The role of Cysteine 6.47 in class A GPCRs

Abstract Background: The CWxP motif of transmembrane helix 6 (x: any residue) is highly conserved in class A GPCRs. Within this motif, W6.48 is a big star in the theory of the global “toggle switch” because of its key role in the activation mechanism of GPCRs upon ligand binding. With all footlights focused on W6.48, the reason why the preceding residue, C6.47, is largely conserved is still unknown. The present study is aimed to fill up this lack of knowledge by characterizing the role of C6.47 of the CWxP motif. Results: A complete analysis of available crystal structures has been made alongside with molecular dynamics simulations of model peptides to explore a possible structural role for C6.47. Conclusions: We conclude that C6.47 does not modulate the conformation of the TM6 proline kink and propose that C6.47 participates in the rearrangement of the TM6 and TM7 interface accompanying activation.

Hypertension. Angio-oedème sous inhibition de l'enzyme de conversion de l'angiotensine et de la dipeptidyl-peptidase-4 [Angioedema during ACE and DPP-4 inhibition]

Angioedema is a rare side effect of angiotensin converting enzyme (ACE) inhibitors. Its cause is probably related to the accumulation of bradykinin and substance P, i.e. two proinflammatory peptides normally inactivated by ACE. Angioedema occurs most of the time at the early phase of treatment, but may also develop during long-term treatment. It might involve the gastro-intestinal tract, leading to abdominal pain, vomiting and/or diarrhea, as well as pancreatitis. Dipeptidyl-ptidase-4 (DPP-4) is another enzyme allowing the degradation of bradykinin and substance P. Co-administering an ACE inhibitor and a DPP-4 inhibitor (as an antidiabetic agent) increases significantly the risk of angioedema.

Schizophrenia and oxidative stress: glutamate cysteine ligase modifier as a susceptibility gene.

Oxidative stress could be involved in the pathophysiology of schizophrenia, a major psychiatric disorder. Glutathione (GSH), a redox regulator, is decreased in patients' cerebrospinal fluid and prefrontal cortex. The gene of the key GSH-synthesizing enzyme, glutamate cysteine ligase modifier (GCLM) subunit, is strongly associated with schizophrenia in two case-control studies and in one family study. GCLM gene expression is decreased in patients' fibroblasts. Thus, GSH metabolism dysfunction is proposed as one of the vulnerability factors for schizophrenia.

The role of ubiquitin specific peptidase 15 (USP 15) in human glioblastoma

Glioblastoma (GBM) is the most common and most aggressive malignant primary brain tumour. Despite the aggressiveness of the applied therapy, the prognosis remains poor with a median survival to of about 15 months. It is important to identify new candidate genes that could have clinical application in this disease. Previous gene expression studies from human GBM samples in our laboratory, revealed Ubiquitin Specific Peptidase 15 (USP15) as a gene with low expression, significantly associated with genomic deletions of the chromosomal region encompassing the USP15 locus. USP15 belongs to the ubiquitin-specific protease (USPs) family of which the main role is the reversion of ubiquitination and thereby stabilization of substrates. Previously, USP15 has been suggested to have a tumour suppressor function via its substrates APC and Caspase 3. We established GBM cell lines that stably express USP15 wt or its catalytic mutant. USP15 expression impairs cell growth by inhibiting cell cycle progression. On the other hand USP15 depletion in GBM cell lines induces cell cycle progression and proliferation. In order to identify the molecular pathways in which USP15 is implicated we aimed to identify protein-binding partners in the GBM cell line LN-229 by Mass spectrometry. As a result we identified eight new proteins that interact with USP15. These proteins are involved in important cellular processes like cytokinesis, cell cycle, cellular migration, and apoptosis. Three of these protein interactions were confirmed by co-immunoprecipitation in four GBM cell lines LN-229, LN428, LN18, LN-Z308. One of the binding proteins is HECTD1 E3 ligase of which the murine homologue promotes the APC-Axin interaction to negatively regulate the Wnt pathway. USP15 can de-ubiquitinate HECTD1 in the LN229 cell line while its depletion led to decrease of HECTD1 in GBM cell lines suggesting stabilizing role for USP15. Moreover, HECTD1 stable expression in LN229 inhibits cell cycle, while its depletion induces cell cycle progression. These results suggest that the USP15-HECTD1 interaction might enhance the antiproliferative effect of HECTD1 in GBM cell lines. Using the TOPflash/FOPflash luciferase system we showed that HECTD1 and USP15 overexpression can attenuate WNT pathway activity, and decrease the Axin2 expression. These data indicate that this new protein interaction of USP15 with HECTD1 results in negative regulation of the WNT pathway in GBM cell lines. Further investigation of the regulation of this interaction or of the protein binding network of HECTD1 in GBM may allow the discovery of new therapeutic targets. Finally PTPIP51 and KIF15 are the other two identified protein partners of USP15. These two proteins are involved in cell proliferation and their depletion in LN-229 cell line led to induction of cell cycle progression. USP15 displays a stabilizing role for them. Hence, these results show that the tumour suppressive role of USP15 in GBM cell line via different molecular mechanisms indicating the multidimensional function of USP15. Résumé Le glioblastome (GBM) est la tumeur primaire la plus fréquente et la plus agressive du cervau caractérisée par une survie médiane d'environ à 15 mois. De précédant travaux effectués au sein de notre laboratoire portant sur l'étude de l'expression de gènes pour des échantillons humains de GBM ont montré que le gène Ubiquitin Specific Peptidase 15 (USP1S) était significativement associée à une délétion locales à 25% des cas. Initialement, les substrats protéiques APC et CaspaseS de USP15 ont conduit à considérer cette protéine comme un suppresseur de tumeur. USP15 appartient à la famille protèsse spécifique de l'ubiquitine (USPs) dont le rôle principal est la réversion de l'ubiquitination et la stabilisation de substrats. Par conséquent, nous avons établi des lignées de cellules de glioblastome qui expriment de manière stable USP15 ou bien son mutant catalytique. Ainsi, nous avons ainsi démontré que l'expression de l'USP15 empêche la croissance cellulaire en inhibant la progression du cycle cellulaire. Inversement, la suppression de l'expression du gène USP15 dans les lignées cellulaires de glioblastome induit la progression du cycle cellulaire et la prolifération. Afin d'identifier les voies moléculaires dans lesquelles sont impliquées USP15, nous avons cherché à identifier les partenaires de liaisons protéiques par spectrométrie de masse dans la lignée cellulaire LN-229. Ainsi, huit nouvelles protéines interagissant avec USP15 ont été identifiées dont la ligase E3 HECTD1. L'homologue murin de Hectdl favorise l'interaction APC-Axin en régulant négativement la voie de signalisation de Wnt. USP15 interagit en désubiquitinant HECTD1 dans la lignée cellulaire LN-229 et provoque ainsi l'atténuation de l'activité de cette voie de signalisation. En conclusion, HECTD1, en interagissant avec USP15, joue un rôle de suppresseur de tumeur dans les lignées cellulaire de GBM.

Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response.

Cysteine cathepsin protease activity is frequently dysregulated in the context of neoplastic transformation. Increased activity and aberrant localization of proteases within the tumour microenvironment have a potent role in driving cancer progression, proliferation, invasion and metastasis. Recent studies have also uncovered functions for cathepsins in the suppression of the response to therapeutic intervention in various malignancies. However, cathepsins can be either tumour promoting or tumour suppressive depending on the context, which emphasizes the importance of rigorous in vivo analyses to ascertain function. Here, we review the basic research and clinical findings that underlie the roles of cathepsins in cancer, and provide a roadmap for the rational integration of cathepsin-targeting agents into clinical treatment.

Free Prostate-specific Antigen Forms and Kallikrein-related Peptidase 2: Tools for Prostate Cancer Diagnostics

Prostate-specific antigen (PSA) is a marker that is commonly used in estimating prostate cancer risk. Prostate cancer is usually a slowly progressing disease, which might not cause any symptoms whatsoever. Nevertheless, some cases of cancer are aggressive and need to be treated before they become life-threatening. However, the blood PSA concentration may rise also in benign prostate diseases and using a single total PSA (tPSA) measurement to guide the decision on further examinations leads to many unnecessary biopsies, over-detection, and overtreatment of indolent cancers which would not require treatment. Therefore, there is a need for markers that would better separate cancer from benign disorders, and would also predict cancer aggressiveness. The aim of this study was to evaluate whether intact and nicked forms of free PSA (fPSA-I and fPSA-N) or human kallikrein-related peptidase 2 (hK2) could serve as new tools in estimating prostate cancer risk. First, the immunoassays for fPSA-I and free and total hK2 were optimized so that they would be less prone to assay interference caused by interfering factors present in some blood samples. The optimized assays were shown to work well and were used to study the marker concentrations in the clinical sample panels. The marker levels were measured from preoperative blood samples of prostate cancer patients scheduled for radical prostatectomy. The association of the markers with the cancer stage and grade was studied. It was found that among all tested markers and their combinations especially the ratio of fPSA-N to tPSA and ratio of free PSA (fPSA) to tPSA were associated with both cancer stage and grade. They might be useful in predicting the cancer aggressiveness, but further follow-up studies are necessary to fully evaluate the significance of the markers in this clinical setting. The markers tPSA, fPSA, fPSA-I and hK2 were combined in a statistical model which was previously shown to be able to reduce unnecessary biopsies when applied to large screening cohorts of men with elevated tPSA. The discriminative accuracy of this model was compared to models based on established clinical predictors in reference to biopsy outcome. The kallikrein model and the calculated fPSA-N concentrations (fPSA minus fPSA-I) correlated with the prostate volume and the model, when compared to the clinical models, predicted prostate cancer in biopsy equally well. Hence, the measurement of kallikreins in a blood sample could be used to replace the volume measurement which is time-consuming, needs instrumentation and skilled personnel and is an uncomfortable procedure. Overall, the model could simplify the estimation of prostate cancer risk. Finally, as the fPSA-N seems to be an interesting new marker, a direct immunoassay for measuring fPSA-N concentrations was developed. The analytical performance was acceptable, but the rather complicated assay protocol needs to be improved until it can be used for measuring large sample panels.

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10% at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65% of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p<0.05), when compared to the other CYS concentrations. The BTS (10.20±0.39) treated group showed a lower rate of estrus return when compared to other (BTSCYS; 86.05±039), and it showed also the highest total number of piglets borne per treatment (12.71±3.38 vs. 9.00±3.38, respectively). In conclusion, the addition of CYS in the BTS semen extender did not maintain spermatic viability of boar cooled spermatozoa and it results in a higher percentage of return to estrus and lower number of piglets borne.

A new allele of peptidase-B in cattle

Electrophoretic analyses of peptidase-B were carried out on red cell hemolysates from Holstein, Mantiqueira and Gyr cattle, using cornstarch, known in Brazil as Penetrose-30. We describe a new peptidase-B allele, denoted Pep-B1, in Mantiqueira cattle, belonging to the Bos taurus group, which are the result of a cross of native cattle of Portuguese origin introduced in Brazil during colonial times (16th century) with Holstein and Caracu cattle. The genetic control of peptidase-B was determined by typing parents and progeny segregating for all three alleles, confirming that peptidase B is controlled by a single autosomal locus with three codominant alleles, denoted Pep-B1, Pep-B2 and Pep-B3 The use of the citrate-phosphate buffer system, at pH 5.9, on 14% gel, under the electrophoretic conditions standardized in this study permitted good visualization of all peptidase-B variants.

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml) or simple sugars (320-350 µg/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

SENSORY ANALYSIS OF A MODEL SYSTEM USING 5'-IMP AND CYSTEINE AT DIFFERENT pH

Sensory analysis was used to get an overall flavour description of a reaction mixtures containing 5'-IMP and Cysteine. Ribose/cysteine systems were used as reference systems. Results from triangle and aroma profiling show a clear correlation between the terms used and the volatile analysis described in literature for these model systems. For instance reactions at pH 3.0 and 4.5 for 5'-IMP/cysteine systems, which were described as "meaty" and "boiled meat" by panellists, presented, in the literature, the higher number of "meaty" compounds in volatile analysis (1, 7, 8, 20) .

Continuous hydride generation in the presence of L- cysteine for the determination of arsenic, bismuth, antimony and tin in steels by inductively coupled plasma atomic emission spectrometry

Arsenic, bismuth, germanium, antimony and tin were simultaneously determined by continuous hydride generation and inductively coupled plasma-atomic emission spectrometry . I Hydrides were introduced into four different types of gas-liquid separators. Two of the gas-liquid separators were available in-house. A third was developed for this project and a fourth was based on a design used by CET AC. The best signal intensity was achieved by the type II frit-based gas-liquid separator, but the modified Cetac design gave promise for the future, due to low relative standard deviation. A method was developed for the determination of arsenic, bismuth, antimony and tin in low-alloy steels. Four standard reference materials from NIST were dissolved in 10 mL aqua regia without heat. Good agreement was obtained between experimental values and certified values for arsenic, bismuth, antimony and tin. The method was developed to provide the analyst with the opportunity to determine the analytes by using simple aqueous standards to prepare calibration lines. Within the limits of the samples analyzed, the method developed is independent of matrix.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Mechanisms by which cysteine can inhibit or promote the oxidation of low density lipoprotein by copper

Oxidised low density lipoprotein (LDL) may play a role in atherogenesis. We have investigated some of the mechanisms by which the thiol cysteine and the disulphide cystine can influence the oxidation of LDL by copper ions. Cysteine or cystine (100 PM) inhibited the oxidation of native LDL by copper in a simple phosphate buffer. One of the mechanisms by which cysteine (or more likely its oxidation products in the presence of copper) and cystine inhibited LDL oxidation was by decreasing the binding of copper to LDL (97% inhibition). Cysteine, but not cystine, rapidly reduced Cu2+ to Cu+. This may help to explain the antioxidant effect of cysteine as it may limit the amount of Cu2+ that is available to convert alpha-tocopherol in LDL into the prooxidant alpha-tocopherol radical. Cysteine (but not cystine) had a prooxidant effect, however, toward partially oxidised LDL in the presence of a low copper concentration, which may have been due to the rapid breakdown of lipid hydroperoxides in partially oxidised LDL by Cu+ generated by cysteine. To prove that cysteine can cause the rapid breakdown of lipid hydroperoxides in LDL, we enriched LDL with lipid hydroperoxides using an azo initiator in the absence of copper. Cysteine, but not cystine, increased the rate of lipid hydroperoxide decomposition to thiobarbituric acid-reactive substances (TBARS) in the presence of copper. (C) 2003 Elsevier Ireland Ltd. All rights reserved.