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We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host’s inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow–derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-β production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1–IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNβ production in bronchoalveolar lavage fluids from COVID-19–positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-β production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Acetylcholine activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) in microglia attenuates neuroinflammation and regulates TNF‐α release. We used lipopolysaccharide to model inflammation in the microglial cell line EOC20 and examined signaling by the α7 nAChR. Co‐immunoprecipitation experiments confirm that α7 nAChRs bind heterotrimeric G proteins in EOC20 cells. Interaction with Gαi mediates α7 nAChR signaling via enhanced intracellular calcium release and a decrease in cAMP, p38 phosphorylation, and TNF‐α release. These α7 nAChR effects were blocked by the inhibition of Gαi signaling via pertussis toxin, PLC activity with U73122, and α7 nAChR channel activity with the selective antagonist α‐bungarotoxin. Moreover, α7 nAChR signaling in EOC20 cells was significantly diminished by the expression of a dominant‐negative α7 nAChR, α7345‐8A, shown to be impaired in G protein binding. These findings indicate an essential role for G protein coupling in α7 nAChR function in microglia leading to the regulation of inflammation in the nervous system.

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on the heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

TDP-43 is a well known RNA binding protein involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia (FTLD). In physiological conditions, TDP-43 mainly localizes in the nucleus and shuttles, at least in neurons, to the cytoplasm to form TDP-43 RNA granules. In the nucleus, TDP-43 participates to the expression and splicing of RNAs, while in the cytoplasm its functions range from transport to translation of specific mRNAs. However, if loss or gain of these TDP-43 functions are affected in ALS/FTLD pathogenesis is not clear. Here, we report that TDP-43 localizes on ribosomes not only in primary neurons but also in SH-SY5Y human neuroblastoma cells. We find that binding of TDP-43 to the translational machinery is mediated by an interaction with a specific ribosomal protein, RACK1, and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect which is rescued by overexpression of RACK1. Ribosomal loss of RACK1, which excludes TDP-43 from the translational machinery, remarkably reduces formation of TDP-43 cytoplasmic inclusions in neuroblastoma cells. Finally, we corroborate the interaction between TDP-43 and RACK1 on polyribosomes of neuroblastoma cells with mis-localization of RACK1 on TDP-43 positive cytoplasmic inclusions in motor neurons of ALS patients. In conclusions, results from this study suggest that TDP-43 represents a translational repressor not only for specific mRNAs but for overall translation and that its binding to polyribosomes through RACK1 may promote, under conditions inducing ALS pathogenesis, the formation of cytoplasmic inclusions.

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ.

Huntington's disease (HD) is a lethal neurodegenerative disorder for which no cure is available yet. It is caused by abnormal expansion of a CAG triplet in the gene encoding the huntingtin protein (Htt), with consequent expansion of a polyglutamine repeat in mutated Htt (mHtt). This makes mHtt highly unstable and aggregation prone. Soluble mHtt is linked to cytotoxicity and neurotoxicity, whereas mHtt aggregates are thought to be neuroprotective. While Htt and mHtt are ubiquitously expressed throughout the brain and peripheral tissues, HD is characterized by selective degradation of the corpus striatum, without notable alterations in peripheral tissues. Screening for mRNAs preferentially expressed in rodent striatum led to the discovery of a GTP binding protein homologous to Ras family members. Due to these features, the newly discovered protein was termed Ras Homolog Enriched in Striatum (RHES). The aetiological role of RHES in HD has been ascribed to its small ubiquitin-like modifier (SUMO)-E3 ligase function. RHES sumoylates mHtt with higher efficiency than wild-type Htt, thereby protecting mHtt from degradation and increasing the amounts of the soluble form. Although RHES is an attractive target for HD treatment, essential information about protein structure and function are still missing. With the aim of investigating RHES 3D structure and function, bioinformatic analyses and molecular modelling have been performed in the present study, based on which, RHES regions predicted to be involved in the interaction with mHtt or the SUMO-E2 ligase Ubc9 have been identified. These regions have been used to design peptides aimed at inhibiting RHES interactions and, therefore, mHtt sumoylation; in turn, these peptides will be used to develop small molecule inhibitors by both rational design and virtual screening of large compound libraries. Once identified, RHES sumoylation inhibitors may open the road to the development of therapeutic agents against the severe, and currently untreatable, HD.

Investigations on prostate inflammation-related disorders, including acute and chronic prostatitis, chronic pelvic pain syndrome, benign prostate hyperplasia (BPH), and prostate cancer (PCa), are still ongoing to find new, accurate, and noninvasive biomarkers for a differential diagnosis of those pathological conditions sharing some common macroscopic features. Moreover, an ideal biomarker should be useful for risk assessment of prostate inflammation progression to more severe disorders, like BPH or PCa, as well as for monitoring of treatment response and prognosis establishment in carcinoma cases. Recent literature evidence highlighted that changes in the expression of transglutaminases, enzymes that catalyze transamidation reactions leading to posttranslational modifications of soluble proteins, occur in prostate inflammation-related disorders. This review focuses on the role specifically played by transglutaminases 4 (TG4) and 2 (TG2) and suggests that both isoenzymes hold a potential to be included in the list of candidates as novel diagnostic biomarkers for the above-cited prostate pathological conditions.

Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.

The spatiotemporal control of mitotic exit is crucial for faithful chromosome segregation during mitosis. In budding yeast, the mitotic exit network (MEN) drives cells out of mitosis, whereas the spindle position checkpoint (SPOC) blocks MEN activity when the anaphase spindle is mispositioned. How the SPOC operates at a molecular level remains unclear. Here, we report novel insights into how mitotic signalling pathways orchestrate chromosome segregation in time and space. We establish that the key function of the central SPOC kinase, Kin4, is to counterbalance MEN activation by the cdc fourteen early anaphase release (FEAR) network in the mother cell compartment. Remarkably, Kin4 becomes dispensable for SPOC function in the absence of FEAR. Cells lacking both FEAR and Kin4 show that FEAR contributes to mitotic exit through regulation of the SPOC component Bfa1 and the MEN kinase Cdc15. Furthermore, we uncover controls that specifically promote mitotic exit in the daughter cell compartment., The mitotic exit network (MEN) triggers mitotic exit and can be blocked by the spindle position checkpoint (SPOC). Here the authors show that SPOC kinase Kin4 counterbalances MEN activation by the Cdc fourteen early anaphase release (FEAR) network in the mother cell and that in the absence of FEAR mitotic exit requires daughter cell-confined factors.

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [α-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease.

The GTPase Ypt7p from S. cerevisiae is involved in late endosome-to-vacuole transport and homotypic vacuole fusion. We present crystal structures of the GDP- and GppNHp-bound conformation of Ypt7p solved at 1.35 and 1.6 Å resolution, respectively. Despite the similarity of the overall structure to other Ypt/Rab proteins, Ypt7p displays small but significant differences. The Ypt7p-specific residues Tyr33 and Tyr37 cause a difference in the main chain trace of the RabSF2 region and form a characteristic surface epitope. Ypt7p•GppNHp does not display the helix α2, characteristic of the Ras-superfamily, but instead possess an extended loop L4/L5. Due to insertions in loops L3 and L7, the neighboring RabSF1 and RabSF4 regions are different in their conformations to those of other Ypt/Rab proteins.

Inductive interactions between different cell layers have an extremely important role in early embryogenesis. One of the most intensively studied and best characterised of these is the induction of neural tissue from ectodermal cells by the dorsal mesoderm. The competence of ectodermal cells to respond to neural induction varies according to dorsal-ventral position; with dorsal ectoderm (much of which forms the neural plate) having a far higher competence. Here we show that overexpression of the nucleotide exchange factor lfc increases ectodermal competence for neural induction as well as the amount of neural tissue in the whole embryo. Lfc is expressed pan ectodermally soon after gastrulation and may respond to an early determinant of dorsal ectoderm. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) vβ3, β1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.

We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 microg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the Vmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+ (154 +/- 13 microM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

The effects of exogenous prostaglandins, inflammatory mediators known to be increased in pancreatic beta-cells by IL-1 beta, on the replication and long-term insulin secretion by beta-cells were investigated. Prostaglandins E1, E2, and F2 alpha suppressed beta-cell proliferation and long-term insulin secretion, thus mimicking the effects of IL-1 beta. The actions of prostaglandins were not prevented by pertussis toxin pretreatment. Additionally, the cyclooxygenase inhibitor indomethacin could not prevent the effects of IL-1 beta. It is concluded that prostaglandins suppress beta-cell growth and long-term insulin secretion without participation of pertussis-toxin sensitive GTP-binding proteins. In addition, although their synthesis is increased by IL-1 beta, prostaglandins seemingly do not convey the inhibitory actions of this cytokine in the beta-cell.

Phospholipase C (PLC) was purified from squid retina. Soluble Gq α , membrane Gq α and G βγ were isolated from GTP γ S-treated and light-illuminated photoreceptor membranes. The membrane Gq α stimulated phosphatidyl inositoi-phospholipase C (PI-PLC) activity in a dose-dependent manner. Soluble Gq α and membrane G βγ showed no stimulating effects on PLC. GTP γ S-binding was found exclusively in membrane fraction, with very little present in the KCl-soluble fraction which contained soluble Gq α . These results indicate that light-activated rhodopsin activates PLC through membrane-bound Gq α and suggest that the rhodopsin/Gq/PLC cascade might be the pathway of phototransduction in squid photoreceptors.

The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.

The opioid receptors mu, delta and kappa have recently been cloned. Here we show that kappa-agonists inhibit adenylyl cyclase activity in Chinese hamster ovary cells stably transfected with rat kappa-opioid receptor cDNA. Chronic exposure of the cells to kappa-agonists did not lead to significant desensitization of the capacity of the agonists to inhibit adenylyl cyclase. On the other hand, withdrawal of the agonist following the chronic treatment led to the phenomenon of supersensitivity ('overshoot') of adenylyl cyclase activity. Both the inhibition of adenylyl cyclase activity by the acute opioid treatment and the chronic agonist-induced supersensitivity are pertussis toxin sensitive, demonstrating involvement of Gi/Go proteins in both processes.

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called ‘aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.

Complement receptor type 3 (CR3)-mediated cellular responses in guinea pig macrophages were investigated by using zymosan and serum-opsonized zymosan (SOZ) as the multivalent ligand for CR3. The ingestion of zymosan and SOZ was accompanied by O 2 − generation and arachidonate release. These responses were suppressed by prior exposure of macrophages to pertussis toxin (PT). Opsonization of zymosan gave rise to more than 6-fold activation of the ingestion, whereas the magnitude of either arachinonate release or O 2 − generation was unchanged. The Fab' fragment of anti-Z-1, a monoclonal antibody specific for the α chain of guinea pig CR3, inhibited the ingestion of zymosan by 60% without affecting zymosan-induced arachidonate release and O 2 − generation. These data suggested that there might be at least two functionally distinct binding sites for zymosan. O 2 − generation and arachidonate release might be regulated through one site and phagocytosis another. Both sites should be coupled to PT-sensitive GTP binding protein.

4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha 2A-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi from alpha 2A-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.

The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a ‘molecular switch’ mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo–eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

We report the existence of several families of GTP-binding proteins in barley aleurone protoplasts. Partial purified plasma membrane proteins were separated by SDS-PAGE, transferred to a nitrocellulose filter and incubated with either antisera raised against a highly conserved animal G protein α subunit peptide/or Ras protein, or with [α-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [α-32P]GTP was detected on Western blots with proteins of M(r) = 22-24 kDa and 16 kDa. Binding was inhibited by 10-7-10-6 M GTPγS, GTP or GDP; binding was not affected by 10-6-10-5 M ATPγS or ADP. The kinetics, specificity and the effects of phytohormones in a[35S]GTPγS binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts. Chemicals/CAS: GTP-Binding Proteins, EC 3.6.1.-; Plant Growth Regulators

A cDNA encoding a small GTP-binding protein, S10, was cloned from Jurkat cells. The deduced amino acid sequence of S10 had the structural features characteristic to this family of proteins with highest homology to rab subfamily. Northern blot analysis revealed that this gene is expressed only in lymphoid cell lines and a histiocytic leukemia, U937. Hence, it should have a specialized function in cells derived from the hematopoietic stem cell.

The regulatory effects of lithium on the function of pertussis toxin-sensitive GTP-binding (G i )-proteins located on the mitogenic pathway activated by insulin-like growth factor-I (IGF-I) in FRTL-5 cells were studied. Addition of GTP-γ-S to the thyroid stimulating hormone-primed cell membranes resulted in a decreased affinity of IGF-I receptor binding, and the dissociation constant ( K d ) increased from 0.46 nM to 3.1 nM. Moreover, IGF-I stimulated GTP-γ-S binding to a 40-kDa protein, and pertussis toxin (PT) attenuated the stimulatory effect of IGF-I on the same protein. Lithium lowered the affinity of IGF-I receptor binding and the K d (3.4 nM) was in the same range as that in the presence of GTP-γ-S. The inhibitory effect of lithium was markedly abolished by pretreatment with PT. Lithium attenuated the amounts of ADP-rebosylation of the 40-kDa protein by PT. In addition, lithium stimulated Ca 2+ entry, similar to that by IGF-I, and induced cell proliferation via a PT-sensitive step. These findings suggest that lithium may be capable of modulating the function of G i -proteins coupled to IGF-I receptors during the G 1 phase of the FRTL-5 cell cycle.

Bradykinin (BK) induced a transient and pertussis toxin (PT)-insensitive increase in cytosolic Ca2+ ([Ca2+]i) in NG 108-15 neuroblastoma × glioma hybrid cells, whereas leucine-enkephalin (EK), somatostatin, norepinephrine or carbachol showed a weak but PT-sensitive action. When any one of the latter agonists was applied to the cells treated with low doses of BK, however, the level of [Ca2+]i rise caused by the agonist was remarkably increased in a PT-sensitive manner. The decreasing of extracellular Ca2+ only slightly influenced the actions or these agonists. Thus, synergism between a BK receptor and PT-sensitive G-protein-coupled receptors results in marked intracellular Ca2+ mobilization by the latter agonists.

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Members of the Ras superfamily of small GTP-binding proteins, γ-subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post-translational modifications that produce prenylcysteine methylester groups at their carboxyl termini. The thioether-linked polyisoprenoid substituent can be either farnesyl (C15) or geranylgeranyl (C20). Small molecule prenylcysteine derivatives with either the C15 or C20 modification, such as N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), S-trans,trans-farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase. Saccharomyces cerevisiae ste 14 mutants that lack RAS and a-factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives. Moreover, C20-substituted cysteine analogs directly compete for carboxyl methylation with the C15-substituted cysteine analogs and vice versa. Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.

During development of the cellular slime mold Dictyostelium discoideum, cAMP induces chemotaxis and expression of different classes of genes by means of interaction with surface cAMP receptors. We describe a cAMP derivative, 8-p-chlorophenylthioadenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), which inhibits cAMP-induced chemotaxis at low concentrations but induces chemotaxis at supersaturating concentrations. This compound, moreover, selectively activates expression of aggregative genes but not of postaggregative genes. 8-CPT-cAMP induces normal cGMP and cAMP accumulation but in contrast to cAMP, which increases inositol 1,4,5-trisphosphate levels, 8-CPT-cAMP decreases inositol 1,4,5-trisphosphate levels. The derivative induces reduced activation of guanine nucleotide regulatory proteins, which may cause its defective activation of inositol 1,4,5-trisphosphate production. Our data suggest that disruption of inositolphospholipid signaling impairs chemotaxis and expression of a subclass of cAMP-regulated genes.

A431 cells, a human epidermoid carcinoma cell line, express an unusually large number of cell surface receptors for the epidermal growth factor (EGF). The growth rate of A431 cells was estimated by measuring [3H]thymidine incorporation at the logarithmic growth phase. The growth of the cells in protein-free medium was partially inhibited by exposure of the cells to pertussis toxin, islet-activating protein (IAP). The growth in both serum-containing and protein-free medium was inhibited by high concentrations of EGF, and these inhibitions were partially reversed by treatment of the cells with IAP. The effects of IAP were well correlated with the degree of ADP-ribosylation of a membrane 40-kDa protein. Thus, IAP sensitive G-proteins appear to be involved in the signal transduction of both positive and negative regulation of A431 cell growth. The possibility is also discussed that phosphatidylinositol turnover may participate in growth regulation.

Platelet-activating factor (PAF), added to the bathing solution, stimulated the cardiac muscarinic K+ channel (KACh) in the cell-attached patch (no agonist in the pipette). The PAF-induced KACh channel activation was blocked by WEB2086, a PAF-receptor inhibitor, indicating that the PAF-receptor mediated the response. PAF-induced activation was prevented by nordihydroguaieretic acid, a lipoxygenase inhibitor, and AA-861, a 5-lipoxygenase inhibitor, but was not affected by indomethacin, a cyclo-oxygenase inhibitor. The PAF-induced KACh, channel activity disappeared upon formation of inside-out patch. In this inside-out patch, intracellular GTP alone induced maximal channel reactivation, which was inhibited by GDP-βS. These results suggest that 5-lipoxygenase metabolites of PAF-released arachidonic acid cause a persistent stimulation of GK but not the KACh channel itself, resulting in a receptor-independent activation of the KACh channel by GTP.

Rap1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxyl terminus and is adjacent to the site of membrane attachment of the protein.

Chemotaxis, the directional movement towards a chemical compound, is an essential property of many cells and has been linked to the development and progression of many diseases. Eukaryotic chemotaxis is a complex process involving gradient sensing, cell polarity, remodelling of the cytoskeleton and signal relay. Recent studies in the model organism Dictyostelium discoideum have shown that chemotaxis does not depend on a single molecular mechanism, but rather depends on several interconnecting pathways. Surprisingly, small G-proteins appear to play essential roles in all these pathways. This review will summarize the role of small G-proteins in Dictyostelium, particularly highlighting the function of the Ras subfamily in chemotaxis. (c) 2008 Elsevier Inc. All rights reserved.

We studied the selectivity of GABAB receptors for coupling to G proteins by testing the ability of various purified G proteins to increase GABA binding to N-ethylmaleimide (NEM)-treated membranes of bovine brain. The addition of Go, Go* or Gi1 to NEM-treated membranes increased GABA binding in a dose-dependent manner. However, the addition of Gi2 did not elicit a marked increase in GABA binding. When alpha subunits of G proteins were mixed with various brain beta gamma subunit complexes composed of different gamma subunits, and they were added to the NEM-treated membranes, Gi2 with any beta gamma subunits hardly increased GABA binding. On the other hand, Go with any beta gamma subunits caused a marked increase, though Go with a small gamma subunit was more effective than that with a large gamma subunit. These data suggest that the selective coupling of the G proteins to GABAB receptors is determined by the alpha subunit.

In IPC-81 cells, the adenylyl-cyclase activation by cholera toxin produces an elevation of cAMP that causes a rapid cytolysis. A resistant clone with deficient cholera toxin-induced cyclase activity (yet sensitive to cAMP) showed a rapid decrease in the amount of membrane-bound Gs alpha (42-47 kDa) detectable soon after ADP-ribosylation of these proteins; pertussis toxin-sensitive G proteins (41 kDa) were not affected. Resistant cells showed a rapid decrease of Gs alpha that is consistent with the finding that cAMP did not accumulate in these cells. Cholera toxin treatment of resistant cells had long-lasting effects (several weeks) on the level of Gs alpha in the cell membrane. The duration of Gs alpha decrease does not correspond to the probable life of catalytically active cholera toxin in the cells, and suggests a regulated process more complex than a proteolytic degradation targeted on ADP-ribosylated molecules.

The γ subunits of GTP-binding proteins are always associated with β subunits under physiological conditions, and at least two γ subunits exist in the brain. We report here that brain βγ subunit complexes composed of distinct γ subunits can be separated by anion exchange chromatography under nondenaturing conditions. One βγ complex was composed of a 36-kDa β subunit and a 6-kDa γ subunit and the other was composed of 36/35-kDa β subunits and 4.5-kDa γ subunit. The 6-kDa γ subunit was phosphorylated by protein kinase C but the 4.5-kDa γ subunit was not.

GTP-binding proteins with Mr values of 22 000 and 25 000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.

Recently we reported there were at least four types of G O or G O -like proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an α-monomeric form, and the others as αβγ-trimers. The four proteins, of which α-subunits were confirmed to be a family of G O -type by an immunoblot analysis, were thus referred toas α O 1 , G O 2 , G O 3 and G O 4 , respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four α-subunits, together with their fragmentation patterns containing radiolabeled ADP-ribose that had been incorporated by pertussis toxin-catalyzed ADP-ribosylation, suggested that the four G O -α were classified into either of two groups such as α 1 and G O 2 -α or G O 3 -α and G O 4 -α. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between α O 1 and G O 2 -α or G O 3 -α and G O 4 -α. Thus, the four G O -type proteins appeared to be different entities from one another.

The Ras-related human GTPase Rab4a is involved in the regulation of endocytosis through the sorting and recycling of early endosomes. Towards further insight, we have determined the three-dimensional crystal structure of human Rab4a in its GppNHp-bound state to 1.6 Angstroms resolution and in its GDP-bound state to 1.8 Angstroms resolution, respectively. Despite the similarity of the overall structure with other Rab proteins, Rab4a displays significant differences. The structures are discussed with respect to the recently determined structure of human Rab5a and its complex with the Rab5-binding domain of the bivalent effector Rabaptin-5. The Rab4 specific residue His39 modulates the nucleotide binding pocket giving rise to a reduced rate for nucleotide hydrolysis and exchange. In comparison to Rab5, Rab4a has a different GDP-bound conformation within switch 1 region and displays shifts in position and orientation of the hydrophobic triad. The observed differences at the S2-L3-S3 region represent a new example of structural plasticity among Rab proteins and may provide a structural basis to understand the differential binding of similar effector proteins.

International audience; We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains.

Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity. Most rabs are ubiquitously expressed. However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions. This review discusses the role of rabs in epithelial membrane transport.

Rat olfactory epithelium and ciliar cytosol of olfactory cells contained the water-soluble 45 kDa protein which was revealed by antibodies against a peptide fragment of the α-subunits common to the G-proteins. No analogous proteins were found in other rat tissues. According to the photo-affinity labeling, the 45 kDa protein possessed a high affinity to GTP; it also exhibited a low GTP hydrolytic activity.

GTP-binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab-related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca(2+)-dependent or Ca(2+)-independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL (30-47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca(2+)-stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 microM peptide. The peptide (100 microM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence-specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.

Small GTPases of the Rab family regulate membrane transport pathways. More than 50 mammalian Rab proteins are known, many with transport step-specific localisation. Rabs must associate with cellular membranes for activity and membrane attachment is mediated by prenyl (geranylgeranyl) post-translational modification. Mutations in genes encoding proteins essential for the geranylgeranylation reaction, Rab escort protein and Rab geranylgeranyl transferase, underlie genetic diseases. Choroideremia patients have loss of function mutations in REP1 and the murine Hermansky–Pudlak syndrome model gunmetal possesses a splice-site mutation in the α-subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases.

The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role. We have cloned a fission yeast gene (alp41+) which encodes a highly conserved small GTP-binding protein homologous to budding yeast CIN4 and human ARF-like Arl2. alp41+ is essential, disruption of which results in microtubule dysfunction and growth polarity defects. Genetic analysis indicates that Alp41 plays a crucial role in the cofactor-dependent pathway, in which it functions upstream of the cofactor D homologue Alp1D and possibly in concert with Alp21E.

In the pig heart sarcolemma, a 65 kDa protein is found to be ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase (exoenzyme C3). ADP-ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates. The new exoenzyme C3 substrate was partially purified. This protein is supposed to be a GTP-binding one.