scholarly journals Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

1996 ◽  
Vol 16 (6) ◽  
pp. 2689-2699 ◽  
Author(s):  
R Foster ◽  
K Q Hu ◽  
Y Lu ◽  
K M Nolan ◽  
J Thissen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structural Features ◽  
Biochemical Properties ◽  
Small Gtpases ◽  
Evolutionary Divergence ◽  
Gtpase Activity ◽  
Activated State ◽  
Rho Protein ◽  
Rho Family

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.

scholarly journals Cytotoxic Necrotizing Factor Type 2 Produced by Pathogenic Escherichia coli Deamidates a Gln Residue in the Conserved G-3 Domain of the Rho Family and Preferentially Inhibits the GTPase Activity of RhoA and Rac1

1999 ◽  
Vol 67 (12) ◽  
pp. 6550-6557 ◽  
Author(s):  
Motoyuki Sugai ◽  
Kiyotaka Hatazaki ◽  
Akira Mogami ◽  
Hiroyuki Ohta ◽  
Sylvie Y. Pérès ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cultured Cells ◽  
Peptide Mapping ◽  
Small Gtpases ◽  
Amino Acid Sequences ◽  
Gtpase Activity ◽  
Toxic Activity ◽  
Cytotoxic Necrotizing Factor ◽  
Rho Family

ABSTRACT Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122RhoGAP. Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

scholarly journals Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

2002 ◽  
Vol 22 (9) ◽  
pp. 3140-3148 ◽  
Author(s):  
Emilio Hirsch ◽  
Michela Pozzato ◽  
Alessandro Vercelli ◽  
Laura Barberis ◽  
Ornella Azzolino ◽  
...  
Keyword(s):  
Pyramidal Neurons ◽  
Embryonic Stem ◽  
Large Family ◽  
Small Gtpases ◽  
Normal Morphology ◽  
Targeted Deletion ◽  
Cortical Pyramidal Neurons ◽  
Rho Family

ABSTRACT Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.

Phage display identification of age-associated TNFα-mediated cardiac oxidative induction

2004 ◽  
Vol 18 (3) ◽  
pp. 255-260 ◽  
Author(s):  
Jay M. Edelberg ◽  
Alvin Wong ◽  
Jacquelyne M. Holm ◽  
Munira Xaymardan ◽  
Inga Duignan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Binding Sites ◽  
Cyclic Peptide ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Lipid Peroxidation Product ◽  
Surface Binding ◽  
Cardiovascular Pathology

Age-associated alterations in the actions of tumor necrosis factor-α (TNFα) in the heart with impaired cardioprotective pathways and enhanced apoptotic induction may contribute to the increased severity of cardiovascular pathology in older persons. To identify the molecular events mediating these changes in the microvasculature of the aging rodent heart, the biochemical properties of in vivo phage-display cyclic peptide cardiac biopanning were studied. Analysis of individual amino acid positions revealed that the center of the peptide motif (amino acid position 4) had a significantly higher frequency of aromatic amino acid side chains in phage homing to the old hearts compared with young controls (18 mo old, 11% vs. 3 mo old, 3%, P < 0.05). This subset of phage motifs revealed an age-associated homology with oxidoreductase enzymes (homology: 18 mo, 7/7; 3 mo, 0/2), suggesting the substrates and/or binding sites of these enzymes are increased in the aging hearts. Immunostaining for the oxidoreductase substrate 4-hydroxy-2-nonenal (HNE), a cardiotoxic lipid peroxidation product, demonstrated a twofold higher density of HNE(+) cells in PBS-treated hearts of old mice (18 mo) compared with young controls (3 mo) (18 mo, 3.2 ± 2.8 vs. 3 mo, 1.0 ± 0.9 cells/HPF, P < 0.05). Moreover, intracardiac injection of TNFα resulted in a significantly greater increase in HNE staining in the old hearts (18 mo, 16.9 ± 13.8 vs. 3 mo, 9.1 ± 6.0 cells/HPF, P < 0.05). Overall, these studies demonstrate that aging-associated alterations in TNFα-mediated pathways with induction of reactive oxidative species and changes in vascular surface binding sites may contribute mechanistically to the increased cardiovascular pathology of the aging heart.

scholarly journals Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB).

2007 ◽  
Vol 54 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Paweł Filipkowski ◽  
Józef Kur
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Deinococcus Radiodurans ◽  
Thermus Thermophilus ◽  
Biochemical Properties ◽  
Thermus Aquaticus ◽  
Single Stranded Dna ◽  
Calculated Molecular Mass

To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5 degrees C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB).

Identification of key residues in the formate channel FocA that control import and export of formate

2014 ◽  
Vol 395 (7-8) ◽  
pp. 813-825 ◽  
Author(s):  
Doreen Hunger ◽  
Claudia Doberenz ◽  
R. Gary Sawers
Keyword(s):  
Amino Acid ◽  
Quaternary Structure ◽  
Salt Bridge ◽  
Structural Features ◽  
Amino Acid Residues ◽  
Form Part ◽  
Import And Export ◽  
Key Residues ◽  
Acid Exchange

Abstract The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases

2005 ◽  
Vol 288 (4) ◽  
pp. C863-C871 ◽  
Author(s):  
Ayako Makino ◽  
Michael Glogauer ◽  
Gary M. Bokoch ◽  
Shu Chien ◽  
Geert W. Schmid-Schönbein
Keyword(s):  
Signal Transduction ◽  
Shear Stress ◽  
Fluid Shear Stress ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Fluid Shear ◽  
Rho Family

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm2) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes.

scholarly journals S-Palmitoylation-Dependent Regulation of Cardiomyocyte Rac1 Signaling Activity and Cardiac Hypertrophy

2021 ◽  
Author(s):  
Matthew J Brody ◽  
Tanya A. Baldwin ◽  
Arasakumar Subramani ◽  
Onur Kanisicak ◽  
Ronald J Vagnozzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transgenic Mice ◽  
Cardiac Disease ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Transferase Activity ◽  
Related Enzyme ◽  
Rho Family ◽  
Novel Therapeutic Strategies

S-palmitoylation is a reversible lipid modification that regulates trafficking, localization, activity, and/or stability of protein substrates by serving as a fatty acid anchor to cell membranes. However, S-palmitoylation-dependent control of signal transduction in cardiomyocytes and its effects on cardiac physiology are not well understood. We performed an in vivo gain-of-function screen of zinc finger Asp-His-His-Cys (zDHHC) family S-acyl transferases that catalyze S-palmitoylation and identified the Golgi-localized enzyme zDHHC3 as a critical regulator of cardiac maladaptation. The closely-related enzyme, zDHHC7, also induced severe cardiomyopathy but this effect was not observed with overexpression of plasma membrane enzyme zDHHC5, endoplasmic reticulum enzyme zDHHC6, or Golgi enzyme zDHHC13. To identify effectors that may underlie zDHHC3-induced cardiomyopathy we performed quantitative site-specific S-acyl proteomics in zDHHC3-overexpressing cells that revealed the small GTPase Rac1 as a novel substrate. We generated cardiomyocyte-specific transgenic mice overexpressing zDHHC3, which develop severe cardiac disease. Cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice are preceded by enhanced S-palmitoylation of Rac1 and induction of additional Rho family small GTPases including RhoA, Cdc42, and the Rho family-specific chaperone RhoGDI. In contrast, transgenic mice overexpressing an enzymatically-dead mutant of zDHHC3 do not exhibit this profound induction of RhoGTPase signaling or develop cardiac disease. Rac1 S-palmitoylation, plasma membrane localization, activity, and downstream hypertrophic signaling were substantially increased in zDHHC3 overexpressing hearts. Taken together, these data suggest inhibition of zDHHC3/7 S-acyl transferase activity at the cardiomyocyte Golgi or disruption of Rac1 S-palmitoylation as novel therapeutic strategies to treat cardiac disease or other diseases associated with enhanced RhoGTPase signaling.

scholarly journals Rho-Family Small GTPases: From Highly Polarized Sensory Neurons to Cancer Cells

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 92 ◽  
Author(s):  
Takehiko Ueyama
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Small Gtpases ◽  
In Vivo Studies ◽  
Signal Pathways ◽  
Ros Production ◽  
Oxygen Species ◽  
Rho Family Gtpases ◽  
Rho Family

The small GTPases of the Rho-family (Rho-family GTPases) have various physiological functions, including cytoskeletal regulation, cell polarity establishment, cell proliferation and motility, transcription, reactive oxygen species (ROS) production, and tumorigenesis. A relatively large number of downstream targets of Rho-family GTPases have been reported for in vitro studies. However, only a small number of signal pathways have been established at the in vivo level. Cumulative evidence for the functions of Rho-family GTPases has been reported for in vivo studies using genetically engineered mouse models. It was based on different cell- and tissue-specific conditional genes targeting mice. In this review, we introduce recent advances in in vivo studies, including human patient trials on Rho-family GTPases, focusing on highly polarized sensory organs, such as the cochlea, which is the primary hearing organ, host defenses involving reactive oxygen species (ROS) production, and tumorigenesis (especially associated with RAC, novel RAC1-GSPT1 signaling, RHOA, and RHOBTB2).

RhoGDI-3, a promising system to investigate the regulatory function of rhoGDIs: uncoupling of inhibitory and shuttling functions of rhoGDIs

2005 ◽  
Vol 33 (4) ◽  
pp. 623-626 ◽  
Author(s):  
E. Dransart ◽  
A. Morin ◽  
J. Cherfils ◽  
B. Olofsson
Keyword(s):  
Membrane Extraction ◽  
Regulatory Function ◽  
Rho Proteins ◽  
Steady State Kinetics ◽  
Rho Protein ◽  
Rho Family ◽  
First Time ◽  
Kinetics Of

rhoGDIs (Rho GDP dissociation inhibitors) are postulated to regulate the activity and the localization of small G-proteins of the Rho family by a shuttling process involving extraction of Rho from donor membranes, formation of inhibitory cytosolic rhoGDI/Rho complexes, and delivery of Rho to target membranes. However, the role of rhoGDIs in site-specific membrane targeting or extraction of Rho is still poorly understood. We investigated here the in vivo functions of two mammalian rhoGDIs: the specific rhoGDI-3 and the well-studied rhoGDI-1 (rhoGDI) after structure-based mutagenesis. We identified two sites in rhoGDIs, forming conserved interactions with their Rho target, whose mutation results in the uncoupling of inhibitory and shuttling functions of rhoGDIs in vivo. Remarkably, these rhoGDI mutants were detected at Rho-induced membrane ruffles or protrusions, where they co-localized with RhoG or Cdc42, probably identifying for the first time the site of extraction of a Rho protein by a rhoGDI in vivo. We propose that these mutations act by modifying the steady-state kinetics of the shuttling process regulated by rhoGDIs, such that transient steps at the cell membranes now become detectable. They should provide valuable tools for future investigations of the dynamics of membrane extraction or delivery of Rho proteins and their regulation by cellular partners.

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