GTP Hydrolysis Research Articles

Subanalgesic morphine doses augment fentanyl analgesia by interacting with delta opioid receptors in male rats

Opioids are commonly used for the treatment of postoperative and post-traumatic pain; however, their therapeutic effectiveness is limited by undesirable and life-threatening side effects. Researchers have long attempted to develop opioid co-administration therapies that enhance analgesia, but the complexity of opioid analgesia and our incomplete mechanistic understanding has made this a daunting task. We discovered that subanalgesic morphine doses (100ng/kg-10µg/kg) augmented the acute analgesic effect of fentanyl (20µg/kg) following subcutaneous drug co-administration to male rats. In addition, administration of equivalent drug ratios to naïve rat spinal cord membranes induced a twofold increase in G protein activation. The rate of GTP hydrolysis remained unchanged. We demonstrated that these behavioral and biochemical effects were mediated by the delta opioid receptor (DOP). Subanalgesic doses of the DOP-selective agonist SNC80 also augmented the acute analgesic effect of fentanyl. Furthermore, co-administration of the DOP antagonist naltrindole with both fentanyl-morphine and fentanyl-SNC80 combinations prevented augmentation of both analgesia and G protein activation. The mu opioid receptor (MOP) antagonist cyprodime did not block augmentation. Confocal microscopy of the substantia gelatinosa of rats treated with fentanyl, subanalgesic morphine, or this combination showed that changes in MOP internalization did not account for augmentation effects. Together, these findings suggest that augmentation of fentanyl analgesia by subanalgesic morphine is mediated by increased G protein activation resulting from a synergistic interaction between or heterodimerization of MOPs and DOPs. This finding is of great therapeutic significance because it suggests a strategy for the development of DOP-selective ligands that can enhance the therapeutic index of clinically used MOP drugs.

Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

ABSTRACTArf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1–3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

Conformational Features of Ras: Key Hydrogen-Bonding Interactions of Gln61 in the Intermediate State during GTP Hydrolysis.

The Ras protein is one of the most important drug targets for battling cancers. To effectively design novel drugs of Ras, we characterize here its conformational ensembles for the hydrolysis intermediate state RasGDP·Pi and the product state RasGDP by extensive replica-exchange molecular dynamics simulations. Several substates for RasGDP·Pi have been identified, while structural analyses have revealed an unrecognized hydrogen-bonding network that stabilizes the hydrolysis intermediate state. More interestingly, Gln61, which is involved in numerous oncogenic mutations, was found to be engaged in this hydrogen-bonding network, adopting a specific conformation that always points to Pi in contrast to that in the RasGTP state. The simulations also reveal that RasGDP has more than one substate, suggesting a conformational selection mechanism for the interaction between Ras and the guanine nucleotide exchange factors (GEFs). These findings offer new opportunities for the drug design of Ras by stabilizing the hydrolysis intermediate or disrupting its interaction with the GEFs.

Dynamin-dependent vesicle twist at the final stage of clathrin-mediated endocytosis.

Dynamin plays an important role in clathrin-mediated endocytosis (CME) by cutting the neck of nascent vesicles from the cell membrane. Here through using gold nanorods as cargos to image dynamin action during live CME, we show that near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle drastically increases, accompanied with simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.

Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification.

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

Abstract 1260: First-in-class, orally bioavailable KRASG12V(ON) tri-complex inhibitors, as single agents and in combinations, drive profound anti-tumor activity in preclinical models of KRASG12V mutant cancers

Abstract KRASG12V mutant cancers represent a significant unmet medical need with nearly 44,000 new diagnoses annually in the US. The KRASG12V mutation occurs frequently in multiple tumor histotypes; the incidence in NSCLC, CRC and pancreatic cancers is 6%, 10% and 26%, respectively. RAS proteins are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins exist predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF. The intrinsic GTP hydrolysis rate of KRASG12V is significantly lower than that of either KRASG12C or KRASG12D. Therefore, targeting the KRASG12V(ON) state will be critical for maximal suppression of this oncogenic driver. No targeted, direct inhibitors of KRASG12V(ON) have been described to date. We have built a pipeline of small molecule inhibitors targeting multiple oncogenic RAS(ON) mutants. Here we describe the preclinical profile of KRASG12V(ON) inhibitors that promote a tri-complex between the inhibitor, the immunophilin cyclophilin A (CypA), and the active GTP-bound state of KRASG12V. In cancer cell lines bearing KRASG12V mutations, KRASG12V(ON) inhibitors trigger an immediate disruption of RAS-effector interactions, leading to attenuation of RAS pathway signaling, potent (sub-nM EC50) growth suppression, and apoptosis. KRASG12V(ON) inhibitors produce deep, durable, and dose-dependent suppression of tumor RAS pathway activation in vivo following oral administration. An extended duration of tumor pharmacodynamic activity, relative to plasma exposure, is observed that likely reflects retention of the inhibitors in tumor tissue due to high affinity binding to CypA. In human tumor xenograft models of KRASG12V mutant NSCLC, CRC and pancreatic cancers, oral administration of KRASG12V(ON) inhibitors is well-tolerated and drives profound and durable tumor regressions, with complete responses in some animals. KRASG12V(ON) inhibitors also downregulate immune checkpoint proteins PD-L1 and CD73 on KRASG12V mutant cancer cells, changes which can support enhanced anti-tumor immunity. The ability to target the GTP-bound form of mutant KRASG12V permits a broad array of combination opportunities in cancer types where single agent KRASG12V(ON) inhibition may be insufficient, for example with agents targeting pathway nodes both upstream (e.g. SHP2, SOS1) and downstream (e.g. MEK, ERK) of RAS, as well as parallel pathways (e.g. mTORC1). Tri-complex inhibitors that target KRASG12V(ON) are predicted to combat escape mechanisms in RAS-addicted cancer cells characterized by an increased pool of activated KRASG12V(ON). These inhibitors may lead to an attractive, targeted therapeutic option for the treatment of RAS-addicted cancers with a very high unmet medical need. Citation Format: Elena Koltun, Jim Cregg, Meghan A. Rice, Dan M. Whalen, Rebecca Freilich, Jingjing Jiang, Richard Hansen, Alun Bermingham, John E. Knox, Jay Dinglasan, Kyle Seamon, Cristina Blaj, Stephanie S. Chang, Yang Liu, Jun Huang, Kang-Jye Chou, Laura McDowell, Bianca J. Lee, David Wildes, Zhengping Wang, Mallika Singh, Adrian L. Gill, Jacqueline A. Smith. First-in-class, orally bioavailable KRASG12V(ON) tri-complex inhibitors, as single agents and in combinations, drive profound anti-tumor activity in preclinical models of KRASG12V mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1260.

Regulators of G protein Signaling (RGS) proteins in GtoPdb v.2021.2

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [225, 529, 578, 583, 584, 742, 753, 444, 10].

Starvation Stress in the Sbds -Null Zebrafish: Implications for Human Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and an increase risk of acute myeloid leukemia. Patients with SDS exhibit abnormalities in other organs, including bone, liver and immunologic and endocrine systems. Almost all cases of SDS result from biallelic mutations in the SBDS gene1. SBDS is involved in ribosomal biogenesis and stabilization of mitotic spindle microtubules2,3. Recently, it has also been shown a reduction cell proliferation and mTOR and STAT3 expression, both with an important role in cellular growth and tumorigenesis, in SDS derived leukocytes.4Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice5. Zebrafish provide an attractive, alternative model organism to study SDS. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.We created sbds zebrafish knockout strains that phenocopies the human Shwachman-Diamond Syndrome. Survival rate of mutants was normal at 15 days post fertilization (dpf) (100%) but decline significantly at 21 dpf (4%). Western blot analysis showed a progressive decrease of Sbds during the first 8 dpf, with total absence at 10 dpf. These results suggest that larval viability is due to maternally deposited sbds transcript and corresponding protein, which was confirmed by RT-qPCR and immunoblot at earliest developmental stages. Zebrafish sbds mutants showed neutropenia at 5 and 15 days post fertilization (dpf) but normal macrophage number and hemoglobinization. Pancreas, liver, digestive tract, and eye showed histologic evidence of atrophy at 21 dpf. Moreover, we observed an important reduction in the intestinal epithelium at 15 dpf.To further characterize effects of Sbds loss, we tested expression of genes involved in lipid metabolism (acc, ppar α , ppar γ and srbp1) and pancreatic transcription factors (ptf1a and prox1) by qPCR. Our results showed that they were not affected at 10 dpf but upregulated at 18 dpf. After staining 15 dpf larvae with oil red we observed, in the mutants compared to their sibling wild type or heterozygous, significantly reduced accumulation of lipids. In addition, we found an increase of expression of genes involved in autophagy (atg5, atg7 and mTOR), and upregulation of casp9 and tp53 expression.With these results we thought that reduced viability was due to a problem in nutrient absorption and therefore starvation. We carried out two starvation experiments, a diet restriction from 10 to 15 dpf, and from 15 to 21 dpf. Unlike sbds mutants, mortality rates were not affected comparing to control groups in any of these experiments. However, genes related to lipid metabolism and autophagy were upregulated and histology showed a reduction in intestinal epithelium as well. Moreover, oil red staining showed lower lipid accumulation in starved fish as we observed in sbds mutants.With these results, we conclude sbds -null zebrafish showed signs of malnutrition and an effect in fatty acid-metabolism gene expression as seen in starved fish. Our results make it a highly relevant model to investigate new mechanistic hypotheses and develop new small molecule therapies for Shwachman-Diamond syndrome.1. Boocock GR, Morrison JA, Popovic M, et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet . 2003;33(1):97-101.2. Austin KM, Gupta ML, Jr., Coats SA, et al. Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest . 2008;118(4):1511-1518.3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev . 2011;25(9):917-929.4. Bezzerri V, Vella A, Calcaterra E, et al. New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes. 2016;6:33165.5. Zhang S, Shi M, Hui CC, Rommens JM. Loss of the mouse ortholog of the Shwachman-Diamond syndrome gene (Sbds) results in early embryonic lethality. Mol Cell Biol . 2006;26(17):6656-6663. DisclosuresNo relevant conflicts of interest to declare.

Archaeal SRP RNA and SRP19 facilitate the assembly of SRP54-FtsY targeting complex

The signal recognition particle (SRP) plays an essential role in protein translocation across biological membranes. Stable complexation of two GTPases in the signal recognition particle (SRP) and its receptor (SR) control the delivery of nascent polypeptide to the membrane translocon. In archaea, protein targeting is mediated by the SRP54/SRP19/7S RNA ribonucleoprotein complex (SRP) and the FtsY protein (SR). In the present study, using fluorescence resonance energy transfer (FRET), we demonstrate that archaeal 7S RNA stabilizes the SRP54·FtsY targeting complex (TC). Moreover, we show that archaeal SRP19 further assists 7S RNA in stabilizing the targeting complex (TC). These results suggest that archaeal 7S RNA and SRP19 modulate the conformation of the targeting complex and thereby reinforce TC to execute protein translocation via concomitant GTP hydrolysis.

FtsZ induces membrane deformations via torsional stress upon GTP hydrolysis

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.

Abstract P-36: Structural Analysis of Conformational Changes of Bacterial and Eukaryotic Tubulins

Background: Eukaryotic α- and β-tubulin proteins stand out among tubulin-like proteins by their ability to form hollow dynamically unstable microtubules (MT) with 13 protofilaments. Microtubules are part of the cell cytoskeleton and play a key role in chromosome division in mitosis. A considerable amount of anticancer drugs works on microtubules level breaking its dynamic. But the mechanism of dynamic instability and works of these drugs remains unknown. Bacteria of the genus Prostecobacter have unique bacterial tubulins (BtubA/B) capable to form hollow dynamically unstable 5 protofilament MTs (miniMT). Instead of great differences, both tubulins have many common features. Eukaryotic tubulin was known to have structural changes through GTP hydrolysis (compactization for approximately 2 Å and a twist for 0,1˚). «Anchor point» structure in alpha-tubulin was noticed to be a fixed point in this movement. Methods: We performed comparative structural analysis of BtubA/B and α- and β-tubulin proteins using USCF Chimera10 and MEGA X software. This data was obtained due to a comparison of 3 structures of microtubules with different nucleotides [pdb6DPU, 6DPV, 6DPW] and two structures for bacterial tubulins (miniMT [pdb5o09] and BtubA/B-dimer [pdb2BTQ]). Results: We noticed that bacterial tubulins form shorter protofilaments in miniMT than eukaryotic ones. It can be explained as compaction in two sites instead of one site in eukaryotic MT. Also, the most motionless point of min MT turned out the same "anchor point." Phylogenetic analysis showed that this structure is very conservative in these orthologs. Moreover, the final state of both tubulins (GDP) repeats each other. Conclusion: Our results suggest that bacterial tubulin can have movements through GTP hydrolysis similar to eukaryotic one. And it means that despite different amino acid sequences, bacterial and eukaryotic tubulins have similar keys structures for dynamic instability.

Abstract P-33: Structural Basis for the Growing Microtubule End Recognition by End Binding Proteins

Background: Eukaryotic end binding proteins (EBs) can follow the growing microtubule end. EBs play a crucial role in microtubule dynamic instability and promote simultaneously growth rate and catastrophe frequency. It makes EB-like proteins perspective drag targets for a wide number of diseases. But the molecular mechanism of tip tracking by EB-like proteins remains unknown. Studies of mutants have revealed that the conservative amino acid Q102 (numbering relative to the human EB1 protein) plays a key role in the recognition of the growing microtubule end. However, the 3D structure studies revealed that this amino acid has no bonds with tubulin. In this work, we performed structural and phylogenetic analysis of EBs proteins to identify a possible molecular mechanism behind the plus end tracking. Methods: UCSF Chimera10 was used for structural analysis. Phylogenetic analysis was performed with MEGA X software. 3D structures of EBs and microtubules with different states of GTP hydrolysis were used (pdb 3JAK, 3JAS, 3JAT, 3JAW, 3JAL, 3JAR, 6DPU, 6DPV, 6DPW). Results: We have shown that two conservative amino acids (K100, E106) should play an important role in the recognition of the microtubule plus end in addition to Q102. It was concluded that these amino acids together form the plus-end «navigation site» of EBs. Analysis of possible interaction of the «navigation site» amino acids with microtubules in different conformational states suggested that the main mechanism of growing microtubule end recognition is not due to an affinity increase for a certain state of tubulin in microtubules at their end, but it due to a significant affinity decrease in other parts of the microtubule as a result of steric clashes. Conclusion: Thus, the results of the analysis suggested the possible molecular mechanism that provides the tip tracking by EB-like proteins and allowed us to identify the key amino acids of this mechanism.

Tubulin binding potentially clears up Bortezomib and Carfilzomib differential neurotoxic effect

Proteasome inhibitors (PIs) represent the gold standard in the treatment of multiple myeloma. Among PIs, Bortezomib (BTZ) is frequently used as first line therapy, but peripheral neuropathy (PN), occurring approximately in 50% of patients, impairs their life, representing a dose-limiting toxicity. Carfilzomib (CFZ), a second-generation PI, induces a significantly less severe PN. We investigated possible BTZ and CFZ off-targets able to explain their different neurotoxicity profiles. In order to identify the possible PIs off-targets we used the SPILLO-PBSS software that performs a structure-based in silico screening on a proteome-wide scale. Among the top-ranked off-targets of BTZ identified by SPILLO-PBSS we focused on tubulin which, by contrast, did not turn out to be an off-target of CFZ. We tested the hypothesis that the direct interaction between BTZ and microtubules would inhibit the tubulin alfa GTPase activity, thus reducing the microtubule catastrophe and consequently furthering the microtubules polymerization. This hypothesis was validated in a cell-free model, since BTZ (but not CFZ) reduces the concentration of the free phosphate released during GTP hydrolysis. Moreover, NMR binding studies clearly demonstrated that BTZ, unlike CFZ, is able to interact with both tubulin dimers and polymerized form. Our data suggest that different BTZ and CFZ neurotoxicity profiles are independent from their proteasome inhibition, as demonstrated in adult mice dorsal root ganglia primary sensory neurons, and, first, we demonstrate, in a cell free model, that BTZ is able to directly bind and perturb microtubules.

The PKC ‐δ/ NF‐κB/p300/ TNFα signaling cascade and HDAC activity facilitate LPS‐induced Rgs10 silencing in pulmonary macrophages Faris Almutairi 1,2 and Balázs Rada 2 1 Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia, USA. 2 Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Regulator of G protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs), which accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Independent of its canonical function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNFα. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in MHS alveolar macrophage cell line. We show that pharmacological inhibition of PKC-δ/ NF-κB/p300 (NF-κB co-activator)/TNFα signaling cascade and the activities of HDAC enzymes block LPS-induced silencing of Rgs10 in MHS cells as well as microglial BV2 cells. Further, loss of RGS10 by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory genes expression following LPS treatment in MHS cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in macrophages.

The Role of RhoH in TCR Signalling and Its Involvement in Diseases.

As an atypical member of the Rho family small GTPases, RhoH shares less than 50% sequence similarity with other members, and its expression is commonly observed in the haematopoietic lineage. To date, RhoH function was observed in regulating T cell receptor signalling, and less is known in other haematopoietic cells. Its activation may not rely on the standard GDP/GTP cycling of small G proteins and is thought to be constitutively active because critical amino acids involved in GTP hydrolysis are absent. Alternatively, its activation can be regulated by other types of regulation, including lysosomal degradation, somatic mutation and transcriptional repressor, which also results in an altered protein expression. Aberrant protein expression of RhoH has been implicated not only in B cell malignancies but also in immune-related diseases, such as primary immunodeficiencies, systemic lupus erythematosus and psoriasis, wherein its involvement may provide the link between immune-related diseases and cancer. RhoH association with these diseases involves several other players, including its interacting partner, ZAP−70; activation regulators, Vav1 and RhoGDI and other small GTPases, such as RhoA, Rac1 and Cdc42. As such, RhoH and its associated proteins are potential attack points, especially in the treatment of cancer and immune-related diseases.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP3 and polyP4), whereas mxPpx2 preferred long-chain polyP60–70 and polyP700–1000. mxPpx2 was activated by 25–50 mM KCl, but mxPpx1 did not significantly depend on K+. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K+, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K+. The exopolyphosphatase activity of mxPpx1 toward polyP3 was inhibited by polyP700–1000 and that of mxPpx2 toward polyP60–70 and polyP700–1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP3 and polyP4 was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30–75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K+ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K+ for activation.