Dissociation Inhibitor Research Articles

Epitope analysis of human monoclonal antibodies from a patient with autoimmune factor XIII deficiency reveals their inhibitory mechanisms.

Autoimmune coagulation factor XIII (FXIII) deficiency (AiF13D) is a bleeding disorder caused by anti-FXIII autoantibodies. Recently, we generated human monoclonal antibodies (mAbs) from the peripheral blood of an AiF13D patient and classified them into three groups: FXIII-dissociation inhibitor, FXIII-assembly inhibitor, and non-neutralizing/inhibitory mAbs. However, the epitope region and molecular inhibitory mechanism of each mAb remain unknown. Here, we localized the epitope regions of the representative inhibitory mAbs A69K (dissociation inhibitor) and A78L (assembly inhibitor) to the β-barrel-2 domain and boundary of β-barrel-1&2 domains, respectively, of the FXIII-A subunit, by combining a binding assay using its synthesized peptides and a protease-protection assay. Our findings suggest that A69K inhibits the activation-related conformational changes and dissociation of FXIII and that A78L competitively inhibits FXIII-assembly.

RNF115 Inhibits the Post-ER Trafficking of TLRs and TLRs-Mediated Immune Responses by Catalyzing K11-Linked Ubiquitination of RAB1A and RAB13.

The subcellular localization and intracellular trafficking of Toll‐like receptors (TLRs) critically regulate TLRs‐mediated antimicrobial immunity and autoimmunity. Here, it is demonstrated that the E3 ubiquitin ligase RNF115 inhibits the post‐endoplasmic reticulum (ER) trafficking of TLRs and TLRs‐mediated immune responses by catalyzing ubiquitination of the small GTPases RAB1A and RAB13. It is shown that the 14‐3‐3 chaperones bind to AKT1‐phosphorylated RNF115 and facilitate RNF115 localizing on the ER and the Golgi apparatus. RNF115 interacts with RAB1A and RAB13 and catalyzes K11‐linked ubiquitination on the Lys49 and Lys61 residues of RAB1A and on the Lys46 and Lys58 residues of RAB13, respectively. Such a modification impairs the recruitment of guanosine diphosphate (GDP) dissociation inhibitor 1 (GDI1) to RAB1A and RAB13, a prerequisite for the reactivation of RAB proteins. Consistently, knockdown of RAB1A and RAB13 in Rnf115 +/+ and Rnf115 −/− cells markedly inhibits the post‐ER and the post‐Golgi trafficking of TLRs, respectively. In addition, reconstitution of RAB1AK49/61R or RAB13K46/58R into Rnf115 +/+ cells but not Rnf115 −/− cells promotes the trafficking of TLRs from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface, respectively. These findings uncover a common and step‐wise regulatory mechanism for the post‐ER trafficking of TLRs.

Targeted disruption of Rab1a causes early embryonic lethality.

Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5-14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a β-galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for β-galactosidase to determine the tissue-specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild-type and heterozygous Rab1a+/− mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.

SLIM: A Short‐Linked, Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

AbstractThe understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.

Molecular switch-like regulation in motor proteins.

Motor proteins are powered by nucleotide hydrolysis and exert mechanical work to carry out many fundamental biological tasks. To ensure their correct and efficient performance, the motors' activities are allosterically regulated by additional factors that enhance or suppress their NTPase activity. Here, we review two highly conserved mechanisms of ATP hydrolysis activation and repression operating in motor proteins-the glutamate switch and the arginine finger-and their associated regulatory factors. We examine the implications of these regulatory mechanisms in proteins that are formed by multiple ATPase subunits. We argue that the regulatory mechanisms employed by motor proteins display features similar to those described in small GTPases, which require external regulatory elements, such as dissociation inhibitors, exchange factors and activating proteins, to switch the protein's function 'on' and 'off'. Likewise, similar regulatory roles are taken on by the motor's substrate, additional binding factors, and even adjacent subunits in multimeric complexes. However, in motor proteins, more than one regulatory factor and the two mechanisms described here often underlie the machine's operation. Furthermore, ATPase regulation takes place throughout the motor's cycle, which enables a more complex function than the binary 'active' and 'inactive' states.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Rho GDP-dissociation inhibitor α is a potential prognostic biomarker and controls telomere regulation in colorectal cancer.

Rho GDP‐dissociation inhibitor α (RhoGDIα) is an essential regulator for Rho GTPases. Although RhoGDIα may serve as an oncogene in colorectal cancer (CRC), the underlying mechanism is still unclear. We investigated the function, mechanism, and clinical significance of RhoGDIα in CRC progression. We founded that downregulation of RhoGDIα repressed CRC cell proliferation, motility, and invasion. Overexpression of RhoGDIα increased DNA damage response signals at telomeres, and led to telomere shortening in CRC cells, also being validated in 26 pairs of CRC tissues. Mechanistic studies revealed that RhoGDIα could promote telomeric repeat factor 1 (TRF1) expression through the phosphatidylinositol 3‐kinase–protein kinase B signal pathway. Moreover, RhoGDIα protein levels were strongly correlated with TRF1 in CRC tissues. A cohort of 297 CRC samples validated the positive relationship between RhoGDIα and TRF1, and revealed that RhoGDIα and TRF1 levels were negatively associated with CRC patients' survival. Taken together, our results suggest that RhoGDIα regulate TRF1 and telomere length and may be novel prognostic biomarkers in colorectal cancer.

Are There Rab GTPases in Archaea?

A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins.

Protective Role of Rho Guanosine Diphosphate (GDP)‐Dissociation Inhibitor Ly‐GDI in Acute Pulmonary Alveolitis

Ly‐GDI is an inhibitory protein of Rho GTPases. Growing evidences indicate that Ly‐GDI plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly‐GDI in inflammation remains largely unknown. In the current study, we found that Ly‐GDI expression was significantly decreased in the IgG immune complex‐injured lungs. To determine if Ly‐GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly‐GDI (Adeno‐Ly‐GDI). In vivo mouse lung expression of Ly‐GDI resulted in a significant attenuation of IgG immune complex‐induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly‐GDI over‐expression in the lungs produced significant less inflammatory mediators (TNF‐α, IL‐6, MCP‐1, and MIP‐1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno‐GFP. Mechanically, IgG immune complex‐induced NF‐κB activity was markedly suppressed by Ly‐GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly‐GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF‐kB activity.

AB0126 Autoantibodies to d4-gdi from sera of patients with systemic lupus erythematosus are modulators of autophagy

BackgroundIn a previous study we demonstrated that serum autoantibodies from patients with Systemic Lupus Erythematosus (SLE) were able to induce autophagy in peripheral lymphocytes [1]. Moreover, it is firmly established...

ARHGDIA: a novel gene implicated in nephrotic syndrome

BackgroundCongenital nephrotic syndrome arises from a defect in the glomerular filtration barrier that permits the unrestricted passage of protein across the barrier, resulting in proteinuria, hypoalbuminaemia, and severe oedema. While...

ARHGDIA, a mutant TP53‐associated Rho GDP dissociation inhibitor, is over‐expressed in gene expression profiles of TP53 disrupted chronic lymphocytic leukaemia cells

<i><scp>ARHGDIA</scp></i>, a mutant <scp>TP</scp>53‐associated Rho <scp>GDP</scp> dissociation inhibitor, is over‐expressed in gene expression profiles of <i><scp>TP</scp>53</i> disrupted chronic lymphocytic leukaemia cells

Targeting S100P Inhibits Colon Cancer Growth and Metastasis by Lentivirus-Mediated RNA Interference and Proteomic Analysis

S100P was recently found to be overexpressed in a variety of cancers and is considered a potential target for cancer therapy, but the functional role or mechanism of action of S100P in colon cancer is not fully understood. In the present study, we knocked down the gene expression of S100P in colon cancer cells using lentivirus-mediated RNA interference. This step resulted in significant inhibition of cancer cell growth, migration and invasion in vitro and tumor growth and liver metastasis in vivo. Moreover, S100P downstream target proteins were identified by proteomic analysis in colon cancer DLD-1 cells with deletion of S100P. Knockdown of S100P led to downregulation of thioredoxin 1 and β-tubulin and upregulation of Rho guanosine diphosphate (GDP) dissociation inhibitor α (RhoGDIA), all potential therapeutic targets in cancer. Taken together, these findings suggest that S100P plays an important role in colon tumorigenesis and metastasis, and the comprehensive and comparative analyses of proteins associated with S100P could contribute to understanding the downstream signal cascade of S100P, leading to tumorigenesis and metastasis.

Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI)

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

The majority of small G-proteins undergo posttranslational modifications (e.g., isoprenylation) at their C-terminal cysteine residues. Such modifications increase their hydrophobicity, culminating in translocation of the modified proteins to their relevant membranous sites for interaction with their respective effectors. Previously, we reported glucose-dependent activation and membrane association of Rac1 in INS 832/13 cells. We also demonstrated modulatory roles for Rac1/GDP dissociation inhibitor in glucose-stimulated insulin secretion (GSIS) in INS 832/13 cells, further affirming roles for Rac1 in GSIS. Herein, we demonstrate that geranylgeranyltransferase inhibitor-2147 (GGTI-2147), an inhibitor of protein prenylation, markedly increased cytosolic accumulation of Rac1 and elicited significant inhibition of GSIS from INS 832/13 cells. In the current study, we also examined the localization of protein prenyltransferases (PPTases) and regulation of GSIS by PPTases in INS 832/13 cells. Western blot analyses indicated that the regulatory alpha-subunit and the structural beta-subunit of PPTase holoenzyme are predominantly cytosolic in their distribution. Overexpression of an inactive mutant of the regulatory alpha-subunit of PPTase markedly attenuated glucose- but not KCl-induced insulin secretion from INS 832/13 cells. Together, our findings provide the first evidence for the regulation of GSIS by PPTase in INS 832/13 cells. Furthermore, they support our original hypothesis that prenylation of specific G-proteins may be necessary for GSIS.

The Presence of a Leu-Gly-Asn Repeat–Enriched Protein (LGN), a Putative Binding Partner of Transducin, in ROD Photoreceptors

Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.

Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase.

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells whose function is governed by the guanosine diphosphate (GDP) dissociation inhibitor (RabGDI). Using a combination of chemical synthesis and protein engineering, we generated and crystallized the monoprenylated Ypt1:RabGDI complex. The structure of the complex was solved to 1.5 angstrom resolution and provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins. Isoprenoid binding requires a conformational change that opens a cavity in the hydrophobic core of its domain II. Analysis of the structure provides a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

A cell-specific, prenylation-independent mechanism regulates targeting of type II RACs.

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.

5] Antibody and oligonucleotide probes to distinguish intracellular expression and localization patterns of rab GDP-dissociation inhibitor isoforms

This chapter describes a strategy to distinguish the endogenous expression and localization patterns of the guanosine diphosphate ( GDP) dissociation inhibitor (GDI) family members. Methods used to express and purify GDI-2 and GDI- β in bacterial hosts are also described. GDIs likely represent the only proteins fully competent to chaperone Rabs to their correct destination. Consistent with this requirement are several reports documenting additional important functions of GDI on membranes. Hence, GDI-1 contributes to the specific recognition of the Rab membrane targets, stimulates GTP/GDP exchange, and dissociates thereafter back into the cytosol, a step likely promoted by GDP dissociation factor(s) and cellular redox state. Three mammalian GDI genes, GDI-1, GDI-2, and GDI- β , have been cloned to date. They encode ubiquitously expressed, highly homologous protein isoforms, tightly conserved in mammals. The three GDIs are found equally capable of operating with various Rabs in cell-free systems, indicating that, at least in resting cells, no specificity toward an individual Rab or subset of Rab proteins is associated with GDI function. However, given the differential intracellular localization of GDI-1 vs. GDI-2 and the subtle regulation of Rab-GDI complex formation in the context of living cells, functional differences among GDIs can be anticipated.

Search for novel proteins involved in the development of chemoresistance in colorectal cancer and fibrosarcoma cells in vitro using two-dimensional electrophoresis, mass spectrometry and microsequencing.

In search of novel mechanisms that may lead to the development of chemoresistance of malignant tumors of the large bowel we used two-dimensional electrophoresis to identify proteins that were overexpressed in colorectal and fibrosarcoma cell lines that were resistant towards mitoxantrone. This cytostatic drug is known to lead to atypical multidrug resistance, i.e., the classical mechanism of multidrug resistance (MDR) accompanied by the overexpression of P-glycoprotein (P-gp) is ineffective. Using mass spectrometry and microsequencing we found adenine phosphoribosyl transferase and breast cancer specific gene 1 (BCSG1) overexpressed in the resistant colorectal tumor cell line. In the chemoresistant fibrosarcoma cell line we found two proteins that were overexpressed. One was identified as Rho-guanine dinucleotide phosphate (Rho-GDP) dissociation inhibitor and the other had sequence homologies with yeast protein yer-7. The putative role of these proteins is discussed.

Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity

RhoA/B/C and CDC42/Rac, which form two subgroups of the rho guanosine triphosphatase (GTPase) family, regulate various aspects of actin cytoskeleton organisation. In cytosol, guanosine diphosphate (GDP) dissociation inhibitor (GDI) interacts with and maintains rho GTPases in their inactive GDP-bound form. RhoGDI is a ubiquitously expressed GDI, whereas D4/LyGDI is hematopoietic cell-specific and 10-fold less potent than RhoGDI in binding to and regulating rho GTPases. We have combined microanalytical liquid chromatography with the use of specific antibodies in order to separate D4/LyGDI and RhoDGI-complexes from the cytosol of U937 cells and to demonstrate that the two GDIs associate with different rho protein partners. RhoGDI can form a complex with CDC42Hs, RhoA, Rac1 and Rac2, while none of these GTPases was found to interact with D4/LyGDI. In addition, we found that stimulation of U937 cells with phorbol ester leads to phosphorylation of D4/LyGDI. Our results suggest that LyGDI forms complexes with specific rho GTPases expressed in hematopoietic cells where it may regulate specific pathways.