Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca²⁺ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity.Knockout (KO)¹ mouse models represent powerful methods for studying the physiological relevance of a protein. However, to elucidate the effects of KO-induced perturbations on the entire system, systems-wide molecular characterization is needed, as, for instance, provided by (phospho)proteomics. Recent technological and methodological advancements now allow the mapping of protein expression, at least in cell cultures, close to completion (1–3). More challenging, proteomics is also increasingly used to attempt systems-wide proteome characterizations in tissue. This has led to semi-quantitative (4–6) and quantitative (7) reasonably comprehensive proteome data on selected tissues, in both humans and animal models. More recently, proteomics has also been applied for the in-depth profiling of perturbations in the proteome occurring in KO models. For instance, de Graaf et al. (8) used an in-depth proteomic approach to identify the proteins changed by DNA-damage-induced premature aging, using a KO mouse model lacking the excision repair cross-complementing group 1 gene. Another recent study used a mouse model lacking apolipoprotein E in order to identify biomarker candidates for coronary artery disease (9).Adaptation and/or perturbations in the proteome caused by a KO can lead to changes in protein expression, but, at least equally likely, also to rewiring of signaling networks, through changes in post-translational modifications, such as protein phosphorylation. The application of (phospho)proteomics technology on KO or knock-in models is therefore also extremely relevant, albeit even more challenging. Hilger et al. (10) combined proteomics and phosphoproteomics on a cell line in which a phosphatase had been knocked out. To perform such experiments in a more (disease) relevant context, we should invest in functional, tissue-based phosphoproteomics approaches. A few examples of such approaches have very recently been reported. Lundby et al. (11) globally assessed phosphorylation events downstream of systemic adrenergic stimulation in mouse cardiac tissue. We recently reported on the use of a cardiac delimited CaMKII inhibited knock-in mouse to probe for substrates using a focused kinase-inhibition directed approach (12). Moreover, a mouse model lacking nitric oxide synthase (13), as a system of interest for Alzheimer disease, was recently studied via (phospho)proteomics.Here we explored how mature state-of-the-art (phospho)proteomics technology could be used to monitor the adaptation at the proteome level of the mouse cerebellum in a mouse line deficient for cGMP-dependent protein kinase type I (cGKI, also known as PKG-I), a kinase that plays an important role in synaptic plasticity, motor learning, and other brain functions (14). The cGMP-dependent protein kinases are serine/threonine kinases that act as key mediators of nitric oxide (NO) signaling, as well as of the natriuretic peptide pathway (15). In mammals, cGKs are encoded by two different genes: prkg1 coding for cGKI, and prkg2 coding for cGKII (16). The prkg1 gene encodes two cGKI isoforms, cGKIα and cGKIβ (17), which differ in their N-terminal leucine zipper and auto-inhibitory domains. cGKI regulates cardiovascular functions such as smooth muscle and cardiac contractility (16); in the nervous system it modulates synaptic plasticity in the hippocampus (18) and cerebellum (19).In the mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to contribute to learning and memory. In particular, cGKIα is highly expressed in cerebellar Purkinje cells (PCs) (20, 21). Long-term depression (LTD) is an activity-dependent reduction in the efficacy of synaptic transmission and occurs at the PC synapses. Both a pharmacological approach using enzyme inhibitors (22) and a conditional PC-specific cGKI-KO (23) showed that cGKI plays a role in cerebellar LTD. Several proteins have been identified in past years as cGKI substrates in vitro or in cultured cells (15), but only a small portion of these have been confirmed as cGKI substrates in vivo. Therefore, the understanding of cGKI signaling and function depends strongly on the identification of novel in vivo substrates and signaling partners. In this perspective, the currently described approach allows us to discover potentially novel cGKI signaling routes and substrates directly in relevant cerebellar tissue. Our study revealed that cGKI-KO led to differential expression in the cerebellum of a specific group of proteins, of which many were closely connected to cGMP-cGKI signaling. More changes were observed at the phosphoproteome level, with the regulation of phosphorylation of a few hundred proteins. In particular, we hypothesize that some of the down-regulated phosphoproteins, but certainly not all, may be putative substrates of cGKI.     

长春市儿童医院1998～2001年婴幼儿杯状病毒腹泻流行病学研究

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.     

Cellular Responses of the Late Blight Pathogen Phytophthora infestans to Cyclic Lipopeptide Surfactants and Their Dependence on G Proteins

Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein α subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling.Oomycetes cause devastating diseases of plants and animals. They are fungal look-alikes that grow as mycelium and propagate via spores but evolved independently from fungi (23). Among the plant pathogens are over 80 Phytophthora species, with the late blight pathogen Phytophthora infestans being the most renowned (12, 16). Late blight control relies heavily on fungicides that contain copper-, tin-, phenylamide-, or cyanocetamide-oximes as active ingredients. Public concerns about the adverse effects of these fungicides on food safety and the environment have led to an increased demand for novel control strategies, preferably based on natural products. In recent years, the destructive effects of cyclic lipopeptides (CLPs) on zoospores of oomycete plant pathogens have attracted considerable attention (8, 9, 33). CLPs are produced by a variety of bacterial genera including Bacillus and Pseudomonas (13, 29, 30, 33, 37). They are composed of a fatty acid tail linked to an oligopeptide, which is cyclized by a lactone ring between two amino acids in the peptide chain. Based on the length and composition of the fatty acid as well as the number, type, and configuration (L-D form) of the amino acids in the peptide moiety, their activity may change (29, 30, 33). CLPs can be chemically produced, and via structural or genetic modifications, their physicochemical properties and antimicrobial activities can be altered (1). Pseudomonas fluorescens strain SS101 produces nine cyclic lipopeptide surfactants, with massetolide A (Mass A) being the main cyclic lipopeptide (7, 9). The others are derivatives of Mass A differing in the amino acid compositions of the peptide ring (7).One of the main modes of action of natural and synthetic CLPs is interference with the membrane integrity of the target organism, leading to pore formation and cytolysis (4, 5, 18, 19, 27, 34). For example, the CLPs Mass A and viscosin, produced by P. fluorescens strains SS101 and SBW25, respectively, act on membranes of zoospores of plant-pathogenic oomycetes, including Pythium and Phytophthora species, and this leads to the complete elimination of these propagules within 1 min of exposure (6-9). The destructive effects of Mass A on zoospores may explain, at least in part, the activity of P. fluorescens SS101 against Pythium root rot of flower bulb crops (6, 9) and tomato late blight caused by P. infestans (44). However, CLPs not only act on zoospores but may also inhibit mycelial growth of oomycetes and fungi (29, 33). The observations that several CLPs induce systemic resistance in plants against fungal and oomycete pathogens including P. infestans (31, 44) further emphasize their versatile activities and potential for crop protection. To explore and exploit the use of CLPs as a novel supplementary strategy for disease control, fundamental knowledge on their modes of action and the cellular responses of target oomycete pathogens is required.In this study, we investigated the response of P. infestans to the CLP Mass A produced by P. fluorescens SS101. We examined various growth stages of P. infestans and performed dose-response experiments to determine the effects of Mass A on mycelial growth, sporangium formation, cyst germination, and zoospore behavior, including chemotaxis, autoaggregation, and encystment. We also examined the involvement of the α subunit of the heterotrimeric G protein in the cellular responses of P. infestans to Mass A. The results show differential sensitivities of the various growth stages to Mass A and suggest that G-protein signaling plays a role in mediating the response.     

Molecular docking analysis of bioactive compounds from Cissampelos pareira with PPAR gamma

We document the Molecular docking analysis of bioactive compounds from Cissampelos pareira with PPAR gamma for further consideration in drug discovery for T2DM.     

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.     

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.     

Conservation of the response regulator gene gacA in Pseudomonas species

The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.     

Antibiotic production by bacterial biocontrol agents

Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides. There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The significance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments. Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants. Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified to influence antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microflora, and the cell density of the producing strain. This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mucin secretion by the human colon cell line LS174T is regulated by bile salts

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.      

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Introduction

Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods

Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results

The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions

The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.