G protein-coupled receptors: what a difference a 'partner' makes

G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly upregulated or downregulated (>2-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly-investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were upregulated in AVMs vs NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were upregulated (e.g. NRK, JAK2, STK38L) or downregulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusions. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

Expression of protein kinase C isoforms during cardiac ventricular development.

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

A Substrate Trapping Mutant Form of Striatal-Enriched Protein Tyrosine Phosphatase Prevents Amphetamine-Induced Stereotypies and Long-Term Potentiation in the Striatum

Background: Chronic, intermittent exposure to psychostimulant drugs results in striatal neuroadaptations leading to an increase in an array of behavioral responses on subsequent challenge days. A brain-specific striatal-enriched tyrosine phosphatase (STEP) regulates synaptic strengthening by dephosphorylating and inactivating several key synaptic proteins. This study tests the hypothesis that a substrate-trapping form of STEP will prevent the development of amphetamine-induced stereotypies. Methods: A substrate-trapping STEP protein, TAT-STEP (C-S), was infused into the ventrolateral striatum on each of 5 consecutive exposure days and I hour before amphetamine injection. Animals were challenged to see whether sensitization to the stereotypy-producing effects of amphetamine developed. The same TAT-STEP (C-S) protein was used on acute striatal slices to determine the impact on long-term potentiation and depression. Results: Infusion of TAT-STEP (C-S) blocks the increase of amphetamine-induced stereotypies when given during the 5-day period of sensitization. The TAT-STEP (C-S) has no effect if only infused on the challenge day. Treatment of acute striatal slices with TAT-STEP (C-S) blocks the induction of long-term potentiation and potentates long-term depression. Conclusions: A substrate trapping form of STEP blocks the induction of amphetamine-induced neuroplasticity within the ventrolateral striatum and supports the hypothesis that STEP functions as a tonic break on synaptic strengthening.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development

P>A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6b(rd1)). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin 1, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Enhanced Neural Progenitor/Stem Cells Self-Renewal via the Interaction of Stress-Inducible Protein 1 with the Prion Protein

Prion protein (PrPC), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C)-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C), with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126-1136

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Defective transcription/repair factor IN recruitment to specific UV lesions in trichothiodystrophy syndrome

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFHH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 64PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some mutations affect the recruitment of TFHH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFHH complexes carrying an NH2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFUH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFHH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.