Analysis of the Guanine Nucleotide-Bound State of KRAS by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography.

Guanine nucleotides can be quantitatively analyzed by high-performance liquid chromatography (HPLC). Here we describe an ion-pair reversed-phase HPLC (IP-RP-HPLC)-based method, which enables analyzing GDP and GTP bound to small GTPases immunoprecipitated from cells. The activation status of FLAG-KRAS expressed in HEK293T cells can be investigated with the IP-RP-HPLC method. This method also can be adapted to determine the effects of compounds such as the KRAS/G12C inhibitor sotorasib on the activation status of FLAG-KRAS in the cells.

BZW1 is a prognostic and immunological biomarker in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma is the most common malignant tumor of the digestive system and is called the "king of cancer" because it has been labeled with high malignancy, rapid progression, poor survival, and poor prognosis. Previously, it was reported that the basic leucine zipper and W2 domains 1 (BZW1) is involved in the progression of many tumors. However, its research in digestive system tumors such as pancreatic cancer is rarely studied. To explore potential biomarkers related to survival and prognosis of pancreatic cancer and provide a new targeted therapy for it. We first analyzed the mRNA and protein expression of BZW1 in pancreatic cancer. We then explored the correlation of BZW1 with survival prognosis and immune infiltration in pancreatic cancer patients. Finally, we explored BZW1-related gene enrichment analysis, including protein-protein interaction networks, gene ontology functional enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The mRNA and protein expression of the BZW1 gene in pancreatic cancer tissues were higher than those in adjacent normal tissues, and pancreatic cancer patients with high BZW1 expression had a poor prognosis. In addition, the expression of BZW1 was positively or negatively correlated with different immune cells of pancreatic cancer, such as CD4 + T lymphocytes, CD8 + T lymphocytes, B cells, macrophages, neutrophils, etc. Correlation enrichment analysis showed that we obtained 50 available experimentally determined BZW1-binding proteins and 100 targeted genes related to BZW1, and the intersection genes were eukaryotic translation termination factor 1 and Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3. Moreover, there was a positive correlation between BZW1 and eukaryotic translation termination factor 1 and Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 genes in pancreatic cancer. Gene ontology enrichment analysis showed BZW1 was mainly related to biological processes such as "mRNA processing," "RNA splicing," "regulation of translational initiation," and "activation of innate immune response." The results of Kyoto Encyclopedia of Genes and Genomes pathway analysis further indicated that BZW1 may be involved in pancreatic carcinogenesis through the "spliceosome" and "ribosome." The BZW1 gene may be a potential immunotherapy target and a promising prognostic marker for pancreatic cancer.

Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators.

Noncanonical G protein activation and inactivation, particularly for the Gαi/s protein subfamilies, have long been a focus of chemical research. Combinatorial libraries were already effectively applied to identify modulators of the guanine-nucleotide exchange, as can be exemplified with peptides such as KB-752 and GPM-1c/d, the so-called guanine-nucleotide exchange modulators. In this study, we identified novel bicyclic peptides from a combinatorial library screening that show prominent properties as molecular switch-on/off modulators of Gαi signaling. Among the series of hits, the exceptional paradigm of GPM-3, a protein and state-specific bicyclic peptide, is the first chemically identified GAP (GTPase-activating protein) modulator with a high binding affinity for Gαi protein. Computational analyses identified and assessed the structure of the bicyclic peptides, novel ligand-protein interaction sites, and their subsequent impact on the nucleotide binding site. This approach can therefore lead the way for the development of efficient chemical biological probes targeting Gαi protein modulation within a cellular context.

Sports Participation Promotes Beneficial Adaptations in the Erythrocyte Guanylate Nucleotide Pool in Male Athletes Aged 20-90 Years.

Introduction: The guanine nucleotide pool (GTP, guanosine-5'-triphosphate; GDP, guanosine-5'-diphosphate, and GMP, guanosine-5'-monophosphate) is an essential energy donor in various biological processes (eg protein synthesis and gluconeogenesis) and secures several vital regulatory functions in the human body. The study aimed to predict the trends of age-related changes in erythrocyte guanine nucleotides and examine whether competitive sport and related physical training promote beneficial adaptations in erythrocyte guanylate concentrations. Methods: The study included 86 elite endurance runners (EN) aged 20-81 years, 58 sprint-trained athletes (SP) aged 21-90 years, and 62 untrained individuals (CO) aged 20-68 years. Results: The concentration of erythrocyte GTP and total guanine nucleotides (TGN) were highest in the SP group, lower in the EN group, and lowest in the CO group. Both athletic groups had higher guanylate energy charge (GEC) values than the CO group (p = 0.012). Concentrations of GTP, TGN, and GEC value significantly decreased, while GDP and GMP concentrations progressively increased with age. Conclusion: Such a profile of change suggests a deterioration of the GTP-related regulatory function in older individuals. Our study explicitly shows that lifelong sports participation, especially of sprint-oriented nature, allows for maintaining a higher erythrocyte guanylate pool concentration, supporting cells' energy metabolism, regulatory and transcription properties, and thus more efficient overall body functioning.

LncRNA HOTAIR enhances RCC2 to accelerate cervical cancer progression by sponging miR-331-3p

Purpose Long noncoding RNAs (lncRNAs) have been gradually regarded as influential indicators of various cancers. The present study aimed to identify the effects of lncRNA HOTAIR on cervical cancer progression. Methods RNA and protein expressions were quantified by RT-qPCR and western blot assays. Fluorescence in situ hybridization (FISH) assay was carried out to examine the intracellular location of HOTAIR. Cancer cell viability and mobility were detected by CCK-8, colony formation, transwell and wound healing assays. Binding relationships between miR-331-3p and HOTAIR/RCC2 were validated by luciferase reporter assay. Results RT-qPCR assays showed that HOTAIR levels were notably upregulated in cervical cancer tissues and cell lines. Furthermore, a fluorescence in situ hybridization (FISH) assay suggested that HOTAIR was mostly located in the cytoplasm of cancer cells, indicating a sponging function. CCK-8, colony formation, Transwell and wound-healing assays indicated that knockdown of HOTAIR in HeLa and SiHa cells significantly reduced cell growth, migration and invasion. Subsequently, miR-331-3p was proven to be the target molecule of HOTAIR. In addition, results from Pearson's correlation analysis indicated negative correlation between HOTAIR and miR-331-3p in cervical cancer tissues. HOTAIR negatively modulated miR-331-3p expression. Ultimately, the target gene of miR-331-3p was verified to be RCC2, and miR-331-3p negatively modulated RCC2 expression. In addition, analysis on clinical cervical cancer tissues confirmed the negative correlation between miR-331-3p and RCC2. HOTAIR and RCC2 showed oncogenic functions in HeLa and SiHa cells, while miR-331-3p exerted the reverse effect. Conclusions HOTAIR plays a carcinogenic role in cervical cancer by targeting the miR-331-3p/RCC2 axis (AU)

LncRNA UCA1 Participates in De Novo Synthesis of Guanine Nucleotides in Bladder Cancer by Recruiting TWIST1 to Increase IMPDH1/2.

Metabolic dysregulation has been identified as one of the hallmarks of cancer biology. Based on metabolic heterogeneity between bladder cancer tissues and adjacent tissues, we discovered several potential driving factors for the bladder cancer occurrence and development. Metabolic genomics showed purine metabolism pathway was mainly accumulated in bladder cancer. Long noncoding RNA urothelial carcinoma-associated 1 (LncRNA UCA1) is a potential tumor biomarker for bladder cancer diagnosis and prognosis, and it increases bladder cancer cell proliferation, migration, and invasion via the glycolysis pathway. However, whether UCA1 plays a role in purine metabolism in bladder cancer is unknown. Our findings showed that UCA1 could increase the transcription activity of guanine nucleotide de novo synthesis rate limiting enzyme inosine monophosphate dehydrogenase 1 (IMPDH1) and inosine monophosphate dehydrogenase 2 (IMPDH2), triggering in guanine nucleotide metabolic reprogramming. This process was achieved by UCA1 recruiting the transcription factor TWIST1 which binds to the IMPDH1and IMPDH2 promoter region. Increased guanine nucleotide synthesis pathway products stimulate RNA polymerase-dependent production of pre-ribosomal RNA and GTPase activity in bladder cancer cells, hence increasing bladder cancer cell proliferation, migration, and invasion. We have demonstrated that UCA1 regulates IMPDH1/2-mediated guanine nucleotide production via TWIST1, providing additional evidence of metabolic reprogramming.

Azathioprine-induced vanishing bile duct syndrome: The value of early thiopurine metabolism assessment.

About 15% to 28% of patients treated with thiopurines experienced adverse drug reactions, such as haematological and hepatic toxicities. Some of these related to the polymorphic activity of the thiopurine S-methyltransferase (TPMT), the key detoxifying enzyme of thiopurine metabolism. We report here a case of thiopurine-induced ductopenia with a comprehensive pharmacological analysis on thiopurine metabolism. A 34-year-old woman, with a medical history of severe systemic lupus erythematosus with recent introduction of azathioprine therapy, presented with mild fluctuating transaminase blood levels consistent with a hepatocellular pattern, which evolved to a cholestatic pattern over the next weeks. A blood thiopurine metabolite assay revealed low 6-thioguanine nucleotides (6-TGN) level and a dramatically increased 6-methylmercaptopurine ribonucleotides (6-MMPN) level, together with an unfavourable [6-MMPN:6-TGN] metabolite ratio and a high TPMT activity. After a total of about 6 months of thiopurine therapy, a transjugular liver biopsy revealed a ductopenia, and azathioprine discontinuation led to further clinical improvement. In line with previous reports from the literature, our case supports the fact that ductopenia is a rare adverse drug reaction of azathioprine. The mechanism of reaction is unknown but may involve high 6-MMPN blood level, due to unusual thiopurine metabolism (switched metabolism). Early therapeutic drug monitoring with measurement of 6-TGN and 6-MMPN blood levels may help physicians to identify patients at risk of similar duct injury.

Activin Receptor-Like Kinase 3 Directly Couples Gαq (Guanine Nucleotide-Binding Protein Subunit αq)/ Gαq (Guanine Nucleotide-Binding Protein Subunit α11) to Regulate Vascular Contractility.

BACKGROUND: Vascular smooth muscle cell (VSMC) contractility is critical for blood pressure regulation and vascular homeostasis. Identifying the key molecule that maintains VSMC contractility may provide a novel therapeutic target for vascular remodeling. ALK3 (activin receptor-like kinase 3) is a serine/threonine kinase receptor, and deletion of ALK3 causes embryonic lethality. However, little is known about the role of ALK3 in postnatal arterial function and homeostasis. METHODS: We conducted in vivo studies in a tamoxifen-induced postnatal VSMC-specific ALK3 deletion mice suitable for analysis of blood pressure and vascular contractility. Additionally, the role of ALK3 on VSMC was determined using Western blot, collagen-based contraction assay and traction force microscopy. Furthermore, interactome analysis were performed to identify the ALK3-associated proteins and bioluminescence resonance energy transfer assay was used to characterize Gαq activation. RESULTS: ALK3 deficiency in VSMC led to spontaneous hypotension and impaired response to angiotensin II in mice. In vivo and in vitro data revealed that ALK3 deficiency impaired contraction force generation by VSMCs, repressed the expression of contractile proteins, and inhibited the phosphorylation of myosin light chain. Mechanistically, Smad1/5/8 signaling mediated the ALK3-modulated contractile protein expressions but not myosin light chain phosphorylation. Furthermore, interactome analysis revealed that ALK3 directly interacted with and activated Gαq (guanine nucleotide-binding protein subunit αq)/Gα11 (guanine nucleotide-binding protein subunit α11), thereby stimulating myosin light chain phosphorylation and VSMC contraction. CONCLUSIONS: Our study revealed that in addition to canonical Smad1/5/8 signaling, ALK3 modulates VSMC contractility through direct interaction with Gαq/Gα11, and therefore, might serve as a potential target for modulating aortic wall homeostasis.

Staphylococcus aureus MazG hydrolyzes oxidized guanine nucleotides and contributes to oxidative stress resistance.

We previously reported that knockout of the mazG (SA1292) gene decreases Staphylococcus aureus killing activity against silkworms. S. aureus MazG (SaMazG) has a nucleotide pyrophosphatase domain conserved among MazG family proteins, but its biochemical characteristics are unknown. In the present study, we purified recombinant N-terminal His-tagged SaMazG protein and examined its biochemical activity. SaMazG hydrolyzed GTP, UTP, dGTP, and TTP into nucleoside monophosphates. Hydrolytic activity of SaMazG against ATP, CTP, dATP, and dCTP was low or not detected. SaMazG exhibited high hydrolytic activity against 8-oxo-GTP and 8-oxo-dGTP, oxidized guanine nucleotides, with a Vmax/Km ratio more than 15-fold that of GTP. Furthermore, the S. aureus mazG knockout mutant was sensitive to hydrogen peroxide compared with the parent strain. These results suggest that SaMazG is a nucleotide pyrophosphatase hydrolyzing oxidized guanine nucleotides that contributes to the oxidative stress resistance of S. aureus.

Urinary insulin signaling pathway related proteins may serve as potential biomarkers for monitoring diabetes mellitus without hypertension and hyperlipidemia.

The insulin signaling pathway plays an important role in the development of diabetes mellitus. The expression of insulin signaling pathway related proteins in the urine of diabetic patients has not been reported. The aim of this study was to analyze and verify the expression of insulin signaling pathway related proteins in the urine of diabetic patients without hypertension and hyperlipidemia, and to explore their clinical application value. Based on data-independent acquisition proteomics technology and bioinformatics, the urinary protein expression profile of diabetic patients without hypertension and hyperlipidemia was established. Western blot and enzyme-linked immunoassay were performed to verify the expression of insulin signaling pathway related proteins in the urine of diabetic patients. Sixteen proteins related to the insulin signaling pathway were screened in urine, and 7 of them were differentially expressed in the urine of diabetic patients without hypertension and hyperlipidemia. Further quantitative analysis showed that the downregulation of protein kinase CAMP-dependent type II regulatory subunit α, growth factor receptor bound protein 2, and guanine nucleotide-binding protein G(s) in the urine of diabetic patients without hyperlipidemia and hypertension was consistent with the preliminary screening results. In this exploratory study, we detected the expression of insulin signaling pathway related proteins in the urine of diabetic patients without hypertension and hyperlipidemia. protein kinase CAMP-dependent type II regulatory subunit α, growth factor receptor bound protein 2, and guanine nucleotide-binding protein G(s) in the urine of diabetic patients were downregulated, which was associated with diabetes. They may be promising noninvasive biomarkers for monitoring diabetes.

A RhoA structure with switch II flipped outward revealed the conformational dynamics of switch II region.

Small GTPase RhoA switches from GTP-bound state to GDP-bound state by hydrolyzing GTP, which is accelerated by GTPases activating proteins (GAPs). However, less study of RhoA structural dynamic changes was conducted during this process, which is essential for understanding the molecular mechanism of GAP dissociation. Here, we solved a RhoA structure in GDP-bound state with switch II flipped outward. Because lacking the intermolecular interactions with guanine nucleotide, we proposed this conformation of RhoA could be an intermediate after GAP dissociation. Further molecular dynamics simulations found the conformational changes of switch regions are indeed existing in RhoA and involved in the regulation of GAP dissociation and GEF recognition. Besides, the guanine nucleotide binding pocket extended to switch II region, indicating a potential "druggable" cavity for RhoA. Taken together, our study provides a deeper understanding of the dynamic properties of RhoA switch regions and highlights the direction for future drug development.

A Focused Review of Ras Guanine Nucleotide-Releasing Protein 1 in Immune Cells and Cancer.

Four Ras guanine nucleotide-releasing proteins (RasGRP1 through 4) belong to the family of guanine nucleotide exchange factors (GEFs). RasGRPs catalyze the release of GDP from small GTPases Ras and Rap and facilitate their transition from an inactive GDP-bound to an active GTP-bound state. Thus, they regulate critical cellular responses via many downstream GTPase effectors. Similar to other RasGRPs, the catalytic module of RasGRP1 is composed of the Ras exchange motif (REM) and Cdc25 domain, and the EF hands and C1 domain contribute to its cellular localization and regulation. RasGRP1 can be activated by a diacylglycerol (DAG)-mediated membrane recruitment and protein kinase C (PKC)-mediated phosphorylation. RasGRP1 acts downstream of the T cell receptor (TCR), B cell receptors (BCR), and pre-TCR, and plays an important role in the thymocyte maturation and function of peripheral T cells, B cells, NK cells, mast cells, and neutrophils. The dysregulation of RasGRP1 is known to contribute to numerous disorders that range from autoimmune and inflammatory diseases and schizophrenia to neoplasia. Given its position at the crossroad of cell development, inflammation, and cancer, RASGRP1 has garnered interest from numerous disciplines. In this review, we outline the structure, function, and regulation of RasGRP1 and focus on the existing knowledge of the role of RasGRP1 in leukemia and other cancers.

Guanine nucleotide-binding protein 2, GNBP2, accelerates the progression of clear cell renal cell carcinoma via regulation of STAT3 signaling transduction pathway.

BACKGROUND: Guanine nucleotide-binding protein 2 (GNBP2) is a GTPase that has critical roles in host immunity and some types of cancer, but its function in clear cell renal cell carcinoma (ccRCC) is not fully understood. OBJECTIVE: This work explored the role of GNBP2 in ccRCC progression and the underlying molecular mechanism. METHODS: Two public human cancer databases TNMplot and TISIDB were employed to analyze the expression pattern of GNBP2 during ccRCC progression and the correlation between GNBP2 expression and clinical features of ccRCC patients. GNBP2 functions in ccRCC cells were determined by EdU staining, flow cytometry, scratch wound assay, transwell assay, and xenograft model. Gene expression was evaluated using qPCR, Western blot, immunofluorescence staining, and immunohistochemical staining. RESULTS: GNBP2 expression was significantly elevated in ccRCC tissues and increased gradually with the increasing tumor grades. Patients with higher GNBP2 expression had shorter overall survival times. Knockdown of GNBP2 suppressed tumor cell proliferation and cell cycle progression and reduced the capability of migration and invasion, while GNBP2 overexpression exhibited protumor effects. GNBP2 silencing by RNA interference significantly inhibited the tumor growth of tumor-bearing nude mice and decreased the proliferation marker Ki67. Mechanistically, GNBP2 downregulation suppressed the STAT3 signaling transduction, as it reduced the phosphorylation of STAT3 and modulated the expression of the target genes, including c-Myc, MMP2, N-cadherin, and E-cadherin. CONCLUSION: These findings reveal that GNBP2 promotes ccRCC progression by regulating STAT3 signaling transduction, indicating that GNBP2 might be a promising molecular target for ccRCC therapy.

A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.

The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

[GNB2L1 gene expression and clinical value in hepatocellular carcinoma based on bioinformatics].

Objective: To analyze guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) expression based on bioinformatics, so as to evaluate its role and its relationship with survival rate during the occurrence and development of hepatocellular carcinoma. Methods: GEPIA, UALCAN and HPA databases were used to analyze the expression level of GNB2L1 and its relationship with HCC survival rate. Mutations in the GNB2L1 gene and their impact on survival were analyzed using the cBioPortal database. LinkedOmics database was used to analyze GNB2L1-related genes in HCC. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed simultaneously. STEING database was used to construct the GNB2L1 protein interaction network. TIMER database was used to analyze the relationship between GNB2L1 gene expression and immune infiltration in hepatocellular carcinoma. Differential expression of GNB2L1 in plasma platelets of HCC patients and healthy controls was analyzed using mRNA-based sequencing technology. Data between groups were compared using an independent-samples t-test. Results: GNB2L1 expression level was significantly increased in HCC tissues (P<0.05), and its expression was significantly correlated with body weight, classification and stage (P<0.05). The overall survival rate was higher in GNB2L1 low expression group (P<0.001). GNB2L1 and its related genes were related to biological process regulation, metabolic process, protein binding, oxidative phosphorylation, JAK-STAT signaling pathway, Ras signaling pathway and so on. GNB2L1 had interaction with RPS12, RPS11 and RPL19, and participated in multiple biological processes such as liver regeneration and positive regulation of endogenous apoptotic signaling pathway. GNB2L1 expression was significantly positively correlated with the infiltration degree of various immune cells in HCC (P<0.05). Cox regression analysis showed that GNB2L1 was an independent risk factor for lower survival rate in patients with HCC [Hazard ratio (95% confidence interval)=1.456 (1.034~2.051), P=0.031]. GNB2L1expression levels were significantly higher in platelets of HCC patients than that of healthy controls (10.40±1.36 vs. 9.58±0.51, t=2.194, P=0.037). Conclusion: GNB2L1 has high expression and close relationship to survival rate in HCC. Therefore, GNB2L1 may be a potential biomarker of HCC.

Common variants in GNL3 gene contributed the susceptibility of hand osteoarthritis in Han Chinese population.

Osteoarthritis (OA) is one of the most popular degenerative joint diseases. The nucleolar GTP binding protein 3 (GNL3) gene encodes guanine nucleotide binding protein-like 3, which is related in cell proliferation, differentiation, and cell cycle regulation. Our study aimed to examine the contribution of GNL3 gene polymorphisms to the risk of hand OA and its related clinical features. A total of 3387 study participants including 1160 patients with hand OA and 2227 controls were recruited in this study. Eleven SNPs in GNL3 gene were selected for genotyping. Genetic association signals were examined using Plink. Relationships between significant SNPs and clinical features of hand OA were also explored. SNP rs11177 was found to be strongly associated with susceptibility of hand OA (P = 4.32 × 10-5). The minor allele of rs11177 was associated with increased susceptibility of hand OA. In addition, significant associations were also identified between genotypes of rs11177 and clinical features of hand OA patients including K-L grade (P < 0.01) and categorized pain scores (P < 0.01). Significant eQTL signals for rs11177 on GNL3 in multiple types of human tissues were also identified in GTEx database. Our results have established the link between GNL3 gene and susceptibility of hand OA.

State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides.

The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.