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Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5-[(β,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

The 2D geological map serves as a synthesis of geological investigations and expert knowledge, making it a crucial data source for bedrock 3D modeling. Nevertheless, insufficient geological information mining has been a problem in previous research utilizing 2D geological maps for bedrock modeling. To address this issue, this paper proposes a 3D bedrock modeling method that incorporates multiple information mining based on 2D geological maps. The method involves extracting surface undulation, occurrence, stratigraphic age, and other information from the 2D geological map multiple times using map-cut cross sections and virtual drills. This information is then stored using the generalized trigonal prismatic (GTP) element. Additionally, the paper introduces connection rules to handle different geological phenomena, such as stratigraphic pinch-out, stratigraphic inversion, stratigraphic duplication, and unconformity contact, and their corresponding GTP types. Finally, the GTPs are connected according to these rules, resulting in the construction of a 3D bedrock model. To validate the method's consistency with expert speculation regarding the expression of geological body structures and occurrence, the paper compares a slice section of the example modeling results with expert hand-drawn section from the same location. The results demonstrate that the proposed modeling method effectively explores the geological information present in the planar geological map and clearly expresses a variety of complex geological formations, enabling the construction of a high-quality 3D bedrock model. [ABSTRACT FROM AUTHOR]

Increased interest in the aging and Alzheimer's disease (AD)-related impairments in autophagy in the brain raise important questions about regulation and treatment. Since many steps in endocytosis and autophagy depend on GTPases, new measures of cellular GTP levels are needed to evaluate energy regulation in aging and AD. The recent development of ratiometric GTP sensors (GEVALS) and findings that GTP levels are not homogenous inside cells raise new issues of regulation of GTPases by the local availability of GTP. In this review, we highlight the metabolism of GTP in relation to the Rab GTPases involved in formation of early endosomes, late endosomes, and lysosomal transport to execute the autophagic degradation of damaged cargo. Specific GTPases control macroautophagy (mitophagy), microautophagy, and chaperone-mediated autophagy (CMA). By inference, local GTP levels would control autophagy, if not in excess. Additional levels of control are imposed by the redox state of the cell, including thioredoxin involvement. Throughout this review, we emphasize the age-related changes that could contribute to deficits in GTP and AD. We conclude with prospects for boosting GTP levels and reversing age-related oxidative redox shift to restore autophagy. Therefore, GTP levels could regulate the numerous GTPases involved in endocytosis, autophagy, and vesicular trafficking. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs. [ABSTRACT FROM AUTHOR]

Abstract Background Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. Methods The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. Results The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. Conclusion RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

In eukaryotes, single cells in a population display different transcriptional profiles. One of the factors regulating this heterogeneity is the chromatin state in each cell. However, the mechanisms of epigenetic chromatin regulation of specific chromosomal regions remain unclear. Therefore, we used single‐cell tracking system to analyze IMD2. IMD2 is located at the subtelomeric region of budding yeast, and its expression is epigenetically regulated by heterochromatin fluctuations. Treatment with mycophenolic acid, an inhibitor of de novo GTP biosynthesis, triggered a decrease in GTP, which caused heterochromatin fluctuations at the IMD2 locus. Interestingly, within individually tracked cells, IMD2 expression state underwent repeated switches even though IMD2 is positioned within the heterochromatin region. We also found that 30% of the cells in a population always expressed IMD2. Furthermore, the addition of nicotinamide, a histone deacetylase inhibitor, or guanine, the GTP biosynthesis factor in salvage pathway of GTP biosynthesis, regulated heterogeneity, resulting in IMD2 expression being uniformly induced or suppressed in the population. These results suggest that gene expression heterogeneity in the IMD2 region is regulated by changes in chromatin structure triggered by slight decreases in GTP. [ABSTRACT FROM AUTHOR]

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Metastatic progression is regulated by metastasis promoter and suppressor genes. NME1, the prototypic and first described metastasis suppressor gene, encodes a nucleoside diphosphate kinase (NDPK) involved in nucleotide metabolism; two related family members, NME2 and NME4, are also reported as metastasis suppressors. These proteins physically interact with members of the GTPase dynamin family, which have key functions in membrane fission and fusion reactions necessary for endocytosis and mitochondrial dynamics. Evidence supports a model in which NDPKs provide GTP to dynamins to maintain a high local GTP concentration for optimal dynamin function. NME1 and NME2 are cytosolic enzymes that provide GTP to dynamins at the plasma membrane, which drive endocytosis, suggesting that these NMEs are necessary to attenuate signaling by receptors on the cell surface. Disruption of NDPK activity in NME-deficient tumors may thus drive metastasis by prolonging signaling. NME4 is a mitochondrial enzyme that interacts with the dynamin OPA1 at the mitochondria inner membrane to drive inner membrane fusion and maintain a fused mitochondrial network. This function is consistent with the current view that mitochondrial fusion inhibits the metastatic potential of tumor cells whereas mitochondrial fission promotes metastasis progression. The roles of NME family members in dynamin-mediated endocytosis and mitochondrial dynamics and the intimate link between these processes and metastasis provide a new framework to understand the metastasis suppressor functions of NME proteins. [ABSTRACT FROM AUTHOR]

Barbara Pospieszna,1 Krzysztof Kusy,1 Ewa Maria Slominska,2 Monika Ciekot-Sołtysiak,1 Jacek Zieliński1 1Department of Athletics, Strength and Conditioning, Poznan University of Physical Education, Poznan, Poland; 2Department of Biochemistry, Medical University of Gdansk, Gdansk, PolandCorrespondence: Barbara Pospieszna, Department of Athletics, Strength and Conditioning, Poznan University of Physical Education, Królowej Jadwigi 27/39, Poznan, 61-871, Poland, Tel +48-618-355-270, Email bpospieszna@awf.poznan.plIntroduction: 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.Keywords: guanosine triphosphate, GTP, guanylate energy charge, GEC, purine metabolites, endurance, sprint, master athletes

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death. Drug therapy for breast cancer is currently selected based on the subtype classification; however, many anticancer drugs are highly cytotoxic. Since intracellular levels of GTP are elevated in many cancer cells that undergo a specific cell proliferation cycle, GTP has potential as a target for cancer therapy. The present study focused on nucleosides and nucleotides and examined intracellular GTP-dependent changes in cell proliferation rates in normal (MCF-12A) and cancer (MCF-7) breast cell lines. Decreased cell proliferation due to a reduction in intracellular GTP levels by mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, was observed in both cell lines. The inhibitory effects of MPA on cell proliferation were suppressed when it was applied in combination with Guanosine (Guo), a substrate for GTP salvage synthesis, while the single exposure to Guo suppressed the proliferation of MCF-7 cells only. Although the underlying mechanisms remain unclear, since the inhibitory effects of Guo on cell proliferation did not correlate with GTP or ATP intracellular levels or the GTP/ATP ratio, there may be another cause besides GTP metabolism. Guo inhibited the proliferation of MCF-7, a human breast cancer cell line, but not MCF-12A, a human normal breast cell line. Further studies are needed to investigate the potential of applying Guo as a target for the development of a novel cancer treatment system. • Guanosine inhibits breast cancer cell proliferation only. • The inhibitory effect of guanosine on cell proliferation is counteracted by the coexistence of mycophenolic acid. • Guanosine has potential to be a novel target for cancer therapy. [ABSTRACT FROM AUTHOR]

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high‐order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5‐phosphate 4‐kinase β (PI5P4Kβ) evolved into a GTP sensor from ATP‐utilizing kinase. Mechanistically, PI5P4Kβ has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP‐dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations—the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kβ is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell. [ABSTRACT FROM AUTHOR]

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Background: The risk of military and civilian radiation exposure is increasing, and determining the effects of exposure is a high priority. Irradiation of the nearby blood supply after a nuclear event may impede mobilization of blood products for resuscitation at a time of great need. RBCs are administered to patients with trauma and hemorrhage to transport and deliver oxygen and avoid tissue hypoxia. Here we determine the effects of ionizing radiation on the energy metabolome of RBCs isolated from cold stored whole blood to determine if their stability is compromised by radiation exposure. Study Design and Methods: Whole blood from healthy volunteers was subjected to 0, 25, or 75 Gy of X‐irradiation, and stored at 4°C. RBCs were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. The levels of extracted Krebs cycle intermediates, nicotinamide adenine dinucleotides, and phosphorylated derivatives of adenosine and guanosine were determined by tandem mass spectroscopy. Results: Irradiation at either 25Gy or 75Gy had no significant effect on any parameter measured compared to control (0Gy). However, there was a significant change over time in storage for ATP, GDP, and guanosine. Discussion: Irradiation at doses up to 75Gy had no effect on the energy metabolome of RBCs prepared from blood stored at 4°C for up to 21 days, suggesting that the RBC energy metabolome is not affected by radiation exposure and the blood can still be used for resuscitation in trauma patients. [ABSTRACT FROM AUTHOR]

Objective: This study investigated the impacts of SIRT1 activation on rheumatoid arthritis (RA)-related angiogenesis. Methods: HUVECs were cultured by different human serum. Intracellular metabolites were quantified by UPLC-MS. Next, HUVECs and rat vascular epithelial cells under different inflammatory conditions were treated by a SIRT1 agonist resveratrol (RSV). Cytokines and biochemical indicators were detected by corresponding kits. Protein and mRNA expression levels were assessed by immunoblotting and PCR methods, respectively. Angiogenesis capabilities were evaluated by migration, wound-healing and tube-formation experiments. To down-regulate certain signals, gene-specific siRNA were applied. Results: Metabolomics study revealed the accelerated glycolysis in RA serum-treated HUVECs. It led to ATP accumulation, but did not affect GTP levels. RSV inhibited pro-angiogenesis cytokines production and glycolysis in both the cells, and impaired the angiogenesis potentials. These effects were mimicked by an energy metabolism interrupter bikini in lipopolysaccharide (LPS)-primed HUVECs, largely independent of HIF-1α. Both RSV and bikinin can inhibit the activation of the GTP-dependent pathway Rho/ROCK and reduce VEGF production. Abrogation of RhoA signaling reinforced HIF-1α silencing-brought changes in LPS-stimulated HUVECs, and overshadowed the anti-angiogenesis potentials of RSV. Conclusion: Glycolysis provides additional energy to sustain Rho/ROCK activation in RA subjects, which promotes VEGF-driven angiogenesis and can be inhibited by SIRT1 activation. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the N-ϵ-(γ-glutamyl) lysine bonds between side chains of glutamine and lysine residues resulting in proteolytically resistant cross-links. Increased TG2 activity and levels are involved in the pathophysiology of various diseases, including liver injury, cystic fibrosis, celiac sprue, metastatic cancers, and several neurodegenerative conditions. Inhibiting TG2 activity is considered a potential strategy to combat these diseases. Although guanine nucleotide (GTP) could inhibit TG2, its inhibitory activity decreased with increased calcium concentration. Search for GTP analogs that could strongly bind and inhibit TG2 activity is intense. This study screened the PubChem database for about two thousand GTP-like compounds for TG2. Using docking and molecular dynamics simulations we identified three compounds (C4959, C4215, and C9560) that could selectively interact with TG2. These three compounds have less affinity for several other intracellular and extracellular GTP-binding proteins suggesting selectivity for TG2. Interestingly, C9560 showed stronger interactions and better binding energy with TG2 than C4959 and C4215, suggesting that C9560 can form a more stable complex with TG2. Our study indicates that C9560, a GTP analog, could be exploited as a promising candidate to inhibit TG2-mediated fibrotic conditions. [ABSTRACT FROM AUTHOR]

Probing binding modes of GDP, GTP and GNP to NRAS are of significance for understanding the regulation mechanism on the activity of RAS proteins. Four separate Gaussian accelerated molecular dynamics (GaMD) simulations were performed on the apo, GDP-, GTP- and GNP-bound NRAS. Dynamics analyses suggest that binding of three ligands highly affects conformational states of the switch domains from NRAS, which disturbs binding of NRAS to its effectors. The analyses of free energy landscapes (FELs) indicate that binding of GDP, GTP and GNP induces more energetic states of NRAS compared to the apo NRAS but the presence of GNP makes the switch domains more ordered than binding of GDP and GNP. The information of interaction networks of ligands with NRAS reveals that the π-π interaction of residue F28 and the salt bridge interactions of K16 and D119 with ligands stabilize binding of GDP, GTP and GNP to NRAS. Meanwhile magnesium ion plays a bridge role in interactions of ligands with NRAS, which is favourable for associations of GDP, GTP and GNP with NRAS. This work is expected to provide useful information for deeply understanding the function and activity of NRAS. [ABSTRACT FROM AUTHOR]

Background: Metabolic reprogramming is a hallmark of cancer. Metabolic rate‐limiting enzymes and oncogenic c‐Myc (Myc) play critical roles in metabolic reprogramming to affect tumourigenesis. However, a systematic assessment of metabolic rate‐limiting enzymes and their relationship with Myc in human cancers is lacking. Methods: Multiple Pan‐cancer datasets were used to develop the transcriptome, genomic alterations, clinical outcomes and Myc correlation landscapes of 168 metabolic rate‐limiting enzymes across 20 cancers. Real‐time quantitative PCR and immunoblotting were, respectively, used to examine the mRNA and protein of inosine monophosphate dehydrogenase 1 (IMPDH1) in human colorectal cancer (CRC), azoxymethane/dextran sulphate sodium‐induced mouse CRC and spontaneous intestinal tumours from APCMin/+ mice. Clone formation, CCK‐8 and subcutaneous xenograft model were applied to investigate the possible mechanisms connecting IMPDH1 to CRC growth. Co‐immunoprecipitation and protein half‐life assay were used to explore the mechanisms underlying the regulation of IMPDH1. Results: We explored the global expression patterns, dysregulation profiles, genomic alterations and clinical relevance of 168 metabolic rate‐limiting enzymes across human cancers. Importantly, a series of enzymes were associated with Myc, especially top three upregulated enzymes (TK1, RRM2 and IMPDH1) were positively correlated with Myc in multiple cancers. As a proof‐of‐concept exemplification, we demonstrated that IMPDH1, a rate‐limiting enzyme in GTP biosynthesis, is highly upregulated in CRC and promotes CRC growth in vitro and in vivo. Mechanistically, IMPDH2 stabilizes IMPDH1 by decreasing the polyubiquitination levels of IMPDH1, and Myc promotes the de novo GTP biosynthesis by the transcriptional activation of IMPDH1/2. Finally, we confirmed that the Myc–IMPDH1/2 axis is dysregulated across human cancers. Conclusions: Our study highlights the essential roles of metabolic rate‐limiting enzymes in tumourigenesis and their crosstalk with Myc, and the Myc–IMPDH1/2 axis promotes tumourigenesis by altering GTP metabolic reprogramming. Our results propose the inhibition of IMPDH1 as a viable option for cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract Antibiotics combat bacteria through their bacteriostatic (by growth inhibition) or bactericidal (by killing bacteria) action. Mechanistically, it has been proposed that bactericidal antibiotics trigger cellular damage, while bacteriostatic antibiotics suppress cellular metabolism. Here, we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic‐induced bacterial protective response: the stringent response. Chloramphenicol targets the ribosome to inhibit the growth of the Gram‐positive bacterium Bacillus subtilis. Intriguingly, we found that chloramphenicol becomes bactericidal in B. subtilis mutants unable to produce (p)ppGpp. We observed a similar (p)ppGpp‐dependent bactericidal effect of chloramphenicol in the Gram‐positive pathogen Enterococcus faecalis. In B. subtilis, chloramphenicol treatment induces (p)ppGpp accumulation through the action of the (p)ppGpp synthetase RelA. (p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action. This GTP regulation is critical for preventing chloramphenicol from killing B. subtilis, as bypassing (p)ppGpp‐dependent GTP regulation potentiates chloramphenicol killing, while reducing GTP synthesis increases survival. Finally, chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin, reminiscent of the clinical phenomenon of antibiotic antagonism. Taken together, our findings suggest a role of (p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram‐positive bacteria, which can be exploited to potentiate the efficacy of existing antibiotics.

Green tea polyphenols (GTP) have been shown to ameliorate lipid metabolic disorders by regulating intestinal bacteria. Given the significant role of intestinal bacteriophages in shaping the gut microbiota, this study investigates GTP's influence on gut bacteriophage-bacteria interactions and lipid metabolism using metagenomics and metabonomics. The research results indicated that GTP significantly reduced body weight, serum triglycerides, leptin, insulin resistance, interleukin-6, and TNF-α levels while increasing adiponectin in ob/ob mice fed high-fat diet, aiding intestinal repair. GTP improved gut health by decreasing Enterobacter , Siphoviridae and Enterobacteria _phage_sfv, increasing Bifidobacterium and intestinal metabolites SCFA and hippuric acid. Correlation analysis showed negative correlations between Enterobacter sp. 50,588,862 and Enterobacteria _phages, Shigella _phages with 4-hydroxyphenylpyruvate and hippuric acid. Bifidobacterium choerinum and Bifidobacterium sp. AGR2158 were positively correlated with fatty acids and bile acids. In conclusion, GTP reduced fat accumulation and inflammation, enhanced gut barrier function in obese mice, closely associated with changes in the gut bacteriophage community. [Display omitted] • GTP's impact on gut bacteria and phages, altering lipid metabolism in ob/ob mice. • Used bioinformatics to link gut health biomarkers with differential microorganism. • Found potential targets for treating metabolic issues in obesity. [ABSTRACT FROM AUTHOR]

Abstract Background Metabolic reprogramming is a hallmark of cancer. Metabolic rate‐limiting enzymes and oncogenic c‐Myc (Myc) play critical roles in metabolic reprogramming to affect tumourigenesis. However, a systematic assessment of metabolic rate‐limiting enzymes and their relationship with Myc in human cancers is lacking. Methods Multiple Pan‐cancer datasets were used to develop the transcriptome, genomic alterations, clinical outcomes and Myc correlation landscapes of 168 metabolic rate‐limiting enzymes across 20 cancers. Real‐time quantitative PCR and immunoblotting were, respectively, used to examine the mRNA and protein of inosine monophosphate dehydrogenase 1 (IMPDH1) in human colorectal cancer (CRC), azoxymethane/dextran sulphate sodium‐induced mouse CRC and spontaneous intestinal tumours from APCMin/+ mice. Clone formation, CCK‐8 and subcutaneous xenograft model were applied to investigate the possible mechanisms connecting IMPDH1 to CRC growth. Co‐immunoprecipitation and protein half‐life assay were used to explore the mechanisms underlying the regulation of IMPDH1. Results We explored the global expression patterns, dysregulation profiles, genomic alterations and clinical relevance of 168 metabolic rate‐limiting enzymes across human cancers. Importantly, a series of enzymes were associated with Myc, especially top three upregulated enzymes (TK1, RRM2 and IMPDH1) were positively correlated with Myc in multiple cancers. As a proof‐of‐concept exemplification, we demonstrated that IMPDH1, a rate‐limiting enzyme in GTP biosynthesis, is highly upregulated in CRC and promotes CRC growth in vitro and in vivo. Mechanistically, IMPDH2 stabilizes IMPDH1 by decreasing the polyubiquitination levels of IMPDH1, and Myc promotes the de novo GTP biosynthesis by the transcriptional activation of IMPDH1/2. Finally, we confirmed that the Myc–IMPDH1/2 axis is dysregulated across human cancers. Conclusions Our study highlights the essential roles of metabolic rate‐limiting enzymes in tumourigenesis and their crosstalk with Myc, and the Myc–IMPDH1/2 axis promotes tumourigenesis by altering GTP metabolic reprogramming. Our results propose the inhibition of IMPDH1 as a viable option for cancer treatment.

Aim: This study aims to build the capacity of the people at grass root level as gatekeepers of mental health. It will assess the effectiveness of the Gatekeeper Training Program (GTP) on gatekeeper behaviour, awareness, attitude, and mental help seeking intention. Design: An evaluative research approach in two phases. Phase 1: Cross-sectional house-to-house exploratory survey. Phase 2: A quasi-experimental design with multiple follow ups at 0, 6 and 12 months. Method: Data will be collected using standardized tools like Mental Health Knowledge Questionnaire (MHKQ), Community Attitude towards Mentally Ill (CAMIS), Mental Help Seeking Intention (MHSIS) and Gatekeeper Behavior Scale (GBS). For Phase 1, a house-to-house survey will be conducted among the selected colonies of Koraga tribe to determine their awareness, attitude, and mental help seeking intention regarding common mental health problems. Phase 2 includes identification of the leaders/representatives of the selected tribal colonies, and involving them in GTP. Pre-test and multiple post-test will be conducted in Phase 2 at 0, 6, 12 months. The study is funded by Indian Council of Medical Research from 16 August 2021 for 3 years duration. Discussion: Treatment gap in psychiatric disorders remains an issue of great concern. Evidence based research promotes task shifting approaches in dealing with mental health problems in the community. Capacity building programs like GTP for the underprivileged section of the society are important especially in low and middle income group of countries. Impact: This need based GTP, will ensure mental health first aid in the society. Early identification of people with mental health problems at their doorsteps has huge impact on the prognosis of the illness, closing the treatment gap and stigma reduction.

Daunorubicin and doxorubicin are among the most potent anti-cancer drugs and bind to DNA through intercalation. In this paper, we demonstrate that formaldehyde can efficiently and specifically conjugate daunorubicin and doxorubicin to GTP, resulting in the formation of daunorubicin-GTP-1 and doxorubicin-GTP-1 conjugates. The linkage occurs between the 2-NH 2 of guanine and the 3'-NH 2 of daunosamine. We characterized these daunorubicin/doxorubicin-GTP conjugates using various methods, including UV-Vis, fluorescence, CD, FT-IR, and mass spectrometry. Our results also indicate that these daunorubicin/doxorubicin-GTP conjugates bind to DNA via intercalation. Furthermore, we observed rapid accumulation of these conjugates in human cancer cells and observed cytotoxic effects in both doxorubicin-sensitive SK-OV-3 and doxorubicin-resistant NCI/ADR-RES cells, suggesting that these daunorubicin and doxorubicin derivatives can overcome doxorubicin resistance., (© 2024 Wiley-VCH GmbH.)

Guanine nucleotide-binding (G) proteins, namely, phosphate-binding (P) loop GTPases, play a critical role in life processes among different species. Based on the structural characteristics, G proteins can be divided into heterotrimeric G proteins, small G proteins and multiple unique unconventional G proteins. The highly conserved unconventional G protein YchF is composed of a core G domain, an inserted coiled-coil domain, and a TGS domain from the N-terminus to the C-terminus. In this review, we compared the structural characteristics of the G domain in rice OsYchF1 with those of Rattus norvegicus heterotrimeric G protein α-subunit and human small G protein Ras-related G protein C and analyzed the binding modes of these G proteins with GTP or ATP by performing molecular dynamics simulations. In summary, it will provide new insights into the enormous diversity of biological function of G proteins.

Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets. GTP is essential for nuclear acid biosynthesis. In addition, through its effects on G proteins, GTP regulates a large array of cellular processes including protein synthesis, cytoskeleton biogenesis and function, and intracellular signaling. The development of genetically encoded GTP sensors has led to the realization that GTP molecules are not homogenously distributed in the cell but rather form gradients which regulate cellular processes. De novo biosynthesis acts as the primary source of GTP in most cell types. It is regulated via both transcriptional and post-translational mechanisms that are far from being completely elucidated. Inhibitors of the de novo GTP biosynthesis are used in organ transplantation as immunosuppressive drugs and in antiviral therapy. Although targeting GTP biosynthesis is an attractive anticancer strategy, no GTP-depleting agents are approved for cancer treatment. [ABSTRACT FROM AUTHOR]

RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies. [Display omitted] • The first structure of the activated GTP binding RhoA-nanobody complex is resolved. • The underlying mechanism of selective binding of Rh57 to active RhoA-GTP but not RhoA-GDP is explained. • The possible mechanism of Rh57-RhoA binding while not interfering with the downstream PRK1 signaling pathway is explained. [ABSTRACT FROM AUTHOR]

The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix. [ABSTRACT FROM AUTHOR]

GTP is an important signaling molecule involved in the growth, development, and stress adaptability of plants. The functions are mediated via binding to GTPases which are in turn regulated by GTPase-activating proteins (GAPs). Satellite reports have suggested the positive roles of GAPs in regulating ABA signaling and pathogen resistance in plants. However, the molecular mechanisms that bring forth the pathogen resistance have remained unclear. In this study, we demonstrated that the expression of AtGAP1 was inducible by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The overexpression of AtGAP1 in Arabidopsis promoted the expression of PR1 and the resistance to Pst DC3000. Proteomic analyses revealed the enhanced accumulation of cell-wall-modifying proteins as a result of AtGAP1 overexpression. By microscopic analyses, we showed that the overexpression of AtGAP1 resulted in increased thickness of the mesophyll cell wall and reduced stomatal aperture, which are effective strategies for restricting the entry of foliar pathogens. Altogether, we demonstrated that AtGAP1 increases the resistance to Pst DC3000 in Arabidopsis by promoting cellular strategies that restrict the entry of pathogens into the cells. These results point to a future direction for studying the modes of action of GAPs in regulating plant cell structures and disease resistance. [ABSTRACT FROM AUTHOR]

Obg proteins belong to P‐loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound "on" state and guanine diphosphate (GDP)‐bound "off" state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine‐rich N‐terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C‐terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg‐like proteins can provide a link between different global pathways that are necessary for fine‐tuning cellular processes to maintain the cellular energy status. [ABSTRACT FROM AUTHOR]

Over the last decade, cancer immunotherapy has progressed from an academically interesting field to one of the most promising forms of new treatments in which not the cancer but the immune system is treated. In particular, genetic modification for purposeful redirection of autologous T cells is providing hope to many treatment-resistant patients. This personalized form of medicine is radically different from more traditional oncologic drugs. With these evolving medical advancements and more cellular therapies becoming available, some regulatory agencies have created new regulatory requirements to manage the production of these types of products. The regulations are specifically suited for the manufacture of gene and cell therapy products, as they use a risk-based approach towards product development and manufacturing, when there is limited characterization available. The correct interpretation of how and when requirements apply is crucial, since theoretical approaches to implementing GMP can easily lead to disproportionate and unwarranted restrictions that may not address the specific risks that regulators were intending to control. This is especially relevant for cell collection and biopreservation preceding the manufacturing process for products manufactured from autologous T cells. Both the fresh and cryopreserved apheresis materials can be filed as minimally manipulated starting materials to the authorities. The preservation of such cellular material can then routinely be managed using the available regulations for tissues and cells, allowing for a more fit-for-purpose approach to the control measures implemented. [ABSTRACT FROM AUTHOR]

The nucleoside antibiotic toyocamycin (TM), which is produced by Streptomyces diastatochromogenes 1628, exhibits potent activity against a broad range of phytopathogenic fungi. TM was synthesized through a multi‐step reaction, using guanosine triphosphate (GTP) as precursor. Based on a comparison of proteomics data from S. diastatochromogenes 1628 and rifamycin‐resistant mutant 1628‐T15 with high yield of TM, we determined that the differentially expressed protein X0NBV6 called ribose‐phosphate pyrophosphokinase (RHP), which is a rate‐limiting enzyme involved in the de novo biosynthesis of GTP, exhibits a higher expression level in mutant 1628‐T15. In this study, to elucidate the relationships between RHP, GTP, and TM production, the gene rhpsd encoding RHP was cloned and overexpressed in S. diastatochromogenes strain 1628. The recombinant strain S. diastatochromogenes 1628‐RHP exhibited better performance at the transcriptional level of the rhpsd gene, as well as RHP enzymatic activity, intracellular GTP concentration, and TM production, compared to S. diastatochromogenes 1628. Finally, the yield of TM produced by S. diastatochromogenes 1628‐RHP (340.2 mg/L) was 133.3% higher than that produced by S. diastatochromogenes1628. Moreover, the transcriptional level of toy genes involved in TM biosynthesis was enhanced due to the overexpression of the rhpsd gene. [ABSTRACT FROM AUTHOR]

Context: The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second-most common form of congenital hyperinsulinism, has been associated with dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP. Objective: To identify the underlying genetic etiology in 2 siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation. Methods: The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated. Results: A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26-aa in-frame deletion in the first repeat domain of the protein, which abolishes transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content, which likely leads to a hyperactivation of glutamate dehydrogenase in our patients. Conclusion: We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion. [ABSTRACT FROM AUTHOR]

Severe traumatic brain injury (TBI) is associated with high rates of mortality and long‐term disability linked to neurochemical abnormalities. Although purine derivatives play important roles in TBI pathogenesis in preclinical models, little is known about potential changes in purine levels and their implications in human TBI. We assessed cerebrospinal fluid (CSF) levels of purines in severe TBI patients as potential biomarkers that predict mortality and long‐term dysfunction. This was a cross‐sectional study performed in 17 severe TBI patients (Glasgow Coma Scale <8) and 51 controls. Two to 4 h after admission to ICU, patients were submitted to ventricular drainage and CSF collection for quantification of adenine and guanine purine derivatives by HPLC. TBI patients' survival was followed up to 3 days from admission. A neurofunctional assessment was performed through the modified Rankin Scale (mRS) 2 years after ICU admission. Purine levels were compared between control and TBI patients, and between surviving and non‐surviving patients. Relative to controls, TBI patients presented increased CSF levels of GDP, guanosine, adenosine, inosine, hypoxanthine, and xanthine. Further, GTP, GDP, IMP, and xanthine levels were different between surviving and non‐surviving patients. Among the purines, guanosine was associated with improved mRS (p = 0.042; r = −0.506). Remarkably, GTP displayed predictive value (AUC = 0.841, p = 0.024) for discriminating survival versus non‐survival patients up to 3 days from admission. These results support TBI‐specific purine signatures, suggesting GTP as a promising biomarker of mortality and guanosine as an indicator of long‐term functional disability. [ABSTRACT FROM AUTHOR]

Aim: This study aims to build the capacity of the people at grass root level as gatekeepers of mental health. It will assess the effectiveness of the Gatekeeper Training Program (GTP) on gatekeeper behaviour, awareness, attitude, and mental help seeking intention. Design: An evaluative research approach in two phases. Phase 1: Cross-sectional house-to-house exploratory survey. Phase 2: A quasi-experimental design with multiple follow ups at 0, 6 and 12 months. Method: Data will be collected using standardized tools like Mental Health Knowledge Questionnaire (MHKQ), Community Attitude towards Mentally Ill (CAMIS), Mental Help Seeking Intention (MHSIS) and Gatekeeper Behavior Scale (GBS). For Phase 1, a house-to-house survey will be conducted among the selected colonies of Koraga tribe to determine their awareness, attitude, and mental help seeking intention regarding common mental health problems. Phase 2 includes identification of the leaders/representatives of the selected tribal colonies, and involving them in GTP. Pre-test and multiple post-test will be conducted in Phase 2 at 0, 6, 12 months. The study is funded by Indian Council of Medical Research from 16 August 2021 for 3 years duration. Discussion: Treatment gap in psychiatric disorders remains an issue of great concern. Evidence based research promotes task shifting approaches in dealing with mental health problems in the community. Capacity building programs like GTP for the underprivileged section of the society are important especially in low and middle income group of countries. Impact: This need based GTP, will ensure mental health first aid in the society. Early identification of people with mental health problems at their doorsteps has huge impact on the prognosis of the illness, closing the treatment gap and stigma reduction.

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (Cg Rab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of Cg Rab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of Cg Rab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, Cg Rab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant Cg Rab1 protein (r Cg Rab1) was able to bind GTP. The mRNA transcripts of Cg Rab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus , which was 2.43-fold (p < 0.01) of that in the control group. After the expression of Cg Rab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cg cathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in Cg Rab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that Cg Rab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cg cathepsin L1 in oyster C. gigas. • A Rab1 homologue protein (CgRab1) was identified from oyster Crassostrea gigas. • CgRab1 mRNA expression level in haemocytes was up-regulated after V. splendidus stimulation. • The recombinant protein of CgRab1 was able to bind GTP in vitro. • CgRab1 regulated the expression of CgcathepsinL1 and involved in the phagocytosis of haemocytes. [ABSTRACT FROM AUTHOR]

MfpA protein is encoded by Mycobacterium tuberculosis (Mtb) and stands for Mycobacterium fluoroquinolone resistance protein A. This protein provides Mtb intrinsic resistant property from fluoroquinolone antibiotics by inhibiting DNA gyrase that are known to be the primary target of fluoroquinolone drugs. DNA gyrases are important for bacterial chromosomal genesis as they are majorly involved in DNA replication, transcription, and bacterial stress response to several external stimulus. Therefore, in Mtb, it forms an essential integrity and also a desirable target for drug development approaches. This paper implies on determining the essential facts about mfpA including its interaction study, epitope prediction, modeling, and validation, and most importantly it deals with the mutation. Mutational analysis was carried out on the basis of sequential information and there were several mutations that cause a large decrease in the stability of the protein. Total 24 mutations were shortlisted based on ΔΔG value: W154G, F54G, L84G, F9G, W4G, F74G, F64G, F49G, L104G, L94G, L124G, F29G, L39G, L59G, W60G, L114G, , W154S, L19G, L144G, L129G, F34G, W154D, W154A, and W4S. Separate mutation on DXXG GTPase motif was examined to check any effect on protein stability and we found that D33A, D98A, D128A, G36A, G101A, G131A, D33G, D98G, D128G, G36W, G101W, G131W, D33K, D98K, and D128K decrease protein stability the most. Further stress‐dependent analysis on selected residues showed that lower temperature and pH destabilize the protein. The reason behind this increase in protein destability was drastic decrease and disruption of interatomic interactions in mutant MfpA. These analysis provides essential information about the residues that are important for MfpA stability and also enlightens protein vulnerability after mutation. [ABSTRACT FROM AUTHOR]

Structural characterization of microtubules has been the realm of three-dimensional electron microscopy and thus has evolved hand in hand with the progress of this technique, from the initial 3D reconstructions of stained tubulin assemblies, and the first atomic model of tubulin by electron crystallography of 2D sheets of protofilaments, to the ever more detailed cryoelectron microscopy structures of frozen-hydrated microtubules. Most recently, hybrid helical and single particle image processing techniques, and the latest detector technology, have lead to atomic models built directly into the density maps of microtubules in different functional states, shading new light into the critical process of microtubule dynamic instability.

A growing body of evidence implicates requisite roles for GTP and its binding proteins (Rho GTPases) in the cascade of events leading to physiological insulin secretion from the islet beta cell. Interestingly, chronic exposure of these cells to hyperglycaemic conditions appears to result in sustained activation of specific Rho GTPases (e.g. Rac1) leading to significant alterations in cellular functions including defects in mitochondrial function and nuclear collapse culminating in beta cell demise. One of the objectives of this review is to highlight our current understanding of the regulatory roles of GTP and Rho GTPases in normal islet function (e.g. proliferation and insulin secretion) as well potential defects in these signalling molecules and metabolic pathways that could contribute islet beta cell dysfunction and loss of functional beta cell mass leading to the onset of diabetes. Potential knowledge gaps in this field and possible avenues for future research are also highlighted. Abbreviations: ARNO: ADP-ribosylation factor nucleotide binding site opener; CML: carboxyl methylation; Epac: exchange protein directly activated by cAMP; ER stress: endoplasmic reticulum stress; FTase: farnesyltransferase; GAP: GTPase activating protein; GDI: GDP dissociation inhibitor; GEF: guanine nucleotide exchange factor; GGTase: geranylgeranyltransferase; GGpp: geranylgeranylpyrophosphate; GGPPS: geranylgeranyl pyrophosphate synthase; GSIS: glucose-stimulated insulin secretion; HGPRTase: hypoxanthine-guanine phosphoribosyltransferase; IMPDH: inosine monophosphate dehydrogenase; α-KIC: α-ketoisocaproic acid; MPA: mycophenolic acid; MVA: mevalonic acid; NDPK: nucleoside diphosphate kinase; NMPK: nucleoside monophosphate kinase; Nox2: phagocyte-like NADPH oxidase; PAK-I: p21-activated kinase-I; β-PIX: β-Pak-interacting exchange factor; PRMT: protein arginine methyltransferase; Rac1: ras-related C3 botulinum toxin substrate 1; Tiam1: T-cell lymphoma invasion and metastasis-inducing protein 1; Trx-1: thioredoxin-1; Vav2: vav guanine nucleotide exchange factor 2 [ABSTRACT FROM AUTHOR]

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20–30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment. • Detailed in-house production protocol for making resin bound bacterial alkaline phosphatase (BAP). • Cheap and reliable reagent to replace commercial agarose bead conjugated phosphatases. • Nucleotide exchange protocols for small GTPase's such as Rat sarcoma virus proteins (RAS). [ABSTRACT FROM AUTHOR]

The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro , but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants. • EF1a is highly prone to aggregation under heat stress in vivo and in vitro. • Heat tolerance was significantly increased by overexpression of EF1a. • Plants overexpressing mutant versions of EF1α compromised heat tolerance. • Soluble EF1a is critical to plant heat response. [ABSTRACT FROM AUTHOR]

The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix.

Multiplayer online battle arena (MOBA), the most popular video game genre, has increased in terms of the number of users online. This increase in popularity depicts the in-game immersion in MOBA games, which determines Flow experience. Despite its popularity, there is a dearth of research on the cause of immersion of MOBA games. This study hypothesized that players acquired automatic thoughts, actions, and sensations about the game even when not playing, identified as Game Transfer Phenomena (GTP), causing Flow. Forty-five (45) college students were recruited to play MOBA games for 20 ​h in one week, referred to as immersive gaming in this study. Flow state scale and GTP scale were administered twice before and after immersive gaming. Pretest and posttest comparisons showed no difference in the Flow and GTP. On the other hand, the cross-lagged analysis shows that GTP causes Flow among MOBA gamers. Players with a high level of GTP experience more intensive levels of Flow. The implications of the development of Flow are discussed in the study.