Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

CCND1/CyclinD1 status in metastasizing bladder cancer: a prognosticator and predictor of chemotherapeutic response

The CCND1 gene encodes the protein CyclinD1, which is an important promoter of the cell cycle and a prognostic and predictive factor in different cancers. CCND1 is amplified to a substantial proportion in various tumors, and this may contribute to CyclinD1 overexpression. In bladder cancer, information about the clinical relevance of CCND1/CyclinD1 alterations is limited. In the present study, amplification status of CCND1 and expression of CyclinD1 were evaluated by fluorescence in situ hybridization and immunohistochemistry on tissue microarrays from 152 lymph node-positive urothelial bladder cancers (one sample each from the center and invasion front of the primary tumors, two samples per corresponding lymph node metastasis) treated by cystectomy and lymphadenectomy. CCND1 amplification status and the percentage of immunostained cancer cells were correlated with histopathological tumor characteristics, cancer-specific survival and response to adjuvant chemotherapy. CCND1 amplification in primary tumors was homogeneous in 15% and heterogeneous in 6% (metastases: 22 and 2%). Median nuclear CyclinD1 expression in amplified samples was similar in all tumor compartments (60-70% immunostained tumor nuclei) and significantly higher than in non-amplified samples (5-20% immunostained tumor nuclei; P<0.05). CCND1 status and CyclinD1 expression were not associated with primary tumor stage or lymph node tumor burden. CCND1 amplification in primary tumors (P=0.001) and metastases (P=0.02) and high nuclear CyclinD1 in metastases (P=0.01) predicted early cancer-related death independently. Subgroup analyses showed that chemotherapy was particularly beneficial in patients with high nuclear CyclinD1 expression in the metastases, whereas expression in primary tumors and CCND1 status did not predict chemotherapeutic response. In conclusion, CCND1 amplification status and CyclinD1 expression are independent risk factors in metastasizing bladder cancer. High nuclear CyclinD1 expression in lymph node metastases predicts favorable response to chemotherapy. This information may help to personalize prognostication and administration of adjuvant chemotherapy.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Maximal acceleration of calcium release refractoriness by β-adrenergic stimulation requires dual activation of protein kinase A and CaMKII in mouse ventricular myocytes

Time-dependent refractoriness of calcium (Ca2+) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca2+ spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca2+ spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca2+ sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark- to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca2+/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen) without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca2+ release refractoriness in mouse myocytes.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Acute Regulation of Renal Na+/H+ Exchanger NHE3 by Dopamine: Role of Protein Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56 delta (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, co-immunoprecipitation, blot overlay and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small t antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition. Key words: Natriuresis, Sodium transport, Signal transduction.

The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion

AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.

Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

Neospora caninum: functional inhibition of protein disulfide isomerase by the broad-spectrum anti-parasitic drug nitazoxanide and other thiazolides

Nitazoxanide (NTZ) and several NTZ-derivatives (thiazolides) have been shown to exhibit considerable anti-Neospora caninum tachyzoite activity in vitro. We coupled tizoxanide (TIZ), the deacetylated metabolite, to epoxy-agarose-resin and performed affinity chromatography with N. caninum tachyzoite extracts. Two main protein bands of 52 and 43kDa were isolated. The 52kDa protein was readily recognized by antibodies directed against NcPDI, and mass spectrometry confirmed its identity. Poly-histidine-tagged NcPDI-cDNA was expressed in Escherichia coli and recombinant NcPDI (recNcPDI) was purified by Co2+-affinity chromatography. By applying an enzyme assay based on the measurement of insulin crosslinking activity, recNcPDI exhibited properties reminiscent for PDIs, and its activity was impaired upon the addition of classical PDI inhibitors such as bacitracin (1-2mM), para-chloromercuribenzoic acid (0.1-1mM) and tocinoic acid (0.1-1mM). RecNcPDI-mediated insulin crosslinking was inhibited by NTZ (5-100 microM) in a dose-dependent manner. In addition, the enzymatic activity of recNcPDI was inhibited by those thiazolides that also affected parasite proliferation. Thus, thiazolides readily interfere with NcPDI, and possibly also with PDIs from other microorganisms susceptible to thiazolides.

Reduction in rat phosphatidylethanolamine binding protein-1 (PEBP1) after chronic corticosterone treatment may be paralleled by cognitive impairment: a first study

Chronic stress is associated with hippocampal atrophy and cognitive dysfunction. This study investigates how long-lasting administration of corticosterone as a mimic of experimentally induced stress affects psychometric performance and the expression of the phosphatidylethanolamine binding protein (PEBP1) in the adult hippocampus of one-year-old male rats. Psychometric investigations were conducted in rats before and after corticosterone treatment using a holeboard test system. Rats were randomly attributed to 2 groups (n = 7) for daily subcutaneous injection of either 26.8 mg/kg body weight corticosterone or sesame oil (vehicle control). Treatment was continued for 60 days, followed by cognitive retesting in the holeboard system. For protein analysis, the hippocampal proteome was separated by 2D electrophoresis (2DE) followed by image processing, statistical analysis, protein identification via peptide mass fingerprinting and gel matching and subsequent functional network mapping and molecular pathway analysis. Differential expression of PEBP1 was additionally quantified by Western blot analysis. Results show that chronic corticosterone significantly decreased rat hippocampal PEBP1 expression and induced a working and reference memory dysfunction. From this, we derive the preliminary hypothesis that PEBP1 may be a novel molecular mediator influencing cognitive integrity during chronic corticosterone exposure in rat hippocampus.

Evaluation of reproducibility of protein identification results after multidimensional human serum protein separation

We developed a gel- and label-free proteomics platform for comparative studies of human serum. The method involves the depletion of the six most abundant proteins, protein fractionation by Off-Gel IEF and RP-HPLC, followed by tryptic digestion, LC-MS/MS, protein identification, and relative quantification using probabilistic peptide match score summation (PMSS). We evaluated performance and reproducibility of the complete platform and the individual dimensions, by using chromatograms of the RP-HPLC runs, PMSS based abundance scores and abundance distributions as objective endpoints. We were interested if a relationship exists between the quantity ratio and the PMSS score ratio. The complete analysis was performed four times with two sets of serum samples containing different concentrations of spiked bovine beta-lactoglobulin (0.1 and 0.3%, w/w). The two concentrations resulted in significantly differing PMSS scores when compared to the variability in PMSS scores of all other protein identifications. We identified 196 proteins, of which 116 were identified four times in corresponding fractions whereof 73 qualified for relative quantification. Finally, we characterized the PMSS based protein abundance distributions with respect to the two dimensions of fractionation and discussed some interesting patterns representing discrete isoforms. We conclude that combination of Off-Gel electrophoresis (OGE) and HPLC is a reproducible protein fractionation technique, that PMSS is applicable for relative quantification, that the number of quantifiable proteins is always smaller than the number of identified proteins and that reproducibility of protein identifications should supplement probabilistic acceptance criteria.

Bothnia dystrophy is caused by domino-like rearrangements in cellular retinaldehyde-binding protein mutant R234W

Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy. Here we report the crystal structures of both wild-type human CRALBP and of its mutant R234W as binary complexes complemented with the endogenous ligand 11-cis-retinal, at 3.0 and 1.7 A resolution, respectively. Our structural model of wild-type CRALBP locates R234 to a positively charged cleft at a distance of 15 A from the hydrophobic core sequestering 11-cis-retinal. The R234W structural model reveals burial of W234 and loss of dianion-binding interactions within the cleft with physiological implications for membrane docking. The burial of W234 is accompanied by a cascade of side-chain flips that effect the intrusion of the side-chain of I238 into the ligand-binding cavity. As consequence of the intrusion, R234W displays 5-fold increased resistance to light-induced photoisomerization relative to wild-type CRALBP, indicating tighter binding to 11-cis-retinal. Overall, our results reveal an unanticipated domino-like structural transition causing Bothnia-type retinal dystrophy by the impaired release of 11-cis-retinal from R234W.