Exchange Of GDP For GTP Research Articles

The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Chromosomal translocation-derived aberrant Rab22a drives metastasis of osteosarcoma.

Osteosarcoma is a type of aggressive malignant bone tumour that frequently metastasizes to lungs, resulting in poor prognosis. However, the molecular mechanisms of lung metastasis of osteosarcoma remain poorly understood. Here we identify exon-intron fusion genes in osteosarcoma cell lines and tissues. These fusion genes are derived from chromosomal translocations that juxtapose the coding region for amino acids 1-38 of Rab22a (Rab22a1-38) with multiple inverted introns and untranslated regions of chromosome 20. The resulting translation products, designated Rab22a-NeoFs, acquire the ability to drive lung metastasis of osteosarcoma. The Rab22a1-38 moiety governs the function of Rab22a-NeoFs by binding to SmgGDS-607, a GTP-GDP exchange factor of RhoA. This association facilitates the release of GTP-bound RhoA from SmgGDS-607, which induces increased activity of RhoA and promotes metastasis. Disrupting the interaction between Rab22a-NeoF1 and SmgGDS-607 with a synthetic peptide prevents lung metastasis in an orthotopic model of osteosarcoma. Our findings may provide a promising strategy for a subset of osteosarcoma patients with lung metastases.

A phase I, open-label, dose-escalation trial of BI 1701963 as monotherapy and in combination with trametinib in patients with KRAS mutated advanced or metastatic solid tumors.

TPS3651 Background: Activating mutations of KRAS drive many types of cancer. Activation of KRAS relies on guanine nucleotide exchange factors, such as SOS1, to mediate exchange of GDP for GTP. BI 1701963 is a small-molecule protein–protein interaction inhibitor that prevents the interaction between KRAS and SOS. Binding of BI 1701963 to the catalytic site of SOS1 inhibits binding of SOS1 to RAS–GDP, thereby hindering the exchange from RAS–GDP (inactive form) to RAS–GTP (active form). In preclinical studies this has been shown to lead to cytostasis in cancer cells addicted to KRAS signaling. Methods: NCT04111458 is a first-in-human trial of BI 1701963 in patients aged ≥18 years with tumors harboring KRAS mutations. Primary objectives are to determine the maximum tolerated dose (MTD) and recommended Phase II dose of BI 1701963 as monotherapy and in combination with trametinib, based on dose-limiting toxicities (DLTs). Secondary objectives are to evaluate safety, tolerability, pharmacokinetics/-dynamics and preliminary efficacy. The study will have two arms (mono- and combination therapy), and be divided into dose escalation (Part A), confirmation (Part B) and expansion (Part C, combination only) phases. Inclusion criteria include activating KRAS mutation, ≥1 evaluable lesion (RECIST 1.1), ECOG PS ≤1 and adequate organ function. Exclusion criteria include history of: RAS, MAPK or SOS1 targeting therapies; retinal vein occlusion; retinal pigment epithelial detachment; and decreased cardiac function. Parts B and C will be conducted in patients with advanced NSCLC. Treatment will continue until confirmed clinical benefit, defined toxicities, or withdrawal of consent. The primary endpoints are: Part A, the MTD, and the number of patients with DLTs during Cycle 1; Part B (monotherapy), the number of patients with DLTs; Part C, objective response (OR, RECIST 1.1). Starting doses of BI 1701963 in Part A will be 50 mg once daily (QD) orally (monotherapy) and 100 mg QD (combination, once proved safe as monotherapy) and will be escalated until the MTD is reached. The starting dose of trametinib will be 1 mg QD, escalated to the MTD or a max. of 2 mg QD. Dose cohorts will include ≥3 patients, with two therapeutic relevant doses (TRDs) established in each arm. In Part B, patients will be randomized to groups receiving one of the TRDs. If an OR is observed at a TRD in the combination arm, additional patients will be recruited into expansion cohorts receiving the relevant dose. As of Feb 11, 2020 three patients have been treated. Clinical trial information: NCT04111458 .

Inactive 5‐HT7 Receptors Associate with Inactive and Active Gs Heterotrimers

G protein‐coupled receptors (GPCRs) interact cooperatively with agonists and heterotrimeric G proteins to form ternary complexes, leading to activation of G proteins. Following exchange of GDP for GTP the complex is thought to rapidly disassemble as nucleotide‐bound G proteins adopt conformations that have low affinity for GPCRs, and heterotrimers dissociate into Gα and Gβγ subunits. Unlike most GPCRs, serotonin 5‐HT7 receptors are known to preassemble with GDP‐bound Gs heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound Gs heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT7‐Gs coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT7‐Gs complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by Gs affects interactions with receptors, we replaced endogenous nucleotides with the non‐hydrolyzable analogs GTPγS or GDPβS in permeabilized, apyrase‐treated HEK293 cells. Unexpectedly, ligand‐mediated effects on 5‐HT7‐Gs complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT7‐Gs dissociation still occurred when only GDPβS was present, indicating that dissociation does not require Gs activation. Similarly, inverse agonist‐induced 5‐HT7‐Gs association still occurred when only GTPγS was present, suggesting that activated Gs heterotrimers retain affinity for inactive 5‐HT7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT7 receptors stabilized active Gs heterotrimers. These results reveal unconventional coupling of inactive 5‐HT7 receptors to both inactive and active Gs heterotrimers. This represents a significant departure from the standard allosteric model of GPCR‐G protein coupling, wherein G proteins interact primarily with active GPCRs, and GTP‐bound Gα subunits dissociate from both receptors and Gβγ subunits.Support or Funding InformationSupported by GM130142 (to N.A.L.)

Lysophosphatidic acid promotes lung carcinoma cell migration via ARHGEF17, a RhoGEF directly controlled by Gβγ

Cell migration plays an essential role in multiple physiological processes, such as the immune response. However, this process contributes to pathological situations such as inflammation and metastatic cancer. During cell migration, cells reshape their actin cytoskeleton, a process finely regulated by Rho family GTPases (RhoGTPases). RhoGEFs activate RhoGTPases by promoting the exchange of GDP for GTP. Thus, RhoGEFs represent a crucial point between activation of chemotactic receptors and remodeling of the actin cytoskeleton. In this sense, several RhoGEFs have been characterized as important for cancer cell migration. Here, we demonstrate that RhoGEF‐ARHGEF17 is important for lung carcinoma cell migration stimulated with lysophosphatidic acid (LPA). In addition, pull down experiments, to directly assess GEF activation, demonstrated that ARHGEF17 is stimulated by LPA via Gi (sensitive to pertussis toxin) and by Gβγ. Mechanistically, Gβγ recruits ARHGEF17 to the plasma membrane and disrupts intramolecular inhibitory interactions. Our results indicate a possible participation of ARHGEF17 in the metastatic dissemination of lung carcinoma cells, a possibility under investigation.

A mitochondrial recruitment assay to characterize a family of DENN‐domain proteins, Rab GDP/GTP exchange factors.

Intracellular membrane trafficking controls the levels, localization and functional activity of a myriad of proteins and is thus critical for cellular function. Rab GTPases are molecular switches that regulate membrane trafficking by toggling between GTP‐bound (active; membrane‐bound) and GDP‐bound (inactive; cytosolic) states. These GTPases are controlled largely by guanine nucleotide exchange factors (GEFs) that mediate the exchange of GDP for GTP. Mammalian cells possess 61 Rabs but many of their GEFs are unknown. One of the largest family of Rab GEFs contain an evolutionarily conserved protein module called the DENN domain. There are 18 DENN domain proteins but many of their corresponding Rab GTPase partner(s) are either not known or are controversial. This is due to the fact that all studies concerning DENN domains have used biochemical and in‐vitro assays, which do not reflect physiological conditions. Here we describe a cell‐based GEF assay as a method to identify GTPase substrates of DENN domain proteins. By overexpressing DENN domains fused to a mitochondrial targeting sequence we can drive the recruitment of physiological GTPase substrates to the mitochondrial membrane, which can then be detected by immunofluorescence analysis. We validated our assay with a known DENN/Rab pair, DENND1A and Rab35. Using a panel of GFP‐tagged Rabs we have resolved controversies over previously reported DENN/Rab pairs and have identified new Rab substrates for DENN domains. Identifying the full complement of DENN/Rab pairs will provide new insights into membrane trafficking and cellular function.Support or Funding Information1) Canada First Research Excellence Fund, awarded to McGill University for Healthy Brains for Healthy Lives. 2) ALS Canada

Photo-Control of RAS Nucleotide Exchange Reaction using Peptide Inhibitor Modified with Spiropyran Derivative

Ras is a kind of GTPase protein, and it regulates cellular signal transduction. For example, some information such as cell growth and cell differentiation from in vitro to in vivo of downstream by cycling between GTP active form and GDP inactive form. GDP-GTP exchange of Ras is induced by binding of GEF(Guanine Exchange Factor) to Ras. Recently some kinds of peptide which bind to GEF binding region or allosteric binding region. Then, they cause inhibition of nucleotide exchange, were reported. It is known that mutated Ras (G12D etc.) continues transducing permanently, so it is the factor of making cancer cells. Then, it is called cancer gene. From this, the study that develops molecule target medicine only abnormality protein is recognized and controlled is attempted so as not to affect normal cells. Photochromic molecule, spiropyran isomerize between ring-closed spiro (SP) and ring-opened merocyanine(MC) forms photo-reversibly upon visible and ultraviolet lights. It is known that MC form of spiropyran has a polarity, so it exhibits electric interaction each other at peptide. Therefore, peptide also change the secondary structure between alpha-helix and random coil. In this study, we designed and synthesized peptide inhibitor of Ras based on interface structure between Ras and GEF. The synthesized petide RRFCGIYCTNACKTCEGNRR composed of four cysteine residues. These cysteine residues were modified with SP-maleimide(SP-MA) stoichiometrically. The secondary structural change of the SOS mimic peptide induce by photo-isomerization of SP-MA were examined by CD spectrum. The photo-control of nucleotide exchange of Ras using the peptide inhibitor was examined. We also synthesized several peptide inhibitors which have different number of cysteine residues and examined the inhibitory effect of the peptides.

Plant receptor-like kinase signaling through heterotrimeric G-proteins.

Heterotrimeric G-proteins regulate multiple aspects of plant growth, development, and response to biotic and abiotic stresses. While the core components of heterotrimeric G-proteins and their basic biochemistry are similar in plants and metazoans, key differences exist in their regulatory mechanisms. In particular, the activation mechanisms of plant G-proteins appear diverse and may include both canonical and novel modes. Classical G-protein-coupled receptor-like proteins exist in plants and interact with Gα proteins, but their ability to activate Gα by facilitating GDP to GTP exchange has not been demonstrated. Conversely, there is genetic and functional evidence that plant G-proteins interact with the highly prevalent receptor-like kinases (RLKs) and are phosphorylated by them. This suggests the exciting scenario that in plants the G-proteins integrate RLK-dependent signal perception at the plasma membrane with downstream effectors. Because RLKs are active kinases, it is also likely that the activity of plant G-proteins is regulated via phosphorylation/dephosphorylation rather than GTP–GDP exchange as in metazoans. This review discusses our current knowledge of the possible RLK-dependent regulatory mechanisms of plant G-protein signaling in the context of several biological systems and outlines the diversity that might exist in such regulation.

NMR resonance assignments for the active and inactive conformations of the small G protein RalA

The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1H, 15 N and 13C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.

Colorimetric RhoB GTPase Activity Assay.

The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the GTPase-activating proteins (GAPs), which enable hydrolysis of GTP leading to the Rho GTPase inactivation. Here, we describe a detailed protocol to perform a RhoB G-LISA activation assay to detect the level of GTP-loaded RhoB in vitro. This is the first colorimetric assay designed to specifically measure RhoB activation. This method was developed by adapting the RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc.) and allow the precise measurement of RhoB activity in less than 3 hours. This rapid methodology can be broadly used to assess the level of GTP-loaded RhoB in any kind of cellular models, to appreciate either the role RhoB activation in physiological processes, diseases, oncogenic transformation or for drug discovery in high throughput screens.

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D2 (PGD2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented l-PGDS-mediated recycling of DP1, and l-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by l-PGDS. Confocal microscopy revealed that DP1 activation induces l-PGDS/Rab4 co-localization. l-PGDS/Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-l-PGDS and His6-Rab4 indicated that both proteins interact directly. l-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that l-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in l-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTPγS loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

Planar Cell Polarity Effector Proteins Inturned and Fuzzy Form a Rab23 GEF Complex.

SummaryA subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells [1, 2, 3, 4, 5, 6]. Rab11 and Rab8, together with their GDP-GTP exchange factors (GEFs), TRAPP-II and Rabin8, promote recruitment of the ciliary vesicle to the mother centriole and its subsequent maturation, docking, and fusion with the cell surface [2, 3, 4, 5]. Rab23 has been linked to cilium formation and membrane trafficking at mature cilia [1, 7, 8]; however, the identity of the GEF pathway activating Rab23, a member of the Rab7 subfamily of Rabs, remains unclear. Longin-domain-containing complexes have been shown to act as GEFs for Rab7 subfamily GTPases [9, 10, 11, 12]. Here, we show that Inturned and Fuzzy, proteins previously implicated as planar cell polarity (PCP) effectors and in developmentally regulated cilium formation [13, 14], contain multiple longin domains characteristic of the Mon1-Ccz1 family of Rab7 GEFs and form a specific Rab23 GEF complex. In flies, loss of Rab23 function gave rise to defects in planar-polarized trichome formation consistent with this biochemical relationship. In cultured human and mouse cells, Inturned and Fuzzy localized to the basal body and proximal region of cilia, and cilium formation was compromised by depletion of either Inturned or Fuzzy. Cilium formation arrested after docking of the ciliary vesicle to the mother centriole but prior to axoneme elongation and fusion of the ciliary vesicle and plasma membrane. These findings extend the family of longin domain GEFs and define a molecular activity linking Rab23-regulated membrane traffic to cilia and planar cell polarity.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GαT helical domain (αHD) contacting the Gβγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gβγ during receptor-catalyzed nucleotide exchange.

Structural basis for the inhibition of translation through eIF2\u03b1 phosphorylation

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58–63 with eIF2Bδ leading to a competition with Met-tRNAi.

Physiological Omics Identifies Mechanisms that Attenuate Renal Injury and Blood Pressure in Dahl salt‐sensitive Arhgef11−/− Rats

Arhgef11 is a Rho guanine nucleotide exchange factor previously implicated in kidney injury in the Dahl salt‐sensitive rat (SS‐WT). Through exchange of GDP for GTP, Arhgef11 regulates cytoskeletal structure, function, and cell‐cell contacts through a variety of stimuli (e.g., G‐protein coupled receptors, growth factors, and shear stress). Genetic studies and reduced Arhgef11 expression in an SS‐Arhgef11SHR‐minimal congenic strain (SHR allele substituted for S allele) significantly decreased proteinuria, fibrosis, and improved renal hemodynamics, without impacting BP compared to the SS‐WT. We hypothesized that actin cytoskeleton regulation through Arhgef11 impacts functional changes in protein reuptake and sodium handling in proximal tubules. Here, we studied the SS‐Arhgef11−/− rat model at 4–12 weeks of age under low and high salt (0.3% or 2% NaCl). On low‐salt, starting at week 6 and continuing through week 12, SS‐Arhgef11−/− animals (n=17) demonstrated significantly reduced proteinuria (week 6, −43.9.0 ± 13.6 mg/24 hours, p=0.0079 and week 12, −102.8 ± 13.6, p<0.0001) compared to SS‐WT animals (n=16). On high‐salt, beginning at week 6, SS‐Arhgef11−/− animals (n=15) demonstrated a significant attenuation of proteinuria from weeks 8 to 12 (week 8, −83.9.0 ± 13.9 mg/24 hours, p<0.0001 and week 12, −118.2 ± 13.9, p<0.0001), along with a substantial reduction in BP (week 12, −42 ± 5 mm Hg, p<0.0001) compared to SS‐WT (n=17). Kidney pathology indices were all significantly improved in SS‐Arhgef11−/− compared to SS‐WT animals (all p<0.05), including glomerular injury/hypertrophy, tubulointerstitial injury/fibrosis, and immune cell infiltration (via flow cytometry). To better understand the molecular mechanisms associated with renal protection from loss of Arhgef11, both RNA sequencing and MS/MS proteomics was performed on kidney from SS‐Arhgef11−/− and SS‐WT at week 4 (before onset of renal injury/proteinuria between groups) and at week 12 (low‐salt). At week 4, 416 genes were differentially expressed between groups, whereas 1017 genes and 363 proteins were observed at week 12 (low‐salt). As anticipated, the ‐omics datasets suggest that loss of Arhgef11 (SS‐Arhgef11−/−) initiates early transcriptome/protein changes in the actin cytoskeleton. Of note, reuptake (endocytosis) of filtered albumin by megalin and cubilin and a large number of solute carrier genes [(n=57–63, 2.2 fold‐enrichment (4.22×10−12)] were upregulated. Many of the genes and proteins in this pathway are involved in tubular sodium transport (Slc5a= Na+/glucose; Slc6a= Na+/Cl−; Slc8a=Na+/Ca2+; Slc9a3= Na+/H+; and Slc34a= Na+‐Pi). In summary, in vivo phenotyping and multi‐omics techniques provides strong evidence that increased Arhgef11 expression in the Dahl S rat leads to actin cytoskeleton mediated changes in cell morphology and cell function (impaired reuptake filtered protein) that promote kidney injury, hypertension, and decline in kidney function.Support or Funding Information1R01HL137673 (MRG). The work performed through the UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including P20GM103476, P20GM104357, and P20GM121334.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability.

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.

Photo-Control of RAS GDP-GTP Exchange using the SOSαH Mimicking Peptides Modified with Spiropyran Derivative

Ras is a kind of GTPase protein, and it regulates cellular signal transduction. For example, some information such as cell growth and cell differentiation from in vitro to in vivo of downstream by cycling between GTP active form and GDP inactive form. GDP-GTP exchange of Ras is induced by binding of GEF(Guanine Exchange Factor) to Ras. Recently some kinds of peptide which interfere binding of GEF resulting in inhibition of GDP-GTP exchange, were reported. Synthetic peptide mimicking αH helix of SOS inhibited the Ras GDP-GTP exchange in the presence of SOS (Son Of Sevenless). Photochromic molecule, spiropyran isomerize between ring-closed spiro (SP) and ring-opened merocyanine (MC) forms photo-reversibly upon visible and ultraviolet lights. It is known that the zwitterion of MC forms exhibit dimerization. Previously, it has been demonstrated that the peptides modified with spiropyran derivatives change their secondary structure accompanied by formation of spiropyran dimerization photoreversibly. In this study, we designed and synthesized the peptides which have two cysteines and analogous to the SOS αH-helix. The two cysteine residues of the peptides were modified with SP-maleimide(SP-MA) stoichiometrically. The spiropyran incorporated into the peptide exhibited SP-MC isomerization upon visible light and ultraviolet light irradiations. The secondary structural change of the SOS mimic peptide induce by photo-isomerization of SP-MA were examined. Furthermore, photoreversible inhibition of Ras GDP-GTP exchange in the presence of SOS with the photochromic peptide was analyzed using fluorescent GDP analogues.

Photo-Control of Small GTPase RAS using Photo Responsive Peptide Inhibitor Which Mimic αH Helix of SOS

Small GTPase Ras is known as a central regulator of several cellular signaling which are related to cell viability, growth, differentiation and apoptosis. Ras is already studied well at molecular level and they have two state, GTP binding (active) state and GDP-binding (inactive) state. These switching of Ras activity was regulated by two factors, GEFs and GAPs. GEFs serves as Ras activator and they replace GDP from Ras and attach GTP to Ras alternatively. While, GAPs serves as Ras inactivator and they accelerate Ras GTPase. Previously it has been reported that SOSαH mimicking peptide which has cyclic structure at N-terminal region exhibited stablea-helix structure, and inhibit Ras GDP-GTP exchange activity competitively with SOS. In this study, we focused on the interaction between Ras and aH-helix which locates Ras interface of SOS (Ras-GEF) in order to photo-regulate Ras function artificially. Several kinds of αH mimicking peptides which have two cysteine to be crosslinked were synthesized. The cysteine residues of the peptides were crosslinked by bifunctional azobenzene derivatives, azobenzene di-maleimide (ABDM) intramolecularly. The ABDM incorporated into the peptides showed cis-trans isomerization upon ultraviolet (UV) light and visible light irradiations. CD spectral analysis revealed that trans-ABDM-αH peptide forms partially ahelix and cis-ABDM-aH peptide exhibit random secondary structure. SOS dependent GDP-GTP exchange reaction of Ras was inhibited by trans- ABDM-aH peptide. Furthermore, Ex vivo experiment revealed the SOS mimicking peptides exhibit cell membrane permeation indicating the photochromic peptides are applicable to regulation of cell function.