Kinase Binding Research Articles

Antioxidant and anti-apoptotic potency of allicin and lycopene against methotrexate-induced cardiac injury in rats.

This study aimed to explore whether allicin (ALC) and lycopene (LP) could offer protection against the harmful effects of methotrexate (MTX), a type of chemotherapy drug known for its severe side effects, on the heart of rats. In this experiment, seven groups of rats (n = 7) were used. The first group was given saline as a control vehicle, the second group was given ALC at a dosage of 20mg/kg orally, the third group was given LP at a dosage of 10mg/kg orally, and the fourth group was given MTX at a dosage of 20mg/kg intraperitoneally on the 15th day of the experiment. The remaining three groups received treatments, including ALC + MTX, LP + MTX, and ALC + LP + MTX. After the administration of MTX, the concentrations of serum cardiac biomarkers, such as Creatine kinase (CK), Lactate dehydrogenase (LDH), and creatine kinase-myoglobin binding (CK-MB) were found to increase. Also, MTX caused a notable rise in malondialdehyde (MDA) levels and significant declines in the levels of glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in the heart tissues of rats. In addition, MTX caused alterations in the cardiac histopathology and enhanced the caspase-3 expression in the cardiac tissues, indicating the occurrence of apoptosis. The antioxidant properties of ALC and/or LP were effectively reduced cardiac toxicity and apoptosis induced by MTX. The administration of ALC and/or LP was found to alleviate these effects caused by MTX.

ATG4B antagonizes antiviral immunity by GABARAP-directed autophagic degradation of TBK1

ABSTRACT TBK1 (TANK binding kinase 1) is an essential kinase of antiviral immunity, yet the regulatory mechanisms responsible for its stringent control via autophagy are not fully understood. Here, we identify the macroautophagy/autophagy-related cysteine protease ATG4B as a negative regulator of human antiviral immune responses by targeting TBK1 for autophagic degradation at the advanced stage of viral infection. Mechanistically, ATG4B serves as an adaptor for recruiting TBK1 to GABARAP (GABA type A receptor-associated protein), which subsequently leads to the TBK1-GABARAP interaction through the LC3-interacting region (LIR) motif of TBK1 ULD domain. Moreover, pharmacological ATG4B inhibitor, a small molecule named S130, contributes to host defense against viral infection and blocks ATG4B-dependent autophagic degradation of TBK1. Accordingly, S130 increases antiviral response and inhibits the VSV infection both in vitro and in vivo. Altogether, our study reveals the regulatory role of ATG4B in modulating TBK1-centered type I interferon (IFN) signaling, and indicates that ATG4B suppression can provide a potential therapy target for viral infection. Abbreviations Baf A1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IFN: interferon; IKBKE/IKKi: inhibitor of nuclear factor kappa B kinase subunit epsilon; IRF3: interferon regulatory factor 3; ISG: interferon-stimulated gene; ISRE: IFN-stimulated response element; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecule patterns; RIGI/DDX58: RNA sensor RIG-I; SeV: Sendai virus; siRNA: small interfering RNA; TBK1: TANK binding kinase 1; WT: wild-type; VSV: vesicular stomatitis virus.

Topology and adenocarcinoma cell localization dataset on the labyrinthin diapeutic biomarker

ObjectiveThe discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. In this regard, labyrinthin has been implicated as a neoantigen found on the cell surface of adenocarcinomas. Data on the (1) topology, (2) amino acid (a.a.) homology analyses and (3) cell surface localization of labyrinthin by fluorescent activated cell sorter (FACS) are studied in support of labyrinthin as a novel, pan-adenocarcinoma marker.ResultsBioinformatics analyses predict labyrinthin as a type II protein with calcium binding domain(s), N-myristoylation sites, and kinase II phosphorylation sites. Sequence homologies for labyrinthin (255 a.a.) were found vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. Labyrinthin was detected by FACS on only non-permeablized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization for greater than 20 min.

Genome-wide association study unravels genomic regions associated with chlorophyll fluorescence parameters in wheat (Triticum aestivum L.) under different sowing conditions.

Genome-wide association study identified 205 significant marker-trait associations for chlorophyll fluorescence parameters in wheat. Candidate gene mining, in silico expression, and promoter analyses revealed the potential candidate genes associated with the studied parameters. The present study investigated the effect of varied sowing conditions (viz., early, timely, and late) on different chlorophyll fluorescence parameters in diverse wheat germplasm set comprising of 198 lines over two cropping seasons (2020-2021 and 2021-2022). Further, a genome-wide association study was conducted to identify potential genomic regions associated with these parameters. The results revealed significant impacts of sowing conditions on all fluorescence parameters, with the maximum and minimum effects on FI (26.64%) and FV/FM (2.12%), respectively. Among the 205 marker-trait associations (MTAs) identified, 11 high-confidence MTAs were chosen, exhibiting substantial effects on multiple fluorescence parameters, and each explaining more than 10% of the phenotypic variation. Through gene mining of genomic regions encompassing high-confidence MTAs, we identified a total of 626 unique gene models. In silico expression analysis revealed 42 genes with an expression value exceeding 2 TPM. Among them, 10 genes were identified as potential candidate genes with functional relevance to enhanced photosynthetic efficiency. These genes mainly encoded for the following important proteins/products-ankyrin repeat protein, 2Fe-2S ferredoxin-type iron-sulfur-binding domain, NADH-ubiquinone reductase complex-1 MLRQ subunit, oxidoreductase FAD/NAD(P)-binding, photosystem-I PsaF, and protein kinases. Promoter analysis revealed the presence of light-responsive (viz., GT1-motif, TCCC-motif, I-box, GT1-motif, TCT-motif, and SP-1) and stress-responsive (viz., ABRE, AuxRR-core, GARE-motif, and ARE) cis-regulatory elements, which may be involved in the regulation of identified putative candidate genes. Findings from this study could directly help wheat breeders in selecting lines with favorable alleles for chlorophyll fluorescence, while the identified markers will facilitate marker-assisted selection of potential genomic regions for improved photosynthesis.

SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection by reducing the intracellular dNTP pool. We have shown that SAMHD1 suppresses nuclear factor kappa-B activation and type I interferon (IFN-I) induction by viral infection and inflammatory stimuli. However, the mechanism by which SAMHD1 inhibits IFN-I remains unclear. Here, we show that SAMHD1 inhibits IFN-I activation induced by the mitochondrial antiviral-signaling protein (MAVS). SAMHD1 interacted with MAVS and suppressed MAVS aggregation in response to Sendai virus infection in human monocytic THP-1cells. This resulted in increased phosphorylation of TANK binding kinase 1 (TBK1), inhibitor of nuclear factor kappa-B kinase epsilon (IKKε), and IFN regulatory factor 3 (IRF3). SAMHD1 suppressed IFN-I activation induced by IKKε and prevented IRF7 binding to the kinase domain of IKKε. We found that SAMHD1 interaction with the inhibitory domain (ID) of IRF7 (IRF7-ID) was necessary and sufficient for SAMHD1 suppression of IRF7-mediated IFN-I activation in HEK293T cells. Computational docking and molecular dynamics simulations revealed possible binding sites between IRF7-ID and full-length SAMHD1. Individual substitution of F411, E416, or V460 in IRF7-ID significantly reduced IRF7 transactivation activity and SAMHD1 binding. Furthermore, we investigated the role of SAMHD1 inhibition of IRF7-mediated IFN-I induction during HIV-1 infection. We found that THP-1cells lacking IRF7 expression had reduced HIV-1 infection and viral transcription compared to control cells, indicating a positive role of IRF7 in HIV-1 infection. Our findings suggest that SAMHD1 suppresses IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis.

Fish SCD1 promotes SGIV infection via modulating the formation of lipid droplets and TBK1/MDA5-activated IFN signal pathway

Stearoyl CoA desaturase (SCD1), the rate-limiting enzyme for monounsaturated fatty acid production, involved in host metabolic regulation. Recent reports indicated that SCD1 played a crucial role in the process of viral infection. To investigate the function of SCD1 in teleost fish and the role it played in fish virus infection, EcSCD1 was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcSCD1 consisted of 1005 nucleotides encoding a protein containing 334 amino acids with a predicted molecular weight of 38.33 kDa, which shared 74.05% and 60.48% identity with zebrafish and human, respectively. EcSCD1 possessed four transmembrane domains and three conserved His-rich regions. In healthy groupers, EcSCD1 was primarily expressed in the liver and brain. Subcellular localization assay revealed that EcSCD1 was mainly distributed in the cytoplasm with uniform distribution or dot-like aggregation forms, and EcSCD1 was co-localized with the endoplasmic reticulum (ER). Singapore grouper iridovirus (SGIV) as well as poly (dA: dT) stimulation induced the expression of EcSCD1. On the other hand, EcSCD1 overexpression increased SGIV infection and SCD1 enzymatic product oleic acid (OA) recovered SGIV infection suppressed by SCD1 inhibitor. In addition, EcSCD1 overexpression facilitated the formation of lipid droplets (LDs), which was crucial for SGIV replication. Furthermore, EcSCD1 overexpression down-regulated the expression of interferon signaling molecules and the activities of interferon (IFN)1/3 and interferon stimulated response element (ISRE) promotors. Moreover, EcSCD1 overexpression restricted TANK binding kinase 1 (TBK1)−/melanoma differentiation-associated protein 5 (MDA5)-, but not mitochondrial antiviral signaling protein (MAVS)-evoked interferon immune responses. Overall, our findings offered novel insights into the regulatory mechanism of EcSCD1 during virus infection.

In silico analysis of autism spectrum disorder through the integration of DNA methylation and gene expression data for biomarker search

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by apparent communication deficits, repetitious unusual rituals, and social dysfunction. The epigenetic mechanism of the disorder has been researched meticulously yet not fully understood. Furthermore, there is yet no diagnostic marker that is associated with the condition. This study aimed at enhancing the understanding of the epigenetic mechanism of ASD and exploring biomarker candidates for the disorder using an in-silico approach.METHODS: ASD associated blood specific DNA methylation datasets were analyzed and overlapped with gene expression data to determine differentially regulated and methylated genes. Enrichment analysis was performed to identify signaling pathways, gene ontology terms, and disease associations. Additionally, metabolites and metabolic pathways associated with differentially methylated genes were demonstrated.RESULTS: The results revealed 42 differentially regulated and methylated genes, the majority of which were previously not associated with ASD. Particularly downregulated NFATC1 (nuclear factor of activated T cells 1) and upregulated MANBA (mannosidase beta) and ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 1) may be researched further to assess their biomarker features. The MAPK signaling pathway and gene ontology terms for protein/serine kinase activity and protein kinase binding came into prominence as a result of enrichment analysis. Metabolic alterations in ASD including tyrosine, tryptophan and asparagine were identified. Loss of amino acid homeostasis as well as dysregulation in related metabolic pathways were also observed.CONCLUSIONS: This in-silico study brings a comprehensive outlook on epigenetic alterations complementing governance by DNA methylation through integrating data across omics. DNA methylation, gene expression as well as metabolomics signatures of ASD that may be regarded as biomarker candidates were reported. The results of this study can guide future experimental studies, which will eventually lead to diagnostic tools and therapeutics designed for individuals suffering from the conditions of this complex disorder.

β-Glucan Induces Training Immunity to Promote Antiviral Activity by Activating TBK1.

Many studies have shown that β-glucan induces a trained immune phenotype in innate immune cells to defend against bacterial and fungal infections. The specific mechanism involves cellular metabolism and epigenetic reprogramming. However, it is unclear whether β-glucan plays a role in antiviral infection. Therefore, this study investigated the role of trained immunity induced by Candida albicans and β-glucan in antiviral innate immunity. It showed that C. albicans and β-glucan promoted the expression of interferon-β (IFN-β) and interleukin-6 (IL-6) in mouse macrophages triggered by viral infection. In addition, β-glucan pretreatment attenuated the pathological damage induced by the virus in mouse lungs and promoted the expression of IFN-β. Mechanistically, β-glucan could promote the phosphorylation and ubiquitination of TANK Binding Kinase 1 (TBK1), a key protein of the innate immune pathway. These results suggest that β-glucan can promote innate antiviral immunity, and this bioactive material may be a potential therapeutic target for antiviral treatment.

Roles of autophagy-related genes in the therapeutic effects of Xuanfei Pingchuan capsules on chronic obstructive pulmonary disease based on transcriptome sequencing analysis.

Objective: Autophagy plays an important role in the occurrence and development of chronic obstructive pulmonary disease (COPD). We evaluated the effect of Xuanfei Pingchuan capsule (XFPC) on autophagy-related genes of COPD by a bioinformatics analysis and experimental verification. Methods: The best treatment duration was screened by CCK8 assays. HBE cells were divided into three groups: blank, CSE and XFPC. After intervened by XFPC, HBE cells were collected and sent to Shenzhen Huada Gene Company for transcriptome sequencing. Subsequently, differential expression analyses, target gene prediction, and function enrichment analyses were carried out. Expression changes were verified in HBE cells by real-time Quantitative PCR (RT-qPCR) and western blotting (WB). Results: The result of differential expression analysis displayed that 125 target genes of HBE cells were mainly related to mitogen-activated protein kinase (MKK) binding, interleukin 33 binding, 1-Pyrroline-5-carboxylate dehydrogenase activity, and the mitogen-activated protein kinase (MAPK) signal pathway. Among the target genes, the core genes related to autophagy obtained by maximum neighborhood component algorithm were CSF1, AREG, MAPK9, MAP3K7, and AKT3. RT-qPCR and WB methods were used to verify the result, it showed similar expression changes in CSF1, MAPK9, MAP3K7, and AKT3 in bronchial epithelial cells to those in the bioinformatics analysis. Conclusion: Through transcriptome sequencing and validation analysis, we predicted that CSF1, MAPK9, MAP3K7, and AKT3 may be the potential autophagy-related genes that play an important role in the pathogenesis of COPD. XFPC may regulate autophagy by down-regulating the expression of CSF1, MAPK9, MAP3K7, and AKT3, thus achieving the purpose of treating chronic obstructive pulmonary disease.

African Swine Fever Virus H240R Protein Inhibits the Production of Type I Interferon through Disrupting the Oligomerization of STING.

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease in domestic pigs and wild boars. Domestic pigs infected with virulent African swine fever virus (ASFV) isolates have a high mortality, approaching 100%. Identification of ASFV genes related to virulence/pathogenicity and deletion of them are considered to be key steps in the development of live attenuated vaccines, because the ability of ASFV to escape host innate immune responses is related to viral pathogenicity. However, the relationship between the host antiviral innate immune responses and the pathogenic genes of ASFV has not been fully understood. In this study, the ASFV H240R protein (pH240R), a capsid protein of ASFV, was found to inhibit type I interferon (IFN) production. Mechanistically, pH240R interacted with the N-terminal transmembrane domain of stimulator of interferon genes (STING) and inhibited its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Additionally, pH240R inhibited the phosphorylation of interferon regulatory factor 3 (IRF3) and TANK binding kinase 1 (TBK1), leading to reduced production of type I IFN. Consistent with these results, infection with H240R-deficient ASFV (ASFV-ΔH240R) induced more type I IFN than infection with its parental strain, ASFV HLJ/18. We also found that pH240R may enhance viral replication via inhibition of type I IFN production and the antiviral effect of interferon alpha (IFN-α). Taken together, our findings provide a new explanation for the reduction of ASFV's replication ability by knockout of the H240R gene and a clue for the development of live attenuated ASFV vaccines. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease with a high mortality, approaching 100% in domestic pigs. However, the relationship between viral pathogenicity and immune evasion of ASFV is not fully understood, which limits the development of safe and effective ASF vaccines, specifically, live attenuated vaccines. In this study, we found that pH240R, as a potent antagonist, inhibited type I IFN production by targeting STING and inhibiting its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Furthermore, we also found that deletion of the H240R gene reduced viral pathogenicity by enhancing type I IFN production, which decreases ASFV replication. Taken together, our findings provide a clue for the development of an ASFV live attenuated vaccine via deleting the H240R gene.

Deletion of MGF-110-9L gene from African swine fever virus weakens autophagic degradation of TBK1 as a mechanism for enhancing type I interferon production.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a devastating disease for the global pig industry and economic benefit. The limited knowledge on the pathogenesis and infection mechanisms of ASF restricts progress toward vaccine development and ASF control. Previously, we illustrated that deletion of the MGF-110-9L gene from highly virulent ASFV CN/GS/2018 strains (ASFV∆9L) results in attenuated virulence in swine, but the underlying mechanism remains unclear. In this study, we found that the difference in virulence between wild-type ASFV (wt-ASFV) and ASFV∆9L strains was mainly caused by the difference in TANK Binding Kinase 1 (TBK1) reduction. TBK1 reduction was further identified to be mediated by the autophagy pathway and this degradative process requires the up-regulation of a positive autophagy regulation molecule- Phosphatidylinositol-4-Phosphate 3-Kinase Catalytic Subunit Type 2 Beta (PIK3C2B). Moreover, TBK1 over-expression was confirmed to inhibit ASFV replication in vitro. In summary, these results indicate that wt-ASFV counteracts type I interferon (IFN) production by degrading TBK1, while ASFVΔ9L enhanced type I IFN production by weakening TBK1 reduction, clarifying the mechanism that ASFVΔ9L present the attenuated virulence in vitro.

Remibrutinib, a Novel Bruton’s Tyrosine Kinase Inhibitor, Exhibits Improved Target Selectivity and Potency In Vitro (P8-3.003)

<h3>Objective:</h3> To assess the potency and selectivity of Bruton’s tyrosine kinase inhibitors (BTKis) under comparable experimental conditions. <h3>Background:</h3> BTK is a key node in B-cell receptor and Fc receptor signaling. BTKis are an emerging oral option for patients with multiple sclerosis (MS). Several covalent and reversible BTKis are in clinical development for the treatment of MS. For covalent enzyme inhibitors, in vitro assays are influenced by experimental conditions and are time dependent. <h3>Design/Methods:</h3> In vitro binding of covalent BTKis was assessed over time and concentration in human blood. Inhibition of B cells and basophils, including the impact of drug washout on B-cell inhibition, was assessed for covalent and reversible BTKis. Kinase selectivity was studied (first screened kinome wide, then using the dissociation constant for select kinases) via a binding assay to allow for the direct comparison of covalent and reversible BTKis. <h3>Results:</h3> Covalent inhibitors demonstrated time- and concentration-dependent BTK binding in human blood; the IC50 at 1 hour was 21 nM for remibrutinib, 508 nM for evobrutinib, 165 nM for tolebrutinib, and 427 nM for orelabrutinib. These values correlated with B-cell inhibition; the IC₅₀ was 18 nM for remibrutinib, 320 nM for evobrutinib, 74 nM for tolebrutinib, 185 nM for orelabrutinib, and 15 nM for fenebrutinib (reversible). Basophil inhibition potency was comparable. B-cell inhibition by remibrutinib (covalent) was not sensitive to washout, but B-cell inhibition by fenebrutinib was. Kinome selectivity screening at 1 μM showed the following ranking (from least to most off-target kinase binding): remibrutinib, fenebrutinib, evobrutinib, orelabrutinib, and tolebrutinib. A quantitative assessment of binding constants to a subset of kinases confirmed this ranking. <h3>Conclusions:</h3> BTKis undergoing development for MS exhibit varying selectivity across the human kinome, with the highest level of selectivity observed for remibrutinib. This distinction may translate into differences in clinical efficacy and safety. <b>Disclosure:</b> Bernd C. Kieseier, MD has nothing to disclose. Mr. Cenni has received personal compensation for serving as an employee of Novartis. Dr. Pulz has received personal compensation for serving as an employee of Novartis Pharma AG. Dr. Pulz has stock in Novartis Pharma AG. Dr. Angst has received personal compensation for serving as an employee of Novartis. Dr. Angst has stock in Novartis. Mr. Eichlisberger has nothing to disclose. Dr. Ziehn has received personal compensation for serving as an employee of Novartis AG.

P161 Systemic Sclerosis Dermal Fibroblast-derived Exosomes trigger a Type I Interferon Response in Keratinocytes, through Tank Binding Kinase 1

Abstract Background/Aims Activation of Type I IFN response has been shown to be present in blood and tissue in systemic sclerosis (SSc) and correlates with disease pathogenesis and therapy response. It is currently unknown whether the tissue-specific Type I IFN activation is a consequence of the response observed in blood or rather its source. SSc fibroblasts share many cellular and molecular features with cancer-associated fibroblasts (CAF), and there is a wealth of evidence supporting the role of CAF in inducing Type I IFN activation in epithelial cancer cells. Here, we aimed to determine whether keratinocytes in SSc show evidence of Type I IFN activation and the potential role of SSc in the process. Methods Skin biopsies were obtained from healthy and SSc patients’ forearms and processed for immunohistochemistry and subculture of primary fibroblasts and keratinocytes. The human skin keratinocyte cell line (HaCats) were stimulated with conditioned media from healthy (n = 3) and SSc (n = 3) fibroblasts. Exosomes were isolated from healthy and SSc patient fibroblast cultured supernatant by a combination of ultracentrifugation and polymer-based precipitation and used to stimulate HaCats. Cellular exosomes were investigated by immunoblotting and electron microscopy. TANK-binding kinase (TBK) was inhibited using a small molecule inhibitor (GSK8612). RT-PCR based IFN array and western blot for pSTAT1 (Tyr701) were employed to assess Type I IFN activation in these cells. Results Consistent with previous studies we found high levels of pSTAT1 in keratinocytes from SSc patient biopsies. Contrary to observations in dermal fibroblasts, primary keratinocytes isolated from SSc skin biopsies lost IFN activation once cultured in-vitro. Conversely, conditioned media from SSc patient fibroblasts triggered ISG induction; MX1 (2.7-fold, p &amp;lt; 0.05), CXCL10 (2.8-fold, p &amp;lt; 0.01) and CXCL11 (1.9-fold, p &amp;lt; 0.05). Media fractionation by ultracentrifugation indicated that the effect on keratinocytes was driven mainly by pelleted micro-vesicles, suggesting a potential role for cellular exosomes. Fibroblasts from SSc patients shed cellular exosomes of similar quantities and biophysical qualities, compared to healthy. SSc exosomes induced a 2.2-fold increase in phosphorylation of STAT1 (p &amp;lt; 0.05), as well as an induction in ISGs; MX1 (1.45-fold, p &amp;lt; 0.05), CXCL10 (1.84-fold, p &amp;lt; 0.01), CXCL11 (2.11-fold, p &amp;lt; 0.01) and OAS (1.24-fold, p &amp;lt; 0.05). Suppression of TBK activity by 10μM of GSK8612 suppressed the induction of pSTAT1. Consistent with these findings IFN array of HaCats stimulated with SSc exosomes showed significant (p &amp;lt; 0.05) upregulation of 16 ISGs by at least 1.2-fold compared to the effect of healthy control exosomes. Conclusion We have shown for the first time that scleroderma keratinocytes ex-vivo IFN activation is lost in-vivo and can be regained following exposure to fibroblast-derived exosomes though a TBK-dependent mechanism. Our data indicates that exosomes from SSc fibroblasts may carry the “signal zero” of local Type I IFN activation, potentially though activation of toll-like receptors and/or pattern recognition receptors. Disclosure J.A. Bryon: None. C. Wasson: None. R.L. Ross: None. E. Zeqiraj: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.. Grants/research support; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Rnf144b alleviates the inflammatory responses and cardiac dysfunction in sepsis.

Sepsis is an inflammatory disease with high mortality and morbidity. Inflammation plays an essential role in sepsis, and suppressing inflammation has been shown to ameliorate sepsis. Rnf144b is an ubiquitin E3 ligation with anti-inflammation activities. Its precise roles in sepsis remain unknown. Here, we explored the function of Rnf144b in sepsis. We generated conditional knockout mice with Rnf144b deficiency in the myeloid cells. We monitored the Rnf144b expression in peripheral blood mononuclear cells from healthy donor and patients with sepsis, and in lipopolysaccharides (LPS)-treated bone marrow-derived macrophages (BMDMs). The cytokine expression between wild-type BMDMs and Rnf144b-deficient BMDMs after LPS and CpG treatments was compared. The survival rate and cardiac function were monitored. The activation of TANK binding kinase 1 and nuclear factor kappa-B was examined by Western blot and real-time PCR. Up-regulated expression of Rnf144b was observed in peripheral blood mononuclear cells from patients with sepsis. LPS induced the expression of Rnf144b in BMDMs. Rnf144b-deficient BMDMs produced more inflammatory cytokines after LPS or CpG stimulation. Septic mice with Rnf144b deficiency in myeloid cells had higher mortality and exacerbated cardiac dysfunction. Rnf144b interacted with TANK binding kinase 1 and Rnf144b deficiency resulted in impaired activation of TBK1 but enhanced activation of nuclear factor kappa-B. Rnf144b prevents inflammatory responses and cardiac dysfunction in sepsis.

Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Binding of USP4 to cortactin enhances cell migration in HCT116 human colon cancer cells.

Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating β-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on β-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of β-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.

Structural mechanism of a drug-binding process involving a large conformational change of the protein target

Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable.

Integrated gene network analysis sheds light on understanding the progression of Osteosarcoma.

Osteosarcoma is a rare disorder among cancer, but the most frequently occurring among sarcomas in children and adolescents. It has been reported to possess the relapsing capability as well as accompanying collateral adverse effects which hinder the development process of an effective treatment plan. Using networks of omics data to identify cancer biomarkers could revolutionize the field in understanding the cancer. Cancer biomarkers and the molecular mechanisms behind it can both be understood by studying the biological networks underpinning the etiology of the disease. In our study, we aimed to highlight the hub genes involved in gene-gene interaction network to understand their interaction and how they affect the various biological processes and signaling pathways involved in Osteosarcoma. Gene interaction network provides a comprehensive overview of functional gene analysis by providing insight into how genes cooperatively interact to elicit a response. Because gene interaction networks serve as a nexus to many biological problems, their employment of it to identify the hub genes that can serve as potential biomarkers remain widely unexplored. A dynamic framework provides a clear understanding of biological complexity and a pathway from the gene level to interaction networks. Our study revealed various hub genes viz. TP53, CCND1, CDK4, STAT3, and VEGFA by analyzing various topological parameters of the network, such as highest number of interactions, average shortest path length, high cluster density, etc. Their involvement in key signaling pathways, such as the FOXM1 transcription factor network, FAK-mediated signaling events, and the ATM pathway, makes them significant candidates for studying the disease. The study also highlighted significant enrichment in GO terms (Biological Processes, Molecular Function, and Cellular Processes), such as cell cycle signal transduction, cell communication, kinase binding, transcription factor activity, nucleoplasm, PML body, nuclear body, etc. To develop better therapeutics, a specific approach toward the disease targeting the hub genes involved in various signaling pathways must have opted to unravel the complexity of the disease. Our study has highlighted the candidate hub genes viz. TP53, CCND1 CDK4, STAT3, VEGFA. Their involvement in the major signaling pathways of Osteosarcoma makes them potential candidates to be targeted for drug development. The highly enriched signaling pathways include FOXM1 transcription pathway, ATM signal-ling pathway, FAK mediated signaling events, Arf6 signaling events, mTOR signaling pathway, and Integrin family cell surface interactions. Targeting the hub genes and their associated functional partners which we have reported in our studies may be efficacious in developing novel therapeutic targets.

Abstract 2548: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity

Abstract Casitas B-lineage lymphoma (CBL) is a family of RING-type E3 ligases that target proteins for degradation via the proteasomal pathway. CBL-b specifically functions as a negative effector of T cell activation by down regulating the T cell receptor, whereas c-CBL interacts with various receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation. Therefore, the CBL family regulates the biology of immune cells and targeting CBL-b or c-CBL is a potential therapeutic strategy for the treatment of cancer, infection, or autoimmune diseases. In this study, we used TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) to uncover new mechanistic aspects of CBL activation. Our assays were designed and optimized using purified components (E1/E2/c-CBL or CBL-b E3 ligase and kinase substrate Tyro3 or Axl) to recreate the polyubiquitination cascade in vitro. The assays use a Europium cryptate-labeled Ubiquitin (donor) and a Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the technique measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay is homogeneous, making it especially suitable for screening applications and real-time kinetics. Validation of the assay indicated that the presence of a kinase substrate (Tyro3 or Axl) activates CBL-b and c-CBL, which are known to display a similar mechanism of activation. Importantly, our assay design allowed us to show that known CBL-B inhibitor CBL-B-IN-1 is not strictly selective toward CBL-B but also affects c-CBL E3 ligase activity to similar extend since the IC50 of the compound for c-CBL and CBL-B differ by only 2-fold. Finally, it has been proposed that CBL-b phosphorylation at Y106, Y133, and Y363 by Tyro3 kinase is required for its activation. Indeed, in our assay, we observed that CBL-b mutation at Y363F decreases TYRO3 ubiquitination drastically. Surprisingly, phospho-mimetic mutation Y363E did not restore CBL-b-mediated ubiquitination of Tyro3, indicating that substrate binding close to Y363 is the predominant requirement for CBL activation. The weak E3 ligase activity of CBL-b Y363E mutant was insensitive to CBL-B-IN-1 treatment.In conclusion, our data indicates that no significant difference in the parameters of substrate ubiquitination exists between CBL-B and c-CBL, and that ubiquitination of receptor tyrosine kinase Tyro3 by either c-CBL or CBL-B depends on the kinase binding to CBL but not on the phosphorylation of CBL. Citation Format: Kasia Zientara-Rytter, Veronique Baron, Pavel Shashkin, Henry Zhu. Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2548.

C-Myb Dominates TBK1-Mediated Endotoxin Tolerance in Kupffer Cells by Negatively Regulating DTX4.

As a protective mechanism regulating excessive inflammation, endotoxin tolerance plays a vital role in regulating endotoxin shock. Kupffer cells are players in mediating endotoxin tolerance. Nonetheless, the regulatory mechanism regulating endotoxin tolerance is barely known. A nonclassical IKK kinase called TRAF-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) can regulate inflammation. Here, we found that TBK1 is required for endotoxin tolerance in Kupffer cells. TBK1 plays a dominant role in regulating endotoxin tolerance by negatively regulating the induction of p100 processing. Deltex E3 ubiquitin ligase 4 (DTX4), a negative regulator of TBK1, can promote TBK1 K48-mediated ubiquitination and indirectly regulate endotoxin tolerance in Kupffer cells. We demonstrate that the c-Myb transcription factor could negatively regulate DTX4. Overexpression of c-Myb can be used to reduce the ubiquitination of TBK1 by reducing DTX4 transcription and to boost the anti-inflammatory effect of endotoxin tolerance. Thus, this study reveals a novel theory of TBK1-mediated endotoxin tolerance in Kupffer cells.