In this review, we highlight the most recent advances in understanding the role of both MAPK cascades in activating plant defense and in suppressing or fine-tuning immune signaling. We also discuss the molecular mechanisms by which plants stabilize and maintain the activation of MAPK cascades during immune signaling. Based on this review, we reveal the complexity and importance of the MEKK1-MKK1/2-MPK4 cascade and the MAPKKK3/4/5-MKK4/5-MPK3/6 cascade, which are tightly controlled by their interacting partners or substrates, in plant immunity. [ABSTRACT FROM AUTHOR]

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs. Synopsis: Salmonella enters the bloodstream early during infection. Here, the effector protein SteC is found to activate myosin light chain (MLC), thereby facilitating Salmonella dissemination by manipulating macrophage motility. Salmonella enters the bloodstream via infection of intestinal CX3CR1+ macrophages. Salmonella promotes macrophage migration and invasion in an actin- and SteC-dependent manner. SteC acts as an atypical kinase that phosphorylates host MLC using a broad range of NTPs. SteC-mediated MLC activation is essential for Salmonella dissemination within the host. Dissemination of the intestinal pathogen Salmonella enterica within the host depends on motility of infected intestinal macrophages. [ABSTRACT FROM AUTHOR]

Kinases are enzymes that play a critical role in governing essential biological processes. Due to their pivotal involvement in cancer cell signaling, they have become key targets in the development of anti‐cancer drugs. Among these drugs, those containing the 2,4‐dihalophenyl moiety demonstrated significant potential. Here we show how this moiety, particularly the 2‐fluoro‐4‐iodophenyl one, is crucial for the structural stability of the formed drug‐enzyme complexes. Crystallographic analysis of reported kinase‐inhibitor complex structures highlights the role of the halogen bonding that this moiety forms with specific residues of the kinase binding site. This interaction is not limited to FDA‐approved MEK inhibitors, but it is also relevant for other kinase inhibitors, indicating its broad relevance in the design of this class of drugs. [ABSTRACT FROM AUTHOR]

Sleep disorders are the most widespread mental disorders after stroke and hurt survivors' functional prognosis, response to restoration, and quality of life. This review will address an overview of the progress of research on the biological mechanisms associated with stroke-complicating sleep disorders. Extensive research has investigated the negative impact of stroke on sleep. However, a bidirectional association between sleep disorders and stroke exists; while stroke elevates the risk of sleep disorders, these disorders also independently contribute as a risk factor for stroke. This review aims to elucidate the mechanisms of stroke-induced sleep disorders. Possible influences were examined, including functional changes in brain regions, cerebrovascular hemodynamics, neurological deficits, sleep ion regulation, neurotransmitters, and inflammation. The results provide valuable insights into the mechanisms of stroke complicating sleep disorders. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs), particularly phosphorylation, play a pivotal role in expanding the complexity of the proteome and regulating diverse cellular processes. In this study, we present an efficient Escherichia coli phosphorylation system designed to streamline the evaluation of potential substrates for Arabidopsis thaliana plant kinases, although the technology is amenable to any. The methodology involves the use of IPTG-inducible vectors for co-expressing kinases and substrates, eliminating the need for radioactive isotopes and prior protein purification. We validated the system's efficacy by assessing the phosphorylation of well-established substrates of the plant kinase SnRK1, including the rat ACETYL-COA CARBOXYLASE 1 (ACC1) and FYVE1/FREE1 proteins. The results demonstrated the specificity and reliability of the system in studying kinase-substrate interactions. Furthermore, we applied the system to investigate the phosphorylation cascade involving the A. thaliana MKK3-MPK2 kinase module. The activation of MPK2 by MKK3 was demonstrated to phosphorylate the Myelin Basic Protein (MBP), confirming the system's ability to unravel sequential enzymatic steps in phosphorylation cascades. Overall, this E. coli phosphorylation system offers a rapid, cost-effective, and reliable approach for screening potential kinase substrates, presenting a valuable tool to complement the current portfolio of molecular techniques for advancing our understanding of kinase functions and their roles in cellular signaling pathways. [ABSTRACT FROM AUTHOR]

Tauopathies are a spectrum of clinicopathological neurodegenerative disorders with increased aggregates included in glia and/or neurons of hyperphosphorylated insoluble tau protein, a microtubule-associated protein. Progressive supranuclear palsy (PSP) is an atypical dopaminergic-resistant parkinsonian syndrome, considered as a primary tauopathy with possible alteration of tau isoform ratio, and tau accumulations characterized by 4 R tau species as the main neuropathological lesions. In the present review article, we analyzed and discussed viable disease-modifying and some symptomatic pharmacological therapeutics for PSP syndrome (PSPS). Pharmacological therapy for PSPS may interfere with the aggregation process or promote the clearance of abnormal tau aggregates. A variety of past and ongoing disease-modifying therapies targeting tau in PSPS included genetic, microtubule-stabilizing compounds, anti-phosphorylation, and acetylation agents, antiaggregant, protein removal, antioxidant neuronal and synaptic growth promotion therapies. New pharmacological gene-based approaches may open alternative prevention pathways for the deposition of abnormal tau in PSPS such as antisense oligonucleotide (ASO)-based drugs. Moreover, kinases and ubiquitin-proteasome systems could also be viable targets. [ABSTRACT FROM AUTHOR]

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine‐reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell‐active TCIs are however available. We report herein lysine‐reactive activity‐based probes (ABPs; 2–14) based on the chemistry of aryl fluorosulfates (ArOSO2F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase‐binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X‐ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX‐680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine‐reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine‐reactive ArOSO2F‐based TCIs. [ABSTRACT FROM AUTHOR]

Gene regulation in changing environments is critical for maintaining homeostasis. Some animals undergo a stress-resistant diapause stage to withstand harsh environmental conditions encountered during development. MicroRNAs are one mechanism for regulating gene expression during and after diapause. MicroRNAs downregulate target genes posttranscriptionally through the activity of the microRNA-induced silencing complex. Argonaute is the core microRNA-induced silencing complex protein that binds to both the microRNA and to other microRNA-induced silencing complex proteins. The 2 major microRNA Argonautes in the Caenorhabditis elegans soma are ALG-1 and ALG-2 , which function partially redundantly. Loss of alg-1 [ alg-1 (0) ] causes penetrant developmental phenotypes including vulval defects and the reiteration of larval cell programs in hypodermal cells. However, these phenotypes are essentially absent if alg-1 (0) animals undergo a diapause stage called dauer. Levels of the relevant microRNAs are not higher during or after dauer, suggesting that activity of the microRNA-induced silencing complex may be enhanced in this context. To identify genes that are required for alg-1 (0) mutants to develop without vulval defects after dauer, we performed an RNAi screen of genes encoding conserved kinases. We focused on kinases because of their known role in modulating microRNA-induced silencing complex activity. We found RNAi knockdown of 4 kinase-encoding genes, air-2 , bub-1 , chk-1 , and nekl-3 , caused vulval defects and reiterative phenotypes in alg-1 (0) mutants after dauer, and that these defects were more penetrant in an alg-1 (0) background than in wild type. Our results implicate these kinases as potential regulators of microRNA-induced silencing complex activity during postdauer development in C. elegans. [ABSTRACT FROM AUTHOR]

Downstream signalling involving adenylyl cyclases (ACs) and kinases is a key component of several processes in plants including cell division, growth, and response to stress. ACs are enzymes that generate the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP) while kinases are enzymes that catalyze the addition of a phosphate group to other molecules (trans-phosphorylation) or themselves (auto-phosphorylation). Apparently, while there has been an expanded record of various ACs and kinases identified in plants, no plant molecule to date has been shown to possess both the AC and kinase activities/functions and with such activities/functions having the characteristic of cross-talking interactions. Therefore, in an endeavor to find such a molecule, we searched the amino acid sequence of a known Arabidopsis AC, pentatricopeptide repeat (AtPPR) protein, and found a kinase-specific sequence signature (KSSS), which we speculated to be working in synergy with the AC center in this protein during downstream signalling. So, in order to test if this additional center is catalytically active and perhaps also having some cross-talking interactions with the AC center, we cloned, expressed, and affinity purified a truncated version of AtPPR, harboring both the AC and KSSS centers (AtPPR-AC/K). When tested in vitro, the recombinant AtPPR-AC/K showed a Mn2+-dependent AC activity that is positively enhanced by Ca2+ and HCO3− and a trans-/auto-phosphorylation kinase activity capable of utilizing both ATP and GTP as substrates and specific to the serine, threonine, and tyrosine amino acids as target residues. In addition, the kinase activity of AtPPR-AC/K was found to be reduced by cAMP while at the same time, it was totally shut down by Ca2+. This thus qualified both cAMP and Ca2+ as molecular switches or modulators, capable of regulating AtPPR functions through cross-talking interactions between the activities of its two domains. Our work, therefore, has essentially established AtPPR as the first member of a new class of moonlighting proteins with AC and kinase activities that have cross-talking interactions between themselves, conceivably presenting this protein as an ideal candidate for further explorations to improve plants, particularly agricultural crops. [ABSTRACT FROM AUTHOR]

Disubstituted isothiazolo[4,3-b]pyridines are known inhibitors of cyclin G-associated kinase. Since 3-substituted-7-aryl-isothiazolo[4,3-b]pyridines remain elusive, a strategy was established to prepare this chemotype, starting from 2,4-dichloro-3-nitropyridine. Selective C-4 arylation using ligand-free Suzuki-Miyaura coupling and palladium-catalyzed aminocarbonylation functioned as key steps in the synthesis. The 3-N-morpholinyl-7-(3,4-dimethoxyphenyl)-isothiazolo[4,3-b]pyridine was completely devoid of GAK affinity, in contrast to its 3,5- and 3,6-disubstituted congeners. Molecular modeling was applied to rationalize its inactivity as a GAK ligand. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase2 (TG2) has emerged as a key enigmatic protein in the development of various metabolic and age-related diseases. It catalyzes covalent cross-linking of countless proteins and provides strength to the extracellular matrix and resistance to proteolytic degradation via different pathways, including NF-kβ, TGF-β and PI3K/Akt as the major signaling pathways. The etiology of diabetes and associated diseases has been found to be linked to unbalanced TG2 activity that may not only result in impaired or delayed wound healing in diabetics but also worsen degenerative and metabolic disease conditions. TG2 is usually overexpressed in diabetes, fibrosis, cancer, and neurodegenerative disorders. These TG2-linked diseases are usually associated with prolonged activation of inflammatory pathways. Therefore, reducing the inflammatory mechanisms and improving tissue remodeling appear to be the main treatment strategies to exterminate TG2-linked diseases. The present review aims to deliver a detailed overview of the existing understanding of TG2 in diabetes and associated diseases' progression, as well as treatment strategies to regulate TG2 tightly and its potential clinical applications. Our research endorses the notion that TG2 can serve as an effective early-stage diagnostic biomarker for metabolic diseases and a therapeutic target for the development of potential drug. [ABSTRACT FROM AUTHOR]

Kinase fusion genes are the most active fusion gene group in human cancer fusion genes. To help choose the clinically significant kinase so that the cancer patients that have fusion genes can be better diagnosed, we need a metric to infer the assessment of kinases in pan-cancer fusion genes rather than relying on the sample frequency expressed fusion genes. Most of all, multiple studies assessed human kinases as the drug targets using multiple types of genomic and clinical information, but none used the kinase fusion genes in their study. The assessment studies of kinase without kinase fusion gene events can miss the effect of one of the mechanisms that enhance the kinase function in cancer. To fill this gap, in this study, we suggest a novel way of assessing genes using a network propagation approach to infer how likely individual kinases influence the kinase fusion gene network composed of ~5K kinase fusion gene pairs. To select a better seed of propagation, we chose the top genes via dimensionality reduction like a principal component or latent layer information of six features of individual genes in pan-cancer fusion genes. Our approach may provide a novel way to assess of human kinases in cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: The heterogeneous expression of signaling molecules within the tumor, including kinases, is the major contributor to the acquisition of drug resistance and poor survival observed in small-cell lung cancer (SCLC). The addition of immunotherapy to chemotherapy has only marginally prolonged survival in patients with extensive-stage SCLC (ES-SCLC). Recent clinical trials have combined immunotherapy with the pharmacological inhibitors of kinases often dysregulated in SCLC. However, the regime has not been effective in the long term. Here, we review studies and clinical trials exploring dysregulated kinases in SCLC progression and resistance to chemotherapies and immunotherapies. We also discuss the possibility of repurposing kinase inhibitors against SCLC that have already demonstrated promising results for other types of cancers. SCLC is refractory to conventional therapies; targeted therapies and immunological checkpoint inhibitor (ICI) molecules have prolonged survival only marginally. In addition, ICIs help only a subgroup of SCLC patients. Different types of kinases play pivotal roles in therapeutics-driven cellular functions. Therefore, there is a significant need to understand the roles of kinases in regulating therapeutic responses, acknowledge the existing knowledge gaps, and discuss future directions for improved therapeutics for recalcitrant SCLC. Here, we extensively review the effect of dysregulated kinases in SCLC. We further discuss the pharmacological inhibitors of kinases used in targeted therapies for recalcitrant SCLC. We also describe the role of kinases in the ICI-mediated activation of antitumor immune responses. Finally, we summarize the clinical trials evaluating the potential of kinase inhibitors and ICIs. This review overviews dysregulated kinases in SCLC and summarizes their potential as targeted therapeutic agents. We also discuss their clinical efficacy in enhancing anticancer responses mediated by ICIs. [ABSTRACT FROM AUTHOR]

Cancer continues to be a major health concern globally, although the advent of targeted therapy has revolutionized treatment options. Aurora Kinase B is a serine‐threonine kinase that has been explored as an oncology therapeutic target for more than two decades. Aurora Kinase B inhibitors show promising biological results in in‐vitro and in‐vivo experiments. However, there are no inhibitors approved yet for clinical use, primarily because of the side effects associated with Aurora B inhibitors. Several studies demonstrate that Aurora B inhibitors show excellent synergy with various chemotherapeutic agents, radiation therapy, and targeted therapies. This makes it an excellent choice as an adjuvant therapy to first‐line therapies, which greatly improves the therapeutic window and side effect profile. Recent studies indicate the role of Aurora B in some deadly cancers with limited therapeutic options, like triple‐negative breast cancer and glioblastoma. Herein, we review the latest developments in Aurora Kinase B targeted research, with emphasis on its potential as an adjuvant therapy and its role in some of the most difficult‐to‐treat cancers. [ABSTRACT FROM AUTHOR]

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Protein kinases have proven to be a very productive class of therapeutic targets, and over 90 inhibitors are currently in clinical use primarily for the treatment of cancer. Repurposing these inhibitors as antimalarials could provide an accelerated path to drug development. In this study, we identified BI-2536, a known potent human polo-like kinase 1 inhibitor, with low nanomolar antiplasmodial activity. Screening of additional PLK1 inhibitors revealed further antiplasmodial candidates despite the lack of an obvious orthologue of PLKs in Plasmodium. A subset of these inhibitors was profiled for their in vitro killing profile, and commonalities between the killing rate and inhibition of nuclear replication were noted. A kinase panel screen identified PfNEK3 as a shared target of these PLK1 inhibitors; however, phosphoproteome analysis confirmed distinct signaling pathways were disrupted by two structurally distinct inhibitors, suggesting PfNEK3 may not be the sole target. Genomic analysis of BI-2536-resistant parasites revealed mutations in genes associated with the starvation-induced stress response, suggesting BI-2536 may also inhibit an aminoacyl-tRNA synthetase.

Although Fischers extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

Abstract Mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) emerges as a pivotal target in developing anti‐cancer therapies. The limitations of ATP‐competitive inhibitors, due to insufficient potency and selectivity, underscore the urgent need for a covalent irreversible MK2 inhibitor. Our initial analyses of The Cancer Genome Atlas database revealed MK2's overexpression across various cancer types, especially those characterized by inflammation, linking it to poor prognosis and highlighting its significance. Investigating MK2's kinase domain led to the identification of a unique cysteine residue, enabling the creation of targeted covalent inhibitors. Compound 11 was developed, demonstrating robust MK2 inhibition (IC50 = 2.3 nM) and high selectivity. It binds irreversibly to MK2, achieving prolonged signal suppression and reducing pathological inflammatory cytokines in macrophages. Furthermore, compound 11 or MK2 knockdown can inhibit the tumor‐promoting macrophage M2 phenotype in vitro and in vivo. In macrophage‐rich tumor model, compound 11 notably slowed growth in a dose‐dependent manner. These findings support MK2 as a promising anticancer target, especially relevant in cancers fueled by inflammation or dominated by macrophages, and provide compound 11 serving as an invaluable chemical tool for exploring MK2's functions.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway functions as a central hub for transmitting signals from more than 50 cytokines, playing a pivotal role in maintaining hematopoiesis, immune balance, and tissue homeostasis. Dysregulation of this pathway has been implicated in various diseases, including immunodeficiency, autoimmune conditions, hematological disorders, and certain cancers. Proteins within this pathway have emerged as effective therapeutic targets for managing these conditions, with various approaches developed to modulate key nodes in the signaling process, spanning from receptor engagement to transcription factor activation. Following the success of JAK inhibitors such as tofacitinib for RA treatment and ruxolitinib for managing primary myelofibrosis, the pharmaceutical industry has obtained approvals for over 10 small molecule drugs targeting the JAK-STAT pathway and many more are at various stages of clinical trials. In this review, we consolidate key strategies employed in drug discovery efforts targeting this pathway, with the aim of contributing to the collective understanding of small molecule interventions in the context of JAK-STAT signaling. We aspire that our endeavors will contribute to advancing the development of innovative and efficacious treatments for a range of diseases linked to this pathway dysregulation.

Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture.

Abstract Background This study examines the biological implications of an overlap between two sequences in the Arabidopsis genome, the 3’UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci. Results The extent of the overlap between 3’UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150-GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in different light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinosteroid signalling), can also act as partners for AT5G58150. Conclusions AT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis.

Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.

Our recent studies revealed the role of mouse Aprataxin PNK-like Factor (APLF) in development. Nevertheless, the comprehensive characterization of mouse APLF remains entirely unexplored. Based on domain deletion studies, here we report that mouse APLF's Acidic Domain and Fork Head Associated (FHA) domain can chaperone histones and repair DNA like the respective human orthologs. Immunofluorescence studies in mouse embryonic stem cells showed APLF co-localized with γ-tubulin within and around the centrosomes and govern the number and integrity of centrosomes via PLK4 phosphorylation. Enzymatic analysis established mouse APLF as a kinase. Docking studies identified three putative ATP binding sites within the FHA domain. Site-directed mutagenesis showed that R37 residue within the FHA domain is indispensable for the kinase activity of APLF thereby regulating the centrosome number. These findings might assist us comprehend APLF in different pathological and developmental conditions and reveal non-canonical kinase activity of proteins harbouring FHA domains that might impact multiple cellular processes.

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1β production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1β production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions. [ABSTRACT FROM AUTHOR]