GTPBP8 plays a role in mitoribosome formation in human mitochondria.

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.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

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.

Systems modeling of oncogenic G-protein and GPCR signaling reveals unexpected differences in downstream pathway activation.

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.

Knockdown of GNL3 inhibits LUAD cell growth by regulating Wnt-ß-catenin pathway.

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.

TG2 participates in pulmonary vascular remodelling by regulating the senescence of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is a severe cardiovascular disease characterised by pulmonary vascular remodelling. The pivotal role of cellular senescence in vascular remodelling has been acknowledged. Transglutaminase type 2 (TG2), a calcium-dependent enzyme, is intricately linked to both cellular senescence and PH. However, the precise mechanisms underlying the involvement of TG2 in PH remain unclear. In this study, we explored the expression of TG2 and the cellular senescence marker p16INK4a in the pulmonary vasculature of mice with PH induced by hypoxia combined with SU5416. Our findings revealed upregulation of both TG2 and p16INK4a expression in the pulmonary vasculature of PH mice. Additionally, a notable increase in TG2 expression was observed in senescent pulmonary artery smooth muscle cells (PASMC). To delve deeper, we employed proteomic sequencing to reveal seven genes associated with cellular senescence, with a subsequent focus on MAPK14. Our investigation revealed that TG2 regulates senescence in PASMC by modulating the phosphorylation levels of MAPK14. Additionally, in the context of hypoxia combined with SU5416, our observations revealed a noteworthy reduction in both pulmonary vascular remodelling and senescent manifestations in smooth muscle-specific TG2 knockout mice compared with their wild-type counterparts. In summary, our findings indicate that TG2 deficiency lowers the senescence levels of PASMC by inhibiting the activity of MAPK14. This inhibition of senescence in the pulmonary vasculature of PH mice helps to decelerate the progression of pulmonary vascular remodelling and consequently hinders the onset and development of PH.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.

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.

Transglutaminase 2 knockout mice are protected from bleomycin-induced lung fibrosis with preserved lung function and reduced metabolic derangements.

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.

Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.

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.

In silico studies of the open form of human tissue transglutaminase.

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.

HBS1L deficiency causes retinal dystrophy in a child and in a mouse model associated with defective development of photoreceptor cells.

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.

Guanylate binding protein 5 is an immune-related biomarker of oral squamous cell carcinoma: A retrospective prognostic study with bioinformatic analysis.

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.

Antibody-assisted selective isolation of Purkinje cell nuclei from mouse cerebellar tissue.

We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.

Diminished Immune Response and Elevated Abundance in Gut Microbe Dubosiella in Mouse Models of Chronic Colitis with GBP5 Deficiency.

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.

Potential Celiac Disease in Children: Health Status on A Long-Term Gluten-Containing Diet.

Potential celiac disease (PCD) is a clinical condition characterised by the presence of a positive CD-specific serology and a normal intestinal architecture. Asymptomatic PCD patients are generally advised to continue on a gluten-containing diet (GCD), but long-term risks of this approach have never been explored. In the present study, we aimed to investigate nutritional and autoimmune complications possibly developing overtime in a cohort of asymptomatic PCD children on a GCD. We compared children's parameters of growth, nutritional status, and autoimmunity between the time of diagnosis and on the occasion of their last medical check, after a long-term gluten-containing diet. Altogether, we collected data from 171 PCD children with a mean follow-up time of 3 years (range 0.35-15.3 years). During follow-up, although patients did not reduce their amount of daily gluten intake, their anti-tissue transglutaminase (anti-TG2) antibodies spontaneously and significantly decreased. Most parameters analysed had not changed during follow-up (height centile, ferritin, albumin, cholesterol, calcium, alkaline phosphatase, parathormone, and vitamin D) or even improved significantly (weight and BMI centile, haemoglobin, blood iron, HDL, glycaemia, and HbA1C, p < 0.05), always remaining within the limit of normality. Equally, autoantibodies for other concomitant autoimmune disorders did not increase overtime. Similar results were obtained excluding from analysis patients who had stopped producing anti-TG2 and those with a follow-up time < 3 years. Our pilot study has provided reassuring results regarding the maintenance of a gluten-containing diet in asymptomatic PCD children, even when long-term follow-up was considered.

Genetic polymorphisms of inflammatory and bone metabolism related proteins in a population with dental implants of the Basque Country. A case-control study.

BACKGROUND: Peri-implantitis (PI) is a frequent inflammatory disorder characterised by progressive loss of the supporting bone. Not all patients with recognised risk factors develop PI. The aim of this study is to evaluate the presence of single nucleotide polymorphisms (SNP) of inflammatory and bone metabolism related proteins in a population treated with dental implants from the Basque Country (Spain). METHODS: We included 80 patients with diagnosis of PI and 81 patients without PI, 91 women and 70 men, with a mean age of 60.90 years. SNPs of BMP-4, BRINP3, CD14, FGF-3, FGF-10, GBP-1, IL-1α, IL-1ß, IL-10, LTF, OPG and RANKL proteins were selected. We performed a univariate and bivariate analysis using IBM SPSS® v.28 statistical software. RESULTS: Presence of SNPs GBP1 rs7911 (p = 0.041) and BRINP3 rs1935881 (p = 0.012) was significantly more common in patients with PI. Patients with PI who smoked (> 10 cig/day) showed a higher presence of OPG rs2073617 SNP (p = 0.034). Also, BMP-4 rs17563 (p = 0.018) and FGF-3 rs1893047 (p = 0.014) SNPs were more frequent in patients with PI and Type II diabetes mellitus. CONCLUSIONS: Our findings suggest that PI could be favoured by an alteration in the osseointegration of dental implants, based on an abnormal immunological response to peri-implant infection in patients from the Basque Country (Spain).

Macrophagic HDAC3 inhibition ameliorates Dextran Sulfate Sodium induced inflammatory bowel disease through GBP5-NLRP3 pathway.

Inflammatory bowel disease (IBD) is a chronic inflammatory intestinal disease, characterized by dysregulated immune response. HDAC3 is reported to be an epigenetic brake in inflammation, playing critical roles in macrophages. However, its role in IBD is unclear. In our study, we found HDAC3 was upregulated in CX3CR1-positive cells in the mucosa from IBD mice. Conditional knockout (cKO) of Hdac3 in CX3CR1 positive cells attenuated the disease severity of Dextran Sulfate Sodium (DSS)-induced colitis. In addition, inhibition of HDAC3 with RGFP966 could also alleviate the DSS-induced tissue injury and inflammation in IBD. The RNA sequencing results revealed that Hdac3 cKO restrained DSS-induced upregulation of genes in the pathways of cytokine-cytokine receptor interaction, complement and coagulation cascades, chemokine signaling, and extracellular matrix receptor interaction. We also identified that Guanylate-Binding Protein 5 (GBP5) was transcriptionally regulated by HDAC3 in monocytes by RNA sequencing. Inhibition of HDAC3 resulted in decreased transcriptional activity of interferon-gamma-induced expression of GBP5 in CX3CR1-positive cells, such as macrophages and microglia. Overexpression of HDAC3 upregulated the transcriptional activity of GBP5 reporter. Lastly, conditional knockout of Hdac3 in macrophages (Hdac3 mKO) attenuated the disease severity of DSS-induced colitis. In conclusion, inhibition of HDAC3 in macrophages could ameliorate the disease severity and inflammatory response in colitis by regulating GBP5-NLRP3 axis, identifying a new therapeutic avenue for the treatment of colitis.

Structure-based identification of a G protein-biased allosteric modulator of cannabinoid receptor CB1.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.