RESUMO
Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their ß-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the ß subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification.
RESUMO
Atherosclerosis (AS) is a chronic progressive inflammatory disease of the vascular wall and the primary pathological basis of cardiovascular and cerebrovascular disease. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), two highly homologous members of the FAK family kinases, play critical roles in integrin signaling. They also serve as scaffolding proteins that contribute to the assembly of cellular signaling complexes that regulate cell survival, cell cycle progression, and cell motility. Research indicates that the FAK family kinases is involved in the gene regulation of vascular cells and that aberrant expression of this family is associated with pathological changes in vascular disease. These findings establish the FAK family kinases as a critical signaling mediator in atherosclerotic lesions and inhibition of its activity has the potential to attenuate the pathological progression of AS. This review highlights the indispensable role of the FAK family kinases in abnormal vascular smooth muscle cell proliferation, endothelial cell dysfunction, inflammation, and lipid metabolism associated with AS. We also summarize therapeutic targets against the FAK family kinases, providing valuable insights into therapeutic strategies for AS.
RESUMO
Background: Neonatal Opioid Withdrawal Syndrome (NOWS) is a consequence of in-utero exposure to prenatal maternal opioids, resulting in the manifestation of symptoms like irritability, feeding problems, tremors, and withdrawal signs. Opioid use disorder (OUD) during pregnancy can profoundly impact both mother and fetus, disrupting fetal brain neurotransmission and potentially leading to long-term neurological, behavioral, and vision issues, and increased infant mortality. Drug resistance complicates OUD and NOWS treatment, with protein kinase regulation of drug transporters not fully understood. Methods: DNA methylation levels of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, along with protein kinase C (PKC) genes, were assessed in 96 placental samples using the Illumina Infinium MethylationEPIC array (850K). Samples were collected from three distinct groups: 32 mothers with infants prenatally exposed to opioids who needed pharmacological intervention for NOWS, 32 mothers with prenatally opioid-exposed infants who did not necessitate NOWS treatment, and 32 mothers who were not exposed to opioids during pregnancy. Results: We identified 69 significantly differentially methylated SLCs, with 24 hypermethylated and 34 hypomethylated, and 11 exhibiting both types of methylation changes including SLC13A3, SLC15A2, SLC16A11, SLC16A3, SLC19A2, and SLC26A1. We identified methylation changes in 11 ABC drug transporters (ABCA1, ABCA12, ABCA2, ABCB10, ABCB5, ABCC12, ABCC2, ABCC9, ABCE1, ABCC7, ABCB3): 3 showed hypermethylation, 3 hypomethylation, and 5 exhibited both. Additionally, 7 PKC family genes (PRKCQ, PRKAA1, PRKCA, PRKCB, PRKCH, PRKCI, and PRKCZ) showed methylation changes. These genes are associated with 13 pathways involved in NOWS, including ABC transporters, bile secretion, pancreatic secretion, insulin resistance, glutamatergic synapse, and gastric acid secretion. Conclusion: We report epigenetic changes in PKC-related regulation of drug transporters, which could improve our understanding of clinical outcomes like drug resistance, pharmacokinetics, drug-drug interactions, and drug toxicity, leading to maternal relapse and severe NOWS. Novel drugs targeting PKC pathways and transporters may improve treatment outcomes for OUD in pregnancy and NOWS.
RESUMO
Given their central role in signal transduction, protein kinases (PKs) were first implicated in cancer development, caused by aberrant intracellular signaling events. Since then, PKs have become major targets in different therapeutic areas. The preferred approach to therapeutic intervention of PK-dependent diseases is the use of small molecules to inhibit their catalytic phosphate group transfer activity. PK inhibitors (PKIs) are among the most intensely pursued drug candidates, with currently 80 approved compounds and several hundred in clinical trials. Following the elucidation of the human kinome and development of robust PK expression systems and high-throughput assays, large volumes of PK/PKI data have been produced in industrial and academic environments, more so than for many other pharmaceutical targets. In addition, hundreds of X-ray structures of PKs and their complexes with PKIs have been reported. Substantial amounts of PK/PKI data have been made publicly available in part as a result of open science initiatives. PK drug discovery is further supported through the incorporation of data science approaches, including the development of various specialized databases and online resources. Compound and activity data wealth compared to other targets has also made PKs a focal point for the application of artificial intelligence (AI) in pharmaceutical research. Herein, we discuss the interplay of open and data science in PK drug discovery and review exemplary studies that have substantially contributed to its development, including kinome profiling or the analysis of PKI promiscuity versus selectivity. We also take a close look at how AI approaches are beginning to impact PK drug discovery in light of their increasing data orientation.
RESUMO
INTRODUCTION: Pain in both peripheral and axial joints is a major symptom in patients with psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). Emerging evidence demonstrates pain mechanisms, beyond those related to inflammation or joint damage, based on aberrant processing of nociceptive stimuli peripherally as well as centrally. The Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway has been implicated in the processing of pain beyond its role in mediating inflammation and inhibitors of this pathway approved for the treatment of axSpA and PsA have been shown to alleviate a broad array of pain outcomes in both axial and peripheral joints. AREAS COVERED: We review recent definitions and standardization of the nomenclature for categorizing chronic pain according to causality, assessment tools to evaluate nociplastic pain, the pathophysiologic role of JAK-STAT signaling in nociplastic pain, evidence for the presence of nociplastic pain in axSpA and PsA, and the impact of JAK inhibitors (JAKi) on pain outcomes in clinical trials (PubMed: 01/01/2019-04/01-2024). EXPERT OPINION: Nociplastic pain assessment has been confined almost entirely to the use of a limited number of questionnaires in cross-sectional studies of these diseases. Though effective for alleviating pain, it is unclear if JAKi specifically impact nociplastic pain.
RESUMO
This study aims to investigate the effects of the Galectin-3 (Gal-3) inhibitor TD139 on inflammation and the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 pathway in gestational diabetes mellitus (GDM). Human placental tissues were treated with TD139 and TNF-α, assessing Gal-3, ERK/JNK/p38 activation, and inflammatory cytokines. GDM was induced in mice via subcutaneous injections of streptozotocin (STZ). After confirming GDM, mice were treated with 15 mg/kg TD139 on GD 10.5 12.5, 14.5, 16.5, and 18.5. Serum inflammatory cytokines were measured on GD 20.5, and post-delivery placental tissues were analyzed. Data were analyzed using one-way or two-way repeated measures ANOVA with post hoc tests. TD139 suppressed TNF-α-induced increases in Gal-3, IL-1ß, IL-6, MCP-1, and ERK/JNK/p38 activation in placental tissues. In STZ-induced GDM mice, TD139 reduced glucose levels, weight loss, and food and water intake. TD139 significantly lowered TNF-α, IL-1ß, IL-6, and MCP-1 in serum and placental tissues and inhibited the ERK/JNK/p38 pathway. TD139 improved pup numbers in GDM mice compared to untreated ones. TD139 reduces inflammation and inhibits the ERK/JNK/p38 pathway in TNF-α stimulated placental tissues and STZ-induced GDM mice, suggesting its therapeutic potential for managing GDM-related placental inflammation and improving pregnancy outcomes. The study used TNF-α to mimic GDM in placental tissues and an STZ-induced GDM mouse model, which may not fully represent human GDM complexity. Future research should explore alternative models, and broader signaling pathways, and thoroughly evaluate TD139's safety in pregnancy.
RESUMO
Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients.
Assuntos
Apoptose , Cardiomiopatias , Fosfatases de Especificidade Dupla , Camundongos Endogâmicos C57BL , MicroRNAs , Estresse Oxidativo , Sepse , Animais , Masculino , Camundongos , Antagomirs/farmacologia , Antagomirs/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Cardiomiopatias/genética , Modelos Animais de Doenças , Regulação para Baixo , Fosfatases de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Lipopolissacarídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Sepse/complicações , Sepse/metabolismo , Sepse/genéticaRESUMO
Growth factors and their receptor tyrosine kinases play a central role in regulating vital cellular processes such as proliferation, differentiation, division, and cell survival, and they are closely associated with the development of various types of cancer, particularly in the context of angiogenesis. Although several small chemical compounds targeting tyrosine kinase receptors have been approved by the FDA for cancer treatment by inhibiting angiogenesis, there is still a need for more effective medications. in silico studies are now crucial tools for the design of new drugs, offering considerable advantages such as cost and time reduction. In this review, we examined recent in silico research carried out between 2022 and 2024, focusing on new drug candidates synthesized to fight cancer, in particular by targeting tyrosine kinase receptors involved in the process of angiogenesis.
RESUMO
Lck, a member of the Src kinase family, is a non-receptor tyrosine kinase involved in immune cell activation, antigen recognition, tumor growth, and cytotoxic response. The enzyme has usually been linked to T lymphocyte activation upon antigen recognition. Lck activation is central to CD4, CD8, and NK activation. However, recently, it has become clearer that activating the enzyme in CD8 cells can be independent of antigen presentation and enhance the cytotoxic response. The role of Lck in NK cytotoxic function has been controversial in a similar fashion as the role of the enzyme in CAR T cells. Inhibiting tyrosine kinases has been a highly successful approach to treating hematologic malignancies. The inhibitors may be useful in treating other tumor types, and they may be useful to prevent cell exhaustion. New, more selective inhibitors have been documented, and they have shown interesting activities not only in tumor growth but in the treatment of autoimmune diseases, asthma, and graft vs. host disease. Drug repurposing and bioinformatics can aid in solving several unsolved issues about the role of Lck in cancer. In summary, the role of Lck in immune response and tumor growth is not a simple event and requires more research.
RESUMO
Since the initial identification of oncogenic Wnt in mice and Drosophila, the Wnt signaling pathway has been subjected to thorough and extensive investigation. Persistent activation of Wnt signaling exerts diverse cancer characteristics, encompassing tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis. Here we review the principal signaling mechanisms and the regulatory influence of pathway-intrinsic and extrinsic kinases on cancer progression. Additionally, we underscore the divergences and intricate interplays of the canonical and non-canonical Wnt signaling pathways and their critical influence in cancer pathophysiology, exhibiting both growth-promoting and growth-suppressing roles across diverse cancer types.
RESUMO
Protein tyrosine kinases (PTKs) function as key molecules in the signaling pathways in addition to their impact as a therapeutic target for the treatment of many human diseases, including cancer. PTKs are characterized by their ability to phosphorylate serine, threonine, or tyrosine residues and can thereby rapidly and reversibly alter the function of their protein substrates in the form of significant changes in protein confirmation and affinity for their interaction with protein partners to drive cellular functions under normal and pathological conditions. PTKs are classified into two groups: one of which represents tyrosine kinases, while the other one includes the members of the serine/threonine kinases. The group of tyrosine kinases is subdivided into subgroups: one of them includes the member of receptor tyrosine kinases (RTKs), while the other subgroup includes the member of non-receptor tyrosine kinases (NRTKs). Both these kinase groups function as an "on" or "off" switch in many cellular functions. NRTKs are enzymes which are overexpressed and activated in many cancer types and regulate variable cellular functions in response to extracellular signaling-dependent mechanisms. NRTK-mediated different cellular functions are regulated by kinase-dependent and kinase-independent mechanisms either in the cytoplasm or in the nucleus. Thus, targeting NRTKs is of great interest to improve the treatment strategy of different tumor types. This review deals with the structure and mechanistic role of NRTKs in tumor progression and resistance and their importance as therapeutic targets in tumor therapy.
RESUMO
The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.
Assuntos
Desenho de Fármacos , Quinase I-kappa B , NF-kappa B , Inibidores de Proteínas Quinases , Transdução de Sinais , Quinase I-kappa B/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Humanos , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Linhagem Celular Tumoral , Piridinas/farmacologia , Piridinas/química , Piridinas/síntese química , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Modelos MolecularesRESUMO
BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for the treatment of various malignancies; however, its therapeutic potential is limited because of the frequent occurrence of tumor cell resistance. In this study, we determined whether TRAIL resistance acquired by repeated administration could be overcome by HDAC inhibition in human colorectal cancer cells. METHODS: TRAIL-resistant HCT116 human colorectal cancer cells (HCT116-TR) were generated by repeated treatment with 10 and 25 ng/mL TRAIL twice weekly for 28 days. RESULTS: The resulting TRAIL-resistant cells were noncross-resistant to other chemotherapeutic agents. The levels of histone acetylation-related proteins, such as ac-histone H4 and HDAC1, were altered in HCT116-TR cells compared with the parental HCT116 cell line. The combined treatment with TRAIL and HDAC inhibitors significantly increased apoptosis in HCT116-TR cells and indicated a synergistic effect. The mechanism by which HDAC inhibition sensitizes HCT116-TR cells to TRAIL is dependent on the intrinsic pathway. In addition, we found that HDAC inhibition enhanced the sensitivity of cells to TRAIL through mitogen-activated protein kinases/CCAAT/enhancer-binding protein homologs of protein-dependent upregulation of death receptor 5. CONCLUSION: These results suggest that histone acetylation is responsible for acquired TRAIL resistance after repeated exposure and acquired resistance to TRAIL may be overcome by combination therapies with HDAC inhibitors.
RESUMO
Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of ß-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-ß treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-ß-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.
RESUMO
Alzheimer's disease is a multifaceted neurodegenerative disease. Cholinergic dysfunction, amyloid ß toxicity, tauopathies, oxidative stress, neuroinflammation are among the main pathologies of the disease. Ligands targeting more than one pathology, multi-target directed ligands, attract attention in the recent years to tackle Alzheimer's disease. In this review, we aimed to cover different biochemical pathways, that are revealed in recent years for the pathology of the disease, as druggable targets such as cannabinoid receptors, matrix metalloproteinases, histone deacetylase and various kinases including, glycogen synthase kinase-3, mitogen-activated protein kinase and c-Jun N-terminal kinase, and their ligands for the treatment of Alzheimer's disease in the hope of providing more realistic insights into the field.
RESUMO
The dysregulation of kinases has emerged as a major class of targets for anticancer drug discovery given its node roles in the etiology of tumorigenesis, progression, invasion, and metastasis of malignancies, which is validated by the FDA approval of 28 small molecule kinase inhibitor (SMKI) drugs for cancer treatment at the end of 2015. While the preclinical and clinical data of these drugs are widely presented, it is highly essential to give an updated review on the medical indications, design principles and binding modes of these anti-tumor SMKIs approved by the FDA to offer insights for the future development of SMKIs with specific efficacy and safety.
Assuntos
Antineoplásicos , Aprovação de Drogas , Neoplasias , Inibidores de Proteínas Quinases , Bibliotecas de Moléculas Pequenas , United States Food and Drug Administration , Humanos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Estados Unidos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Estrutura Molecular , Sítios de Ligação , Relação Estrutura-AtividadeRESUMO
Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.
Assuntos
Proteínas de Protozoários , Toxoplasma , Toxoplasma/genética , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Toxoplasma/crescimento & desenvolvimento , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Transdução de Sinais , Reprodução AssexuadaRESUMO
Mitogen-activated protein kinase kinases (MAPKKs) play a critical role in the mitogen-activated protein kinase (MAPK) signaling pathway, transducing external stimuli into intracellular responses and enabling plant adaptation to environmental challenges. Most research has focused on the model plant Arabidopsis (Arabidopsis thaliana). The systematic analysis and characterization of MAPKK genes across different plant species, particularly in cotton (Gossypium hirsutum), are somewhat limited. Here, we identified MAPKK family members from 66 different species, which clustered into 5 different sub-groups, and MAPKKs from four cotton species clustered together. Through further bioinformatic and expression analysis, GhMAPKK5 was identified as the most responsive MAPKK member to salt and drought stress among the 23 MAPKKs identified in Gossypium hirsutum. Silencing GhMAPKK5 in cotton through virus-induced gene silencing (VIGS) led to quicker wilting under salt and drought conditions, while overexpressing GhMAPKK5 in Arabidopsis enhanced root growth and seed germination under these stresses, demonstrating GhMAPKK5's positive role in stress tolerance. Transcriptomics and Yeast-Two-Hybrid assays revealed a MAPK cascade signal module comprising GhMEKK (Mitogen-activated protein kinase kinase kinases)3/8/31-GhMAPKK5-GhMAPK11/23. This signaling cascade may play a role in managing drought and salt stress by regulating transcription factor genes, such as WRKYs, which are involved in the biosynthesis and transport pathways of ABA, proline, and RALF. This study is highly important for further understanding the regulatory mechanism of MAPKK in cotton, contributing to its stress tolerance and offering potential in targets for genetic enhancement.
RESUMO
Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate mitogen-activated protein kinase (MAPK) activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high-osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on a high-osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain. IMPORTANCE: Legionella pneumophila grows in a membrane-bound compartment in macrophages during disease. Construction of the compartment requires a dedicated secretion system that translocates virulence proteins into host cells. One of these proteins, LegA7, is shown to activate a stress response pathway in host cells called the mitogen-activated protein kinase (MAPK) pathway. The effects on the mammalian MAPK pathway were reconstructed in yeast, allowing the development of a strategy to identify the role of individual domains of LegA7. A domain similar to cysteine proteases is demonstrated to be critical for impinging on the MAPK pathway, and the catalytic activity of this domain is required for targeting this path. In addition, a conserved series of repeats, called ankyrin repeats, controls this activity. Data are provided that argue the interaction of the ankyrin repeats with unknown targets probably results in activation of the cysteine protease domain.
RESUMO
NXP900 is a selective and potent SRC family kinase (SFK) inhibitor, currently being dosed in a phase 1 clinical trial, that locks SRC in the "closed" conformation, thereby inhibiting both kinase-dependent catalytic activity and kinase-independent functions. In contrast, several multi-targeted kinase inhibitors that inhibit SRC, including dasatinib and bosutinib, bind their target in the active "open" conformation, allowing SRC and other SFKs to act as a scaffold to promote tumorigenesis through non-catalytic functions. NXP900 exhibits a unique target selectivity profile with sub-nanomolar activity against SFK members over other kinases. This results in highly potent and specific SFK pathway inhibition. Here, we demonstrate that esophageal squamous cell carcinomas and head and neck squamous cell carcinomas are exquisitely sensitive to NXP900 treatment in cell culture and in vivo, and we identify a patient population that could benefit from treatment with NXP900.