Guanosine Triphosphate Binding Research Articles

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Binding of Nucleotide Inhibitors to the NS5 RdRp of the ZIKA Virus in the Replication Initiation State.

The bindings of several ribonucleoside triphosphate (NTP) inhibitors to the RNA-dependent RNA polymerase (RdRp) of the Zika virus (ZIKV) are studied herein to identify potential drug-like candidates that can inhibit the replication of the viral genome by RdRp. In this study, a guanosine triphosphate (GTP) bound RdRp structure is generated to model the replication initiation state of RdRp. Subsequently, the bindings of 30 NTP inhibitors to the GTP binding site of RdRp are studied in detail by using the molecular docking method. Based on the docking scores, four NTP inhibitors, such as 2'-Cmethyl- adenosine-5'-triphosphate (mATP), 7-deaza-2'-C-methyladenosine-TP (daza-- mATP), 1-N6-Ethenoadenosine-5'-triphosphate (eATP), and Remdesivir-5'-triphosphate (RTP) are shortlisted for further analysis by employing molecular dynamics simulations and binding free-energy methods. These inhibitors are found to bind to RdRp quite strongly, as evident from their relative binding free energies that lie between -31.54±4.54 to -89.46±4.58 kcal/- mol. As the binding of RTP to the GTP site of RdRp generates the most stable complex, which is about 45 kcal/mol more stable than the binding of GTP to RdRp, it is most likely that RTP may inhibit the replication of the Zika viral genome efficiently. However, experimental studies are required to measure the potency of RTP and other drugs before their clinical use.

Markov State Models Reconcile Conformational Plasticity of GTPase with Its Substrate Binding Event.

Ras GTPase is an enzyme that catalyzes the hydrolysis of guanosine triphosphate (GTP) and plays an important role in controlling crucial cellular signaling pathways. However, this enzyme has always been believed to be undruggable due to its strong binding affinity with its native substrate GTP. To understand the potential origin of high GTPase/GTP recognition, here we reconstruct the complete process of GTP binding to Ras GTPase via building Markov state models (MSMs) using a 0.1 ms long all-atom molecular dynamics (MD) simulation. The kinetic network model, derived from the MSM, identifies multiple pathways of GTP en route to its binding pocket. While the substrate stalls onto a set of non-native metastable GTPase/GTP encounter complexes, the MSM accurately discovers the native pose of GTP at its designated catalytic site in crystallographic precision. However, the series of events exhibit signatures of conformational plasticity in which the protein remains trapped in multiple non-native conformations even when GTP has already located itself in its native binding site. The investigation demonstrates mechanistic relays pertaining to simultaneous fluctuations of switch 1 and switch 2 residues which remain most instrumental in maneuvering the GTP-binding process. Scanning of the crystallographic database reveals close resemblance between observed non-native GTP binding poses and precedent crystal structures of substrate-bound GTPase, suggesting potential roles of these binding-competent intermediates in allosteric regulation of the recognition process.

Autosomal dominant Parkinson's disease caused by the recently identified LRRK2 N1437D mutation in a Chinese family: Clinical features, imaging findings, and functional impact

IntroductionMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of autosomal dominantly inherited Parkinson's disease (PD). Recently, a novel pathogenic variant (N1437D; c.4309A > G; NM_98578) in the LRRK2 gene has been identified in three Chinese families with PD. In this study, we describe a Chinese family with autosomal dominant PD that segregated with the N1437D mutation. A detailed clinical and neuroimaging characterization of the affected family members is reported. We also sought to investigate the functional mechanisms by which the detected mutation could cause PD. MethodsWe characterized the clinical and imaging phenotype of a Chinese pedigree with autosomal dominant PD. We searched for a disease-causing mutation by targeted sequencing and multiple ligation-dependent probe amplification. The functional impact of the mutation was investigated in terms of LRRK2 kinase activity, guanosine triphosphate (GTP) binding, and guanosine triphosphatase (GTPase) activity. ResultsThe disease was found to co-segregate with the LRRK2 N1437D mutation. Patients in the pedigree exhibited typical parkinsonism (age at onset: 54.0 ± 5.9 years). One affected family member – who had evidence of abnormal tau accumulation in the occipital lobe on tau PET imaging – developed PD dementia at follow-up. The mutation markedly increased LRRK2 kinase activity and promoted GTP binding, without affecting GTPase activity. ConclusionsThis study describes the functional impact of a recently identified LRRK2 mutation, N1437D, that causes autosomal dominant PD in the Chinese population. Further research is necessary to investigate the contribution of this mutation to PD in multiple Asian populations.

Chemically Fueled Supramolecular Materials.

In biology, the function of many molecules is regulated through nonequilibrium chemical reaction cycles. The prototypical example is the phosphorylation of an amino acid in an enzyme which induces a functional change, e.g., it folds or unfolds, assembles or disassembles, or binds a substrate. Such phosphorylation does not occur spontaneously but requires a phosphorylating agent with high chemical potential (for example, adenosine triphosphate (ATP)) to be converted into a molecule with lower chemical potential (adenosine diphosphate (ADP)). When this energy is used to regulate an assembly, we speak of chemically fueled assemblies; i.e., the molecule with high potential, the fuel, is used to regulate a self-assembly process. For example, the binding of guanosine triphosphate (GTP) to tubulin induces self-assembly. The bound GTP is hydrolyzed to guanosine diphosphate (GDP) upon assembly, which induces tubulin disassembly. The result is a dynamic assembly endowed with unique characteristics, such as time-dependent behavior and the ability to self-heal. These intriguing, unique properties have inspired supramolecular chemists to create similar chemically fueled molecular assemblies from the bottom up. While examples have been designed, they remain scarce partly because chemically fueled reaction cycles are rare and often complex. Thus, we recently developed a carbodiimide-driven reaction cycle that is versatile and easy to use, quantitatively understood, and does not suffer from side reactions. In the reaction cycle, a carboxylate precursor reacts with a carbodiimide to form an activated species like an anhydride or ester. The activated state reacts with water and thereby reverts to its precursor state; i.e., the activated state is deactivated. Effectively, the precursor catalyzes carbodiimides' conversion into waste and forms a transient activated state. We designed building blocks to regulate a range of assemblies and supramolecular materials at the expense of carbodiimide fuel. The simplicity and versatility of the reaction cycles have democratized and popularized the field of chemically fueled assemblies. In this Account, we describe what we have "learned" on our way. We introduce the field exemplified by biological nonequilibrium self-assembly. We describe the design of the carbodiimide-driven reaction cycle. Using examples from our group and others, we offer design rules for the building block's structure and strategies to create the desired morphology or supramolecular materials. The discussed morphologies include fibers, colloids, crystals, and oil- and coacervate-based droplets. We then demonstrate how these assemblies form supramolecular materials with unique material properties like the ability to self-heal. Besides, we discuss the concept of reciprocal coupling in which the assembly exerts feedback on its reaction cycle and we also offer examples of such feedback mechanisms. Finally, we close the Account with a discussion and an outlook on this field. This Account aims to provide our fundamental understanding and facilitate further progress toward conceptually new supramolecular materials.

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the β2-adrenergic receptor (β2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from β2AR and Gβγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

Structural basis of the oncogenic KRAS mutant and GJ101 complex

KRAS mutations occur in a quarter of all human cancers. When activated in its GTP-bound form, RAS stimulates diverse cellular systems, such as cell division, differentiation, growth, and apoptosis through the activations of various signaling pathways, which include mitogen-activated protein kinase (MAPK), phosphoinositide 3 kinases (PI3K), and RAL-GEFs pathways. We found that GJ101 (65LYDVA69) binds directly to the KRAS mutant (G12V) and showed tumor-suppressive activity. In addition, the GJ101 peptide inhibited KRAS mutant as determined by a [α-32P] guanosine triphosphate (GTP) binding assay and suppressed pancreatic cell line in a cell proliferation assay. Herein, the complex structure of KRAS and GJ101 was clarified by X-ray crystallography. Isothermal titration calorimetry showed that GJ101 binds highly with KRAS mutant and the complex structure of KRAS G12V.GJ101 complex presented that the residue of Q61 directly interacted with L65 of GJ101. Overall, the results suggest GJ101 be considered a developmental starting point for KRAS G12V inhibitor.

Novel Loss of Function (G15D) Mutation on RAC2 in a Family with Combined Immunodeficiency and Increased Levels of Immunoglobulin G, A, and E.

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a small guanine nucleotide binding molecule that is exclusively expressed in hematopoietic cell lineages as a switcher. Based on in vivo and/or in vitro model experiments, RAC2 plays important roles in different cells through proliferation, secretion, and phagocytosis. It also performs a suppressing function in immunoglobulin (Ig) switching in Rac2-/- animals or cells. Several RAC2 natural mutations have been described in patients with primary immunodeficiency. RAC2 mutations can be classified into loss-of-function inactivating (LoF-I) and gain-of-function activating mutations according to their functional effects. Only two LoF-I mutations on RAC2 have been reported, including a dominant D57N mutation in several cases that exhibit granulocyte function defects and a recessive D56X mutation in cases with common variable immunodeficiency. Regardless of the type of mutation, most of the reported RAC2 mutant cases have shown reduced IgG, IgA, and IgM levels. Herein, we report on a family with three members that suffer from persistent HPV infection, recurrent respiratory infections, bronchiectasis, and autoimmune disease. The immunologic profile suggests that the family was affected by combined immunodeficiency (CID) with increased serum levels of IgG, IgA, and IgE. Exome sequencing identified a de novo RAC2 mutation (c.44G > A/p.G15D) that was co-segregated with the disease in the family. Gene functional experiments identified that such mutation results in reduced guanosine triphosphate binding activity and RAC2 protein expression. In patients' lymphocytes, impaired aggregation and proliferation effects, decreased mitochondrial membrane potential, and increased levels of cell apoptosis were observed, although no functional abnormalities were detected in neutrophils. To our knowledge, this study was the first to identify a LoF-I mutation of RAC2 affecting lymphocyte function that consequently led to CID and increased levels of serum IgG, IgE, and IgA. This study presents a novel subtype of RAC2-related immune disorder.

Longin domain GAP complexes in nutrient signalling, membrane traffic and neurodegeneration.

Small GTPases act as molecular switches and control numerous cellular processes by their binding and hydrolysis of guanosine triphosphate (GTP). The activity of small GTPases is coordinated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Recent structural and functional studies have characterized a subset of GAPs whose catalytic units consist of longin domains. Longin domain containing GAPs regulate small GTPases that facilitate nutrient signalling, autophagy, vesicular trafficking and lysosome homeostasis. All known examples in this GAP family function as part of larger multiprotein complexes. The three characterized mammalian protein complexes in this class are FLCN:FNIP, GATOR1 and C9orf72:SMCR8. Each complex carries out a unique cellular function by regulating distinct small GTPases. In this article, we explore the roles of longin domain GAPs in nutrient sensing, membrane dynamic, vesicular trafficking and disease. Through a structural lens, we examine the mechanism of each longin domain GAP and highlight potential therapeutic applications.

Differentially expressed whey proteins of donkey and bovine colostrum revealed with a label-free proteomics approach

This study aimed to analyze and compare the differentially expressed whey proteins (DEWPs) of donkey and bovine colostrum using high-performance liquid chromatography with tandem mass spectrometry-based proteomics. A total of 620 and 696 whey proteins were characterized in the donkey and bovine colostrum, respectively, including 383 common whey proteins. Among these common proteins, 80 were identified as DEWPs, including 21 upregulated and 59 downregulated DEWPs in donkey colostrum compared to bovine colostrum. Gene Ontology analysis revealed that these DEWPs were mainly related to cellular components, such as extracellular exosome, plasma membrane, and mitochondrion; biological processes, such as oxidation-reduction process, cell-cell adhesion, and small guanosine triphosphate (GTP) ase-mediated signal transduction; and molecular functions, such as GTP binding, GTPase activity, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor activity. Metabolic pathway analysis suggested that the majority of the DEWPs were associated with soluble NSF factor attachment protein receptor interactions in vesicular transport, fatty acid biosynthesis, and estrogen signaling pathways. Our results provide a vital insight into the differences between donkey and bovine colostrum, along with important information on the significant components as nutritional and functional factors to be included in infant formula based on multiple milk sources.

GTPBP4 promotes hepatocellular carcinoma progression and metastasis via the PKM2 dependent glucose metabolism

Guanosine triphosphate binding protein 4 (GTPBP4) is a key regulator of cell cycle progression and MAPK activation. However, how its biological properties intersect with cellular metabolism in hepatocellular carcinoma (HCC) development remains poorly unexplained. Here, high GTPBP4 expression is found to be significantly associated with worse clinical outcomes in patients with HCC. Moreover, GTPBP4 upregulation is paralleled by DNA promoter hypomethylation and regulated by DNMT3A, a DNA methyltransferase. Additionally, both gain- and loss-of-function studies demonstrate that GTPBP4 promotes HCC growth and metastasis in vitro and in vivo. Mechanically, GTPBP4 can induce dimeric pyruvate kinase M2 (PKM2) formation through protein sumoylation modification to promote aerobic glycolysis in HCC. Notably, active GTPBP4 facilitates SUMO1 protein activation by UBA2, and acts as a linker bridging activated SUMO1 protein and PKM2 protein to induce PKM2 sumoylation. Furthermore, SUMO-modified PKM2 relocates from the cytoplasm to the nucleus may also could contribute to HCC progression through activating epithelial-mesenchymal transition (EMT) and STAT3 signaling pathway. Shikonin, a PKM2-specific inhibitor, can attenuate PKM2 dependent HCC glycolytic reprogramming, growth and metastasis promoted by GTPBP4, which offers a promising therapeutic candidate for HCC patients. Our findings indicate that GTPBP4-PKM2 regulatory axis plays a vital role in promoting HCC proliferation as well as metastasis by aerobic glycolysis and offer a promising therapeutic target for HCC patients.

Neonatal Onset of Hemophagocytic Lymphohistiocytosis Due to Prenatal Varicella-Zoster Infection in a Neonate with Griscelli Syndrome Type 2.

Type 2 Griscelli syndrome (Type2 GS) is a primary inborn error of the immune system, classified in the immune dysregulation group.1,2 There are three different types of the disease, with different genetic causes responsible for the autosomal recessive inheritance pattern. Although hypopigmentation is common in all variants, neurological involvement or immunodeficiency with varying severity is seen in different types. Molecular motor protein myosin 5 an (MYo5A) [Type1GS], guanosine Triphosphate (GTP) binding protein (RAB27A) [Type2GS], and mutation in human melanophilin (MLPH) [Type 3GS] which is limited to hypopigmentation are reported as the known genetic defects in GS.3 Severe, ineffective, and uncontrolled inflammatory reactions are referred to as the pathogenesis of Hemophagocytic lymphohistiocytosis (HLH). HLH is a life-threatening condition that can be defined as either primary or secondary. Secondary causes happen in the context of autoimmunity, malignancy, spontaneous, or infections.4 Prenatal infections play an important role in causing long-term complications in the fetus. Some of them include toxoplasmosis, rubella, cytomegalovirus, herpes simplex, and other organisms including syphilis, parvovirus, and Varicella zoster, known as TORCH syndrome (5).TORCH has been well described for a long time but there are limited reports of developing HLH in the context of prenatal infections. We described a type 2GS syndrome with neonatal-onset HLH triggered by a prenatal infection.

Structural basis for activation of Arf1 at the Golgi complex.

SUMMARYThe Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

The role of nucleoside triphosphate hydrolase metallochaperones in making metalloenzymes.

Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.

Moving nickel along the hydrogenase-urease maturation pathway.

Hydrogenases and ureases play vital metabolic functions in all three domains of life. However, nickel ions are cytotoxic because they can inactivate enzymes that require less competitive ions (e.g. Mg2+) in the Irving-Williams series to function. Life has evolved elegant mechanisms to solve the problem of delivering the toxic metal to the active site of nickel-containing enzymes inside the cells. Here, we review our current understanding of nickel trafficking along the hydrogenase and urease maturation pathways. Metallochaperones and accessory proteins (SlyD, HypA, HypB, UreD, UreE, UreF, and UreG) form specific protein complexes to allow the transfer of nickel from one protein to another without releasing the toxic metal into the cytoplasm. The role of SlyD is not fully understood, but it can interact with and transfer its nickel to HypB. In the hydrogenase maturation pathway, nickel is transferred from HypB to HypA, which can then deliver its nickel to the hydrogenase large subunit precursor. In Helicobacter pylori, the urease maturation pathway receives its nickel from HypA of the hydrogenase maturation pathway via the formation of a HypA/UreE2 complex. Guanosine triphosphate (GTP) binding promotes the formation of a UreE2G2 complex, where UreG receives a nickel from UreE. In the final step of the urease maturation, nickel/GTP-bound UreG forms an activation complex with UreF, UreD, and apo-urease. Upon GTP hydrolysis, nickel is released from UreG to the urease. Finally, some common themes learned from the hydrogenase-urease maturation pathway are discussed.

Role of ARHGEF3 as a GEF and mTORC2 Regulator.

Guanine nucleotide exchange factors (GEFs) activate GTPases by stimulating the release of guanosine diphosphate to permit the binding of guanosine triphosphate. ARHGEF3 or XPLN (exchange factor found in platelets, leukemic, and neuronal tissues) is a selective guanine nucleotide exchange factor for Rho GTPases (RhoGEFs) that activates RhoA and RhoB but not RhoC, RhoG, Rac1, or Cdc42. ARHGEF3 contains the diffuse B-cell lymphoma homology and pleckstrin homology domains but lacks similarity with other known functional domains. ARHGEF3 also binds the mammalian target of rapamycin complex 2 (mTORC2) and subsequently inhibits mTORC2 and Akt. In vivo investigation has also indicated the communication between ARHGEF3 and autophagy-related muscle pathologies. Moreover, studies on genetic variation in ARHGEF3 and genome-wide association studies have predicted exciting novel roles of ARHGEF3 in controlling bone mineral density, platelet formation and differentiation, and Hirschsprung disease. In conclusion, we hypothesized that additional biochemical and functional studies are required to elucidate the detailed mechanism of ARHGEF3-related pathologies and therapeutics.

Suppression of pheromone biosynthesis and mating behavior by RNA interference of pheromone gland-specific fatty acyl reductase in Maruca vitrata.

In moths, various enzymes, such as fatty acid synthases, fatty acyl desaturases, and fatty acyl reductases (FARs), are involved in pheromone biosynthesis. In particular, pheromone gland-specific FAR (pgFAR) plays an important role in converting the functional group from carboxylic to alcohol during pheromone biosynthesis. A novel pgFAR of Maruca vitrata, Mvi-pgFAR, was identified through transcriptome sequencing of its pheromone gland. To investigate the involvement of Mvi-pgFAR in pheromone biosynthesis, Mvi-pgFAR was cloned from the pheromone gland and suppressed by RNA interference (RNAi). Mvi-pgFAR harbored several conserved motifs related to NAD(P)H-binding, N-glycosylation, and adenosine / guanosine triphosphate binding. Phylogenetic analysis revealed that Mvi-pgFAR with other lepidopteran pgFARs formed an independent clade. Mvi-pgFAR was specifically expressed only in the pheromone gland. Quantitative real-time polymerase chain reaction showed that the diurnal expression levels of Mvi-pgFAR in the pheromone gland were the highest at 2 h before the scotophase. After primarily confirming Mvi-pgFAR suppression by RNAi, (E,E)-10,12-hexadecadienal (E10E12-16:Ald), a major sex pheromone component, was quantified by gas chromatography. When Mvi-pgFAR was successfully suppressed, E10E12-16:Ald production was reduced by up to half of that of the control, and the mating rate was subsequently decreased. Our results demonstrate that Mvi-pgFAR downregulation can suppress mating behavior by changing the relative sex pheromone component ratio, suggesting that Mvi-pgFAR can be used as a novel control target.

In silico comparative analysis of KRAS mutations at codons 12 and 13: Structural modifications of P-Loop, switch I&II regions preventing GTP hydrolysis

KRAS mutation is prevalent in around 30% of all cancers and is an undruggable molecular target. Of seven mutations at codon 12 and 13, only one, the G12C mutant is finally proven to be druggable, as evidenced by the recent USFDA approval of sotorasib. Investigation of other small molecules targeting G12C and G12D are undergoing clinical trials. Understanding the fine structural details is a prerequisite to design specific inhibitors which also requires in depth molecular exploration. We used bioinformatics as a tool to analyze the KRAS protein's GTP (guanosine triphosphate) binding dynamics when mutated. KRAS undergoes significant conformational changes, affecting GTP binding conformation within the active site pocket of KRAS due to high torsional strains, hydrophobicity, and altered Switch I and II regions. GTP molecule for wildtype had a low torsional strain of 10.71, and is the only molecule, in comparison to KRAS mutant bound GTP, to have a glycine at position 10 interacting with its nitrogenous base. All mutant KRAS proteins lacked the interaction of glycine with the nitrogenous base. The binding affinity of wildtype (WT) KRAS for the gamma-phosphate was lower in scoring compared to the mutated KRAS protein in multiple analyses. This study provides an insight to the GTP-KRAS protein binding details that are important to define parameters required to be explored to design the appropriate inhibitor for each different type of mutant KRAS protein.

Cell Division Control Protein 42 Interacts With Hepatitis E Virus Capsid Protein and Participates in Hepatitis E Virus Infection.

Hepatitis E Virus (HEV) causes viral hepatitis in humans worldwide, while a subset of HEV species, avian HEV, causes hepatitis-splenomegaly syndrome in chickens. To date, there are few reports on the host proteins interacting with HEV and being involved in viral infection. Previous pull-down assay combining mass spectrometry indicated that cell division control protein 42 (CDC42), a member belonging to the Rho GTPase family, was pulled down by avian HEV capsid protein. We confirmed the direct interaction between CDC42 and avian and mammalian HEV capsid proteins. The interaction can increase the amount of active guanosine triphosphate binding CDC42 state (GTP-CDC42). Subsequently, we determined that the expression and activity of CDC42 were positively correlated with HEV infection in the host cells. Using the different inhibitors of CDC42 downstream signaling pathways, we found that CDC42-MRCK (a CDC42-binding kinase)-non-myosin IIA (NMIIA) pathway is involved in naked avian and mammalian HEV infection, CDC42-associated p21-activated kinase 1 (PAK1)-NMIIA/Cofilin pathway is involved in quasi-enveloped mammalian HEV infection and CDC42-neural Wiskott-Aldrich syndrome protein-actin-polymerizing protein Arp2/3 pathway (CDC42-(N-)WASP-Arp2/3) pathway participates in naked and quasi-enveloped mammalian HEV infection. Collectively, these results demonstrated for the first time that HEV capsid protein can directly bind to CDC42, and non- and quasi-enveloped HEV use different CDC42 downstream signaling pathways to participate in viral infection. The study provided some new insights to understand the life cycle of HEV in host cells and a new target of drug design for combating HEV infection.