ABSTRACT
Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
Subject(s)
Cryoelectron Microscopy , Guanosine Diphosphate , Guanosine Triphosphate , Mutation , Humans , HEK293 Cells , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/metabolism , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D2/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Protein Binding , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Protein gamma Subunits/metabolism , GTP-Binding Protein gamma Subunits/geneticsABSTRACT
Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.
Subject(s)
Coumaric Acids , GTP-Binding Proteins , Glioblastoma , Nanoparticles , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Coumaric Acids/pharmacology , Humans , Transglutaminases/metabolism , Transglutaminases/genetics , Glioblastoma/metabolism , Glioblastoma/drug therapy , Glioblastoma/pathology , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Cell Line, Tumor , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Nanoparticles/chemistry , Drug Carriers/chemistry , Apoptosis/drug effects , Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Kisspeptin receptor (KISS1R), belonging to the class A peptide-GPCR family, plays a key role in the regulation of reproductive physiology after stimulation by kisspeptin and is regarded as an attractive drug target for reproductive diseases. Here, we demonstrated that KISS1R can couple to the Gi/o pathway besides the well-known Gq/11 pathway. We further resolved the cryo-electron microscopy (cryo-EM) structure of KISS1R-Gq and KISS1R-Gi complexes bound to the synthetic agonist TAK448 and structure of KISS1R-Gq complex bound to the endogenous agonist KP54. The high-resolution structures provided clear insights into mechanism of KISS1R recognition by its ligand and can facilitate the design of targeted drugs with high affinity to improve treatment effects. Moreover, the structural and functional analyses indicated that conformational differences in the extracellular loops (ECLs), intracellular loops (ICLs) of the receptor, and the "wavy hook" of the Gα subunit may account for the specificity of G protein coupling for KISS1R signaling.
Subject(s)
Cryoelectron Microscopy , Receptors, Kisspeptin-1 , Humans , Ligands , Receptors, Kisspeptin-1/metabolism , Receptors, Kisspeptin-1/chemistry , Protein Binding , Kisspeptins/metabolism , Kisspeptins/chemistry , Models, Molecular , HEK293 Cells , Protein Conformation , Signal Transduction , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , Structure-Activity RelationshipABSTRACT
Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.
Subject(s)
Epigenesis, Genetic , Histones , Neoplasms , Protein Glutamine gamma Glutamyltransferase 2 , Protein Processing, Post-Translational , Humans , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Neoplasms/metabolism , Neoplasms/genetics , Neoplasms/enzymology , Neoplasms/pathology , Histones/metabolism , Animals , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Transglutaminases/metabolism , Transglutaminases/genetics , Gene Expression Regulation, Neoplastic , Signal TransductionABSTRACT
Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
Subject(s)
Cell Survival , GTP-Binding Proteins , Protein Conformation , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Humans , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Transglutaminases/metabolism , Transglutaminases/chemistry , Transglutaminases/genetics , Cell Survival/drug effects , Cryoelectron Microscopy , Cell Line, Tumor , Cell Death/drug effects , Scattering, Small Angle , X-Ray Diffraction , Calcium/metabolismABSTRACT
ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown (KD) leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 KD in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and γ-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold multimer, which was validated by co-immunoprecipitation and proximity ligation assay. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 KD. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.
Subject(s)
Cell Cycle Proteins , Centrosome , Microtubules , Nerve Tissue Proteins , Neural Stem Cells , Neurogenesis , Animals , Microtubules/metabolism , Mice , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Neurogenesis/genetics , Neural Stem Cells/metabolism , Centrosome/metabolism , Cell Proliferation , Cell Movement , Cerebral Cortex/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Tubulin/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , ADP-Ribosylation Factors/metabolism , ADP-Ribosylation Factors/geneticsABSTRACT
Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies.
Subject(s)
GTP-Binding Proteins , Mice, Knockout , Animals , Mice , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/immunology , Disease Models, Animal , Virus Replication , Herpesvirus 1, Human/physiology , Herpesvirus 1, Human/genetics , Mice, Inbred C57BL , Lymphocytic choriomeningitis virus/physiology , Lymphocytic choriomeningitis virus/immunology , Virus Diseases/immunology , Virus Diseases/geneticsABSTRACT
BACKGROUND: Lung adenocarcinoma (LUAD) is a leading cause of tumor-associated mortality, and it is needed to find new target to combat this disease. Guanine nucleotide-binding -protein-like 3 (GNL3) mediates cell proliferation and apoptosis in several cancers, but its role in LUAD remains unclear. OBJECTIVE: To explore the expression and function of Guanine nucleotide-binding protein-like 3 (GNL3) in lung adenocarcinoma (LUAD) and its potential mechanism in inhibiting the growth of LUAD cells. METHODS: We evaluated the expression of GNL3 in LUAD tissues and its association with patient prognosis using databases and immunohistochemistry. Cell proliferation was assessed by CCK-8 assay as well as colony formation, while apoptosis was evaluated by FCM. The effect of GNL3 knockdown on the Wnt/ß-catenin axis was investigated by Immunoblot analysis. RESULTS: GNL3 is overexpressed in LUAD tissues and is correlated with poor prognosis. Knockdown of GNL3 significantly inhibited the growth as well as induced apoptosis in A549 as well as H1299 cells. Furthermore, we found that the inhibitory effect of GNL3 knockdown on LUAD cell growth is associated with the downregulation of the Wnt/ß-catenin axis. CONCLUSION: GNL3 is key in the progression of LUAD by metiating Wnt/ß-catenin axis. Targeting GNL3 may represent a novel therapeutic method for LUAD treatment.
Subject(s)
Adenocarcinoma of Lung , Apoptosis , Cell Proliferation , Gene Knockdown Techniques , Lung Neoplasms , Wnt Signaling Pathway , Humans , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/genetics , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/metabolism , Cell Line, Tumor , Prognosis , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , beta Catenin/metabolism , Gene Expression Regulation, Neoplastic , A549 Cells , Nuclear ProteinsABSTRACT
Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.
Subject(s)
GTP-Binding Proteins , Mitochondria , Mitochondrial Ribosomes , Oxidative Phosphorylation , Humans , Mitochondrial Ribosomes/metabolism , Mitochondria/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , HEK293 Cells , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Messenger/genetics , HeLa CellsABSTRACT
Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Animals , Fluorescence Resonance Energy Transfer/methods , Mice , Biosensing Techniques/methods , GTP-Binding Proteins/metabolism , Humans , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Cell Line, Tumor , Receptors, Lysophosphatidic Acid/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathologyABSTRACT
Mathematical models of biochemical reaction networks are an important and emerging tool for the study of cell signaling networks involved in disease processes. One promising potential application of such mathematical models is the study of how disease-causing mutations promote the signaling phenotype that contributes to the disease. It is commonly assumed that one must have a thorough characterization of the network readily available for mathematical modeling to be useful, but we hypothesized that mathematical modeling could be useful when there is incomplete knowledge and that it could be a tool for discovery that opens new areas for further exploration. In the present study, we first develop a mechanistic mathematical model of a G-protein coupled receptor signaling network that is mutated in almost all cases of uveal melanoma and use model-driven explorations to uncover and explore multiple new areas for investigating this disease. Modeling the two major, mutually-exclusive, oncogenic mutations (Gαq/11 and CysLT2R) revealed the potential for previously unknown qualitative differences between seemingly interchangeable disease-promoting mutations, and our experiments confirmed oncogenic CysLT2R was impaired at activating the FAK/YAP/TAZ pathway relative to Gαq/11. This led us to hypothesize that CYSLTR2 mutations in UM must co-occur with other mutations to activate FAK/YAP/TAZ signaling, and our bioinformatic analysis uncovers a role for co-occurring mutations involving the plexin/semaphorin pathway, which has been shown capable of activating this pathway. Overall, this work highlights the power of mechanism-based computational systems biology as a discovery tool that can leverage available information to open new research areas.
Subject(s)
Mutation , Receptors, G-Protein-Coupled , Signal Transduction , Humans , Signal Transduction/genetics , Signal Transduction/physiology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Mutation/genetics , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Systems Biology/methods , Models, Biological , Melanoma/genetics , Melanoma/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolismABSTRACT
Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
Subject(s)
Cholesterol , GTP-Binding Proteins , Protein Binding , Receptor, Cholecystokinin A , Receptor, Cholecystokinin B , Animals , Humans , CHO Cells , Cholesterol/metabolism , Cricetulus , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , HEK293 Cells , Ligands , Mutation , Protein Conformation , Receptor, Cholecystokinin A/metabolism , Receptor, Cholecystokinin A/genetics , Receptor, Cholecystokinin B/metabolism , Receptor, Cholecystokinin B/geneticsABSTRACT
Guanylate binding protein 5 (GBP5) is an emerging immune component that has been increasingly recognized for its involvement in autoimmune diseases, particularly inflammatory bowel disease (IBD). IBD is a complex disease involving inflammation of the gastrointestinal tract. Here, we explored the functional significance of GBP5 using Gbp5 knockout mice and wildtype mice exposed to dextran sulfate sodium (DSS) to generate chronic colitis model. We found that Gbp5 deficiency protected mice from DSS-induced chronic colitis. Transcriptome analysis of colon tissues showed reduced immune responses in Gbp5 knockout mice compared to those in corresponding wildtype mice. We further observed that after repeated DSS exposure, the gut microbiota was altered, both in wildtype mice and Gbp5 knockout mice; however, the gut microbiome health index was higher in the Gbp5 knockout mice. Notably, a probiotic murine commensal bacterium, Dubosiella, was predominantly enriched in these knockout mice. Our findings suggest that GBP5 plays an important role in promoting inflammation and dysbiosis in the intestine, the prevention of which might therefore be worth exploring in regards to IBD treatment.
Subject(s)
Colitis , Dextran Sulfate , Disease Models, Animal , Gastrointestinal Microbiome , Mice, Knockout , Animals , Mice , Chronic Disease , Colitis/microbiology , Colitis/chemically induced , Colitis/immunology , Colitis/genetics , Colitis/metabolism , Dysbiosis/microbiology , Dysbiosis/immunology , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/deficiency , Mice, Inbred C57BLABSTRACT
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
Subject(s)
Calcium , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Transglutaminases/metabolism , Transglutaminases/chemistry , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Humans , Calcium/metabolism , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , Crystallography, X-Ray , Glutens/metabolism , Glutens/chemistry , Models, Molecular , Protein Conformation , Celiac Disease/metabolism , Protein BindingABSTRACT
Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.
Subject(s)
Bleomycin , Lung , Protein Glutamine gamma Glutamyltransferase 2 , Pulmonary Fibrosis , Transglutaminases , Animals , Male , Mice , Glycolysis , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/genetics , Lung/pathology , Lung/metabolism , Lung/drug effects , Mice, Inbred C57BL , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/pathology , Transglutaminases/metabolism , Transglutaminases/geneticsABSTRACT
BACKGROUND: Cancer utilizes immunosuppressive mechanisms to create a tumor microenvironment favorable for its progression. The purpose of this study is to histologically characterize the immunological properties of the tumor microenvironment of oral squamous cell carcinoma (OSCC) and identify key molecules involved in the immunological microenvironment and patient prognosis. METHODS: First, overlapping differentially expressed genes (DEGs) were screened from OSCC transcriptome data in public databases. Correlation analysis of DEGs with known immune-related genes identified genes involved in the immune microenvironment of OSCC. Next, stromal patterns of tumor were classified and immunohistochemical staining was performed for immune cell markers (CD3, CD4, Foxp3, CD8, CD20, CD68, and CD163), programmed death-ligand 1 (PD-L1), and guanylate binding protein 5 (GBP5) in resected specimens obtained from 110 patients with OSCC who underwent resection. Correlations between each factor and their prognostic impact were analyzed. RESULTS: Among the novel OSCC-specific immune-related genes screened (including ADAMDEC1, CXCL9, CXCL13, DPT, GBP5, IDO1, and PLA2G7), GBP5 was selected as the target gene. Histopathologic analysis showed that multiple T-cell subsets and CD20-positive cells were less common in the advanced stages, whereas CD163-positive cells were more common in advanced stages. The immature type in the stromal pattern category was associated with less immune cell infiltration, lower expression of PD-L1 in immune cells, lower expression of GBP5 in the stroma, and shorter overall survival and recurrence-free survival. Expression of GBP5 in the tumor and stroma correlated with immune cell infiltration of tumors and PD-L1 expression in tumor and immune cells. Patients with low tumor GBP5 expression and high stromal expression had significantly longer overall survival and recurrence-free survival. CONCLUSIONS: The stromal pattern category may reflect both invasive and immunomodulatory potentials of cancer-associated fibroblasts in OSCC. GBP5 has been suggested as a potential biomarker to predict the prognosis and therapeutic efficacy of immune checkpoint inhibitors.
Subject(s)
Biomarkers, Tumor , Computational Biology , Mouth Neoplasms , Tumor Microenvironment , Adult , Aged , Female , Humans , Male , Middle Aged , B7-H1 Antigen/metabolism , B7-H1 Antigen/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/metabolism , Computational Biology/methods , Gene Expression Regulation, Neoplastic , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , GTP-Binding Proteins/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mouth Neoplasms/immunology , Mouth Neoplasms/pathology , Mouth Neoplasms/genetics , Mouth Neoplasms/mortality , Mouth Neoplasms/metabolism , Mouth Neoplasms/surgery , Prognosis , Retrospective Studies , Tumor Microenvironment/immunologyABSTRACT
Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.
Subject(s)
Catalytic Domain , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases , Transglutaminases/metabolism , Transglutaminases/chemistry , Transglutaminases/antagonists & inhibitors , Humans , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/antagonists & inhibitors , Computer Simulation , Protein Binding , Models, Molecular , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Docking Simulation , Ligands , Protein ConformationABSTRACT
Inherited retinal diseases encompass a genetically diverse group of conditions caused by variants in genes critical to retinal function, including handful of ribosome-associated genes. This study focuses on the HBS1L gene, which encodes for the HBS1-like translational GTPase that is crucial for ribosomal rescue. We have reported a female child carrying biallelic HBS1L variants, manifesting with poor growth and neurodevelopmental delay. Here, we describe the ophthalmologic findings in the patient and in Hbs1ltm1a/tm1a hypomorph mice and describe the associated microscopic and molecular perturbations. The patient has impaired visual function, showing dampened amplitudes of a- and b-waves in both rod- and cone-mediated responses. Hbs1ltm1a/tm1a mice exhibited profound thinning of the entire retina, specifically of the outer photoreceptor layer, due to extensive photoreceptor cell apoptosis. Loss of Hbs1l resulted in comprehensive proteomic alterations by mass spectrometry analysis, with an increase in the levels of 169 proteins and a decrease in the levels of 480 proteins, including rhodopsin (Rho) and peripherin 2 (Prph2). Gene Ontology biological process and gene set enrichment analyses reveal that the downregulated proteins are primarily involved in phototransduction, cilium assembly and photoreceptor cell development. These findings underscore the importance of ribosomal rescue proteins in maintaining retinal health, particularly in photoreceptor cells.
Subject(s)
Disease Models, Animal , Retinal Dystrophies , Animals , Retinal Dystrophies/pathology , Retinal Dystrophies/genetics , Female , Humans , Mice , Photoreceptor Cells/metabolism , Photoreceptor Cells/pathology , Apoptosis , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/genetics , GTP Phosphohydrolases/deficiency , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/genetics , ChildABSTRACT
Kappa opioid receptor (KOR) agonists represent promising therapeutics for pain relief due to their analgesic properties along with lower abuse potential than opioids that act at the mu opioid receptor. However, typical KOR agonists produce sedation and dysphoria. Previous studies have shown that G protein signaling-biased KOR agonists may present a means to untangle the desired analgesic properties from undesired side effects. In this paper, we report a new series of G protein signaling-biased KOR agonists entailing -S- â -CH2- replacement in a previously reported KOR agonist, triazole 1.1. With an optimized carbon linker in hand, further development of the scaffold was undertaken to investigate the appendages of the triazole core. The structure-activity relationship study of this series is described, including several analogues that display enhanced potency while maintaining G protein-signaling bias compared to triazole 1.1.