IL-17 induces NSCLC cell migration and invasion by elevating MMP19 gene transcription and expression through the interaction of p300-dependent STAT3-K631 acetylation and its Y705-phosphorylation.

The cancer cell metastasis is a major death reason for patients with non-small cell lung cancer (NSCLC). Although researchers have disclosed that interleukin 17 (IL-17) can increase matrix metalloproteinases (MMPs) induction causing NSCLC cell metastasis, the underlying mechanism remains unclear. In the study, we found that IL-17 receptor A (IL-17RA), p300, p-STAT3, Ack-STAT3, and MMP19 were up-regulated both in NSCLC tissues and NSCLC cells stimulated with IL-17. p300, STAT3 and MMP19 overexpression or knockdown could raise or reduce IL-17-induced p-STAT3, Ack-STAT3 and MMP19 level as well as the cell migration and invasion. Mechanism investigation revealed that STAT3 and p300 bound to the same region (-544 to -389 nt) of MMP19 promoter, and p300 could acetylate STAT3-K631 elevating STAT3 transcriptional activity, p-STAT3 or MMP19 expression and the cell mobility exposed to IL-17. Meanwhile, p300-mediated STAT3-K631 acetylation and its Y705-phosphorylation could interact, synergistically facilitating MMP19 gene transcription and enhancing cell migration and invasion. Besides, the animal experiments exhibited that the nude mice inoculated with NSCLC cells by silencing p300, STAT3 or MMP19 gene plus IL-17 treatment, the nodule number, and MMP19, Ack-STAT3, or p-STAT3 production in the lung metastatic nodules were all alleviated. Collectively, these outcomes uncover that IL-17-triggered NSCLC metastasis involves up-regulating MMP19 expression via the interaction of STAT3-K631 acetylation by p300 and its Y705-phosphorylation, which provides a new mechanistic insight and potential strategy for NSCLC metastasis and therapy.

A quest for cytosolic sequons and their functions.

Evolution shapes protein sequences for their functions. Here, we studied the moonlighting functions of the N-linked sequon NXS/T, where X is not P, in human nucleocytosolic proteins. By comparing membrane and secreted proteins in which sequons are well known for N-glycosylation, we discovered that cyto-sequons can participate in nucleic acid binding, particularly in zinc finger proteins. Our global studies further discovered that sequon occurrence is largely proportional to protein length. The contribution of sequons to protein functions, including both N-glycosylation and nucleic acid binding, can be regulated through their density as well as the biased usage between NXS and NXT. In proteins where other PTMs or structural features are rich, such as phosphorylation, transmembrane ɑ-helices, and disulfide bridges, sequon occurrence is scarce. The information acquired here should help understand the relationship between protein sequence and function and assist future protein design and engineering.

Identification and structural characterization of small molecule inhibitors of PINK1.

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.

Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

The differential signaling of multiple FGF ligands through a single fibroblast growth factor (FGF) receptor (FGFR) plays an important role in embryonic development. Here, we use quantitative biophysical tools to uncover the mechanism behind differences in FGFR1c signaling in response to FGF4, FGF8, and FGF9, a process which is relevant for limb bud outgrowth. We find that FGF8 preferentially induces FRS2 phosphorylation and extracellular matrix loss, while FGF4 and FGF9 preferentially induce FGFR1c phosphorylation and cell growth arrest. Thus, we demonstrate that FGF8 is a biased FGFR1c ligand, as compared to FGF4 and FGF9. Förster resonance energy transfer experiments reveal a correlation between biased signaling and the conformation of the FGFR1c transmembrane domain dimer. Our findings expand the mechanistic understanding of FGF signaling during development and bring the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

[Deciphering cellular processes responding to lethality of 17ß-estradiol by quantitative phosphoproteomics].

17ß-Estradiol (E2), an important endocrine hormone in the mammalian body, participates in the regulation of the physiological functions of the reproductive system, mammary glands, bone, and cardiovascular system, among others. Paradoxically, despite the physiological actions of endogenous E2 (0.2-1.0 nmol/L), numerous clinical and experimental studies have demonstrated that high-dose E2 treatment can cause tumor regression and exert pro-apoptotic actions in multiple cell types; however, the underlying mechanism remains undescribed. In particular, little information of the cellular processes responding to the lethality of E2 is available. In the present study, we attempted to characterize the cellular processes responding to high-dose (µmol/L) E2 treatment using quantitative phosphoproteomics to obtain a better understanding of the regulatory mechanism of E2-induced cell death. First, the cell phenotype induced by high-dose E2 was determined by performing Cell Counting Kit-8 assay (CCK8), cell cytotoxicity analysis by trypan blue staining, and microscopic imaging on HeLa cells treated with 1-10 µmol/L E2 or dimethyl sulfoxide (DMSO) for 1-3 d. E2 inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Compared with the DMSO-treated HeLa cells, the cells treated with 5 µmol/L E2 for 2 d demonstrated >74% growth inhibition and approximately 50% cell death. Thus, these cells were used for quantitative phosphoproteomic analysis. Next, a solid-phase extraction (SPE)-based immobilized titanium ion affinity chromatography (Ti4+-IMAC) phosphopeptide-enrichment method coupled with data-independent acquisition (DIA)-based quantitative proteomics was employed for the in-depth screening of high-dose E2-regulated phosphorylation sites to investigate the intracellular processes responding to high-dose E2 treatment. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified over 10000 phosphorylation sites regulated by E2 and DMSO in HeLa cells. In comparison with the DMSO-treated cells, the cells treated with 5 µmol/L E2 showed 537 upregulated phosphorylation sites and 387 downregulated phosphorylation sites, with a threshold of p<0.01 and |log2(fold change)|≥1. A total of 924 phosphorylation sites on 599 proteins were significantly regulated by high-dose E2, and these sites were subjected to enrichment analysis. In addition, 453 differently regulated phosphorylation sites on 325 proteins were identified only in the E2- or DMSO-treated cell samples. These phosphorylation sites may be phosphorylated or dephosphorylated in response to high-dose E2 stimulation and were subjected to parallel enrichment analyses. Taken together, 1218 phosphorylation sites on 741 proteins were significantly regulated by high-dose E2 treatment. The functional phosphoproteins in these two groups were then analyzed using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) to determine the biological processes in which they participate and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Consistent with the cell-phenotype data, cell cycle-related proteins were highly enriched in the two groups of E2-regulated phosphoproteins (p<0.05), indicating that high-dose E2 treatment can regulate cell proliferation. In addition, E2-regulated phosphoproteins were highly enriched in the cellular processes of ribosome biogenesis, nucleocytoplasmic transport, and messenger ribonucleic acid (mRNA) processing/splicing (p<0.05), indicating that the activation of these processes may contribute to high-dose E2-induced cell death. These results further confirm that high-dose E2 treatment inhibits protein translation and induces cell death. Furthermore, the significant upregulation of multiple phosphorylation sites associated with epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPKs) MAPK1, MAPK4, and MAPK14 by high-dose E2 indicates that the EGFR and MAPK signaling pathways are likely involved in the regulation of E2-induced cell death. These phosphorylation sites likely play vital roles in E2-induced cell death in HeLa cells. Overall, our phosphoproteomic data could be a valuable resource for uncovering the regulatory mechanisms of E2 in the micromolar range.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Development of an Imidazopyridazine-Based MNK1/2 Inhibitor for the Treatment of Lymphoma.

The mitogen-activated protein kinase-interacting protein kinases (MNKs) are the only kinases known to phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at Ser209, which plays a significant role in cap-dependent translation. Dysregulation of the MNK/eIF4E axis has been found in various solid tumors and hematological malignancies, including diffuse large B-cell lymphoma (DLBCL). Herein, structure-activity relationship studies and docking models determined that 20j exhibits excellent MNK1/2 inhibitory activity, stability, and hERG safety. 20j exhibits strong and broad antiproliferative activity against different cancer cell lines, especially GCB-DLBCL DOHH2. 20j suppresses the phosphorylation of eIF4E in Hela cells (IC50 = 90.5 nM) and downregulates the phosphorylation of eIF4E and 4E-BP1 in A549 cells. In vivo studies first revealed that ibrutinib enhances the antitumor effect of 20j without side effects in a DOHH2 xenograft model. This study provided a solid foundation for the future development of a MNK inhibitor for GCB-DLBCL treatment.

Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3ß (pGSK3ß) relative to GSK3ß in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3ß inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3ß. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-LCK interactions.

CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

Listeria-vectored cervical cancer vaccine candidate strains reduce MDSCs via the JAK-STAT signaling pathway.

BACKGROUND: Immunosuppressive status is prevalent in cancer patients and increases the complexity of tumor immunotherapy. It has been found that Listeria-vectored tumor vaccines had the potential ability of two-side regulatory effect on the immune response during immunotherapy. RESULTS: The results show that the combined immunotherapy with the LM∆E6E7 and LI∆E6E7, the two cervical cancer vaccine candidate strains constructed by our lab, improves the antitumor immune response and inhibits the suppressive immune response in tumor-bearing mice in vivo, confirming the two-sided regulatory ability of the immune response caused by Listeria-vectored tumor vaccines. The immunotherapy reduces the expression level of myeloid-derived suppressor cells (MDSCs)-inducing factors and then inhibits the phosphorylation level of STAT3 protein, the regulatory factor of MDSCs differentiation, to reduce the MDSCs formation ability. Moreover, vaccines reduce the expression of functional molecules associated with MDSCs may by inhibiting the phosphorylation level of the JAK1-STAT1 and JAK2-STAT3 pathways in tumor tissues to attenuate the immunosuppressive function of MDSCs. CONCLUSIONS: Immunotherapy with Listeria-vectored cervical cancer vaccines significantly reduces the level and function of MDSCs in vivo, which is the key point to the destruction of immunosuppression. The study for the first to elucidate the mechanism of breaking the immunosuppression.

The effect of saraglitazar on TGF-ß-induced smad3 phosphorylation and expression of genes related to liver fibrosis in LX2 cell line.

BACKGROUND AND PURPOSE: Liver fibrosis is a reversible liver injury that occurs as a result of many chronic inflammatory diseases and can lead to cirrhosis, which is irreversible and fatal. So, we studied the anti-fibrotic effects of saroglitazar on LX-2 cell lines, as a dual PPARα/γ agonist. METHODS: Cells, after 80% confluence, were treated with TGF-ß (2 ng/mL) for 24 h. Then cells were treated with saroglitazar at different doses (2.5, 5, 10 µM) for 24 h. After same incubation, the cells of control group, TGF-ß group, and TGF-ß + saroglitazar group were harvested for RNA and protein extraction to determine the effects of saroglitazar. RT-PCR and western blot methods were used to express genes related to fibrosis. RESULTS: Our results show that the relative expression of α-SMA, collagen1α, N-cadherin, NOX (1, 2, and 4), and phosphorylated Smad3 protein was significantly higher in TGF-ß-treated cells compared with the normal group, and E-cadherin expression was decreased in TGF-ß-treated cells. After TGF-ß-treated cells were exposed to saroglitazar, the expression of these genes was significantly reversed (P < 0.05). CONCLUSIONS: Our results clearly show the short-term inhibitory role of saroglitazar in the expression of fibrotic factors using the TGF-ß/Smad signaling pathway. These results suggest that saroglitazar can be considered as a suitable therapeutic strategy for fibrotic patients. Although more studies are needed.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

Discovery of novel quinolin-2-one derivatives as potential GSK-3ß inhibitors for treatment of Alzheimer's disease: Pharmacophore-based design, preliminary SAR, in vitro and in vivo biological evaluation.

Recently, glycogen synthase kinase-3ß (GSK-3ß) has been considered as a critical factor implicated in Alzheimer's disease (AD). In a previous work, a 3D pharmacophore model for GSK-3ß inhibitors was created and the results suggested that derivative ZINC67773573, VIII, may provide a promising lead for developing novel GSK-3ß inhibitors for the AD's treatment. Consequently, in this work, novel series of quinolin-2-one derivatives were synthesized and assessed for their GSK-3ß inhibitory properties. In vitro screening identified three compounds: 7c, 7e and 7f as promising GSK-3ß inhibitors. Compounds 7c, 7e and 7f were found to exhibit superior inhibitory effect on GSK-3ß with IC50 value ranges between 4.68 ± 0.59 to 8.27 ± 0.60 nM compared to that of staurosporine (IC50 = 6.12 ± 0.74 nM). Considerably, compounds 7c, 7e and 7f effectively lowered tau hyperphosphorylated aggregates and proving their safety towards the SH-SY5Y and THLE2 normal cell lines. The most promising compound 7c alleviated cognitive impairments in the scopolamine-induced model in mice. Compound 7c's activity profile, while not highly selective, may provide a starting point and valuable insights into the design of multi-target inhibitors. According to the ADME prediction results, compounds 7c, 7e and 7f followed Lipinski's rule of five and could almost permeate through the BBB. Molecular docking simulations showed that these compounds are well accommodated in the ATP binding site interacting by its quinoline-2-one ring through hydrogen bonding with the key amino acids Asp133 and Val135 at the hinge region. The findings of this study suggested that these new compounds may have potential as anti-AD drugs targeting GSK-3ß.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Discovery of novel macrocyclic derivatives as potent and selective cyclin-dependent kinase 2 inhibitors.

Cyclin-dependent kinase 2 (CDK2) is a member of CDK family of kinases (CDKs) that regulate the cell cycle. Its inopportune or over-activation leads to uncontrolled cell cycle progression and drives numerous types of cancers, especially ovarian, uterine, gastric cancer, as well as those associated with amplified CCNE1 gene. However, developing selective lead compound as CDK2 inhibitors remains challenging owing to similarities in the ATP pockets among different CDKs. Herein, we described the optimization of compound 1, a novel macrocyclic inhibitor targeting CDK2/5/7/9, aiming to discover more selective and metabolically stable lead compound as CDK2 inhibitor. Molecular dynamic (MD) simulations were performed for compound 1 and 9 to gain insights into the improved selectivity against CDK5. Further optimization efforts led to compound 22, exhibiting excellent CDK2 inhibitory activity, good selectivity over other CDKs and potent cellular effects. Based on these characterizations, we propose that compound 22 holds great promise as a potential lead candidate for drug development.

DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-ß expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

Mechanistic insights into G-protein activation via phosphorylation mediated non-canonical pathway.

Activation of heterotrimeric G-proteins (Gαßγ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of Gα that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce Gα activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gαi3, a subtype of Gα-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. In addition to weakened salt bridges at the domain interface, phosphorylations induced the unfolding of helix αF, which contributed to increased flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gαi3. Although the larger domain separation in the phosphorylated system provided an unobstructed path for the nucleotide, the accelerated release of GDP was attributed to increased fluctuations in several conserved regions like P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

Discovery and optimization of 4-pyrazolyl-2-aminopyrimidine derivatives as potent spleen tyrosine kinase (SYK) inhibitors.

Spleen tyrosine kinase (Syk) is a key signal transduction mediator of the B cell receptor (BCR) signaling pathway. Abnormal BCR signaling plays a key role in initiation and development of B-cell-derived hematological malignancies, therefore, Syk represents a potential target for inhibiting the BCR signaling resulting in a therapeutic effect in these cancers. Herein, we describe a novel series of SYK inhibitors with 4-(3'-pyrazolyl)-2-amino-pyrimidine scaffold. Extensive study of structure-activity relationships led to the identification of 1 (NMS-0963), a highly potent Syk inhibitor (IC50 = 3 nM) endowed with high selectivity within a panel of tested kinases and high antiproliferative activity in SYK-dependent BaF3-TEL/SYK cells and in other BCR-dependent hematological tumor cell lines. Additionally, 1 effectively inhibited Syk phosphorylation and downstream signaling mediators of the BCR in treated cells. In in vivo pharmacokinetics studies, 1, displayed good pharmacokinetics properties, with linear exposure with dose and excellent oral bioavailability. These findings suggest that 1 is a promising new Syk inhibitor for treating BCR-dependent hematological cancers.