Month: May 2020

In the present study, SB was found to inhibit the adhesion and aggregation of platelets induced by collagen. The effect of SB might be related to its binding to integrin a2b1, the possible direct target protein of SB found in our previous study. The un-direct targetrelated proteins of SB in platelets were searched in the present study using a differential proteomic study. Proteomic technology has become an indispensable and efficient tool in biochemical research. And, this technology has successfully used to study signal pathways in platelets. Twenty possible target-related proteins of SB in platelets were found in the present study. Among the 20 proteins, 6 proteins including heat shock-related 70 kDa protein 2, LIM Reversine domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C might be the most important ones and have close relationship with integrin a2b1 binding. The possible signal cascades network in platelets after binding of SB to integrin a2b1 was predicted based on previous reports and protein-protein interaction database. For example, previous works indicated that integrin a2b1 binding could cause change in calcium homeostasis and calcium-dependent functions. Previous report also suggested that, by binding to integrin a2b1, endorepellin could trigger transient activation of p38 mitogenactivated protein kinase and heat shock protein 27 and ultimately disassembly of actin stress fibers and focal adhesions. To certify the predicted signal network, against integrin a2b1 on the effects of SB in platelets were checked. The results indicated that SB had dose-dependently binding affinity to integrin a2b1 in vitro and could compete with antibody against integrin a2b1 for the binding site of integrin a2b1 on the membrane of platelets. Furthermore, flow cytometry analysis result confirmed that SB treatment caused dosedependently decrease in intracellular level of Ca level and the effect of SB on Ca level might be related to integrin a2b1 binding. And, the change in coronin-1 B protein level caused by SB treatment was also dependent on integrin a2b1 binding. The revised signal network of SB including binding with integrin a2b1, change in Ca level, reorganization of cytoskeleton as well as change in cytoskeleton-related proteins such as coronin-1 B was established. As to the SB-caused change in ROS level and heat shock protein 70 level, our results indicated that these effects of SB might not be dependent on binding with integrin a2b1. For example, the decrease in ROS might be caused by the potency of SB to act as direct ROS scavenger. Based on its chemical structure characteristic, SB could exhibit direct antioxidative effect in vitro as well as in vivo.

The possibility that reversing the resulting Michael addition and thiocarbamate products with DTT might result in identification of the targets of these reactions using PROP. Some evidence suggests that this is not so. For example, modification of the MEKK1 protein kinase by isothiocyanate is stable to boiling in SDS and DTT, though other cysteine adducts might be less stable. Similarly, numerous cell proteins labeled with biotin-prostaglandin J2, and detected with anti-biotin immunoblotting are not measurably reversed by boiling in SDS and DTT, though again, specific proteins might be. Owing to these observations, we predict that these reactions will not be detectable using PROP. We and others have previously detected regulatory oxidation of other protein kinases. Importantly, in most of these experimental systems, oxidation is induced by hydrogen peroxide at high concentrations, diamide, menadione, or other non-physiologic agents. Our finding that micromolar concentrations of PGJ2 can induce oxidation and inhibition of p38 extends these results to agents involved in the physiologic response to disease or inflammation. Our results suggest that oxidation may serve as a redox switch that provides a feedback mechanism for p38 and other signaling kinases. Specifically, initial MK-2206 2HCl activation of p38 results from phosphorylation within the “activation loop”, that is detectable using anti-phospho p38 antibodies. Subsequently, activated p38 may become oxidized at one or more cysteine residues, resulting in the loss of kinase activity. Importantly, this inactive kinase retains its phosphorylation and may incorrectly be interpreted as an active kinase when using anti-phospho antibody immunoblot as a surrogate measure of “activity”. Signal transduction investigators are cautioned that the simplicity of an anti-phospho-immunoblot may obscure more subtle means by which signal transduction is regulated. We recommend that for a specific signaling pathway to be deemed “activated”, a direct measurement of the catalytic activity in vitro is indicated, together with an evaluation of natural substrates of the kinase within cells. Gap junction channels are formed when connexin subunits situated in adjacent plasma membranes dock together. These transmembrane channels allow the passage of metabolites, ions and second messengers and nucleotides up to 1 kDa in size. Undocked connexins, or hemichannels have also been identified and they provide a means to contact the extracellular environment. Gap junction channels are formed when connexin subunits situated in adjacent plasma membranes dock together.

Cells expressing FNR and Fld showed reduced oxidative damage of lipids indicating that the protection is probably exerted through an antioxidative action towards H2O2. Even though a mechanism of protection through repair can not be discarded and it has been observed for iron-sulfur centers of hydro-lyases in FNR-overexpressing bacterial cells after oxidative stress induction, FNR and Fld are not repairing enzymes per se. However, it is possible that some repairing pathways are working more efficiently in cells expressing FNR and Fld because they are more protected by the actions of these two proteins. This observation reinforces the hypothesis that oxidative stress persists 24 h after induction by hydrogen peroxide. MDA levels were much higher for Cos-7 than Cos-7/pcDNA3 cells. This difference is not really surprising as Cos-7 cell line has a faster rate of growth than all the transfected lines when grown in the presence of G418. Faster growing cells are metabolically more active and, as a consequence, more susceptible to damages by exposure to cytotoxic compounds. Given this observation, we considered important to include both lines as controls in this study to be able to discriminate between the effects of the expression of FNR and Fld per se and including other factors of the process of transfection and selection. Both controls are necessary to allow comparison in the case transfection or transduction systems different from the one used here were employed. The protection from hydrogen peroxide-induced damage resulted of similar magnitude for FNR and Fld. This finding was not expected a priori because FNR and Fld are enzymes with different properties regarding kinetics and, most probably, specificity of interactions with endogenous cellular components. We have measured the diaphorase activity of Cos-7/pFNR and found that it doubles that of the control line Cos-7/pcDNA3 supporting the idea that FNR is functional and elevates basal levels of this enzymatic activity. For Fld we do not have an estimation of the expression of this protein. MTT reduction and LPO were analyzed under MV induced injury. Cells demonstrated a major reduction in viability/growth and almost duplication of LPO content but we could not see any protection in cells expressing FNR and Fld. These findings were surprising since both FNR and Fld have shown to protect a number of eukaryote and prokaryote organisms from MV exposure. Shimizu et al. showed protection from MV damage in Cos-7 cells overexpressing oxidized protein hydrolase. This finding does not exclude, however, that changes associated with the HhAntag691 overexpression of FNR and/or Fld may result in the lack of protection we observed. Other authors using MV to induce oxidative stress have observed that protective enzymes work better if their expression is directed to mitochondria when compared with cytosolic localization. MLS in Cos-7/pFld has not been efficiently excised but this fact did not avoid protection from H2O2.

Syntaxin family SNARE proteins are integral membrane proteins that belong to Q-SNAREs i. e. they contain a glutamine at the central layer of the SNARE motif bundle. In addition to the SNARE motif, syntaxins have an N-terminal domain that is composed of three short helixes and a C-terminal transmembrane domain that is followed by a very short hydrophilic tail. S. cerevisiae expresses two highly homologous syntaxins Sso1p and Sso2p that both mediate membrane fusion during exocytosis at the plasma membrane. The Sso1p the N-terminal domain has been shown to interact with the SNARE motif and regulate the rate of SNARE complex assembly. Together, Sso1p and Sso2p perform an essential function in vegetatively growing haploid and diploid cells where they interact with plasma membrane SNARE proteins Sec9p, Snc1p and Snc2p. However, in meiotic diploid cells there is a specific Dasatinib abmole bioscience requirement for Sso1p for de novo formation of the prospore membrane during meiosis. The functional difference for Sso1p and Sso2p in meiotic cells is not explained by transcriptional regulation, or differences in expression levels. Both proteins are expressed at similar level in meiotic cells, localize to the prospore membrane, and swapping of promoters between SSO1 and SSO2 does not render Sso2p functional in prospore membrane formation. The two N-terminal a-helices Ha and Hb of Sso1p are important for its function during meiosis. In addition to the specific requirement of Sso1p, in sporulating cells the Q-SNARE Sec9p is replaced by a homologous protein Spo20. Recent results indicate that phosphatidic acid and PIP2 are important for membrane fusion during prospore membrane formation. However, the signals that regulate the activity of Sso1p and the initiation of meiotic SNARE complex formation are unknown. Post-translational modifications are central modifiers of protein activity. Mass spectrometry studies have revealed in vivo phosphorylation sites in the amino terminal part of Sso1p and Sso2p. In this study we set out to establish the contribution of these phosphoamino acids on the functional regulation of Sso1 and Sso2 proteins. In addition, we tested, whether, in analogy to meiosis and sporulation, also pseudohyphal and invasive growth, two nutritionally regulated cell differentiation processes display differential requirements for Sso1p and Sso2p. In addition, serine 79 was previously reported as an in vivo phosphorylation site in Sso1p. Subsequent analysis showed that S79 phosphorylation reduced participation of Sso1p in haploid cell SNARE complexes. These amino acids represent potential regulatory means to modulate Sso protein in vivo function and differentiate between these proteins during sporulation. The structure of a cytosolic fragment of Sso1p has been determined. This structure is missing the very amino-terminus that contains several phosphorylation sites in Sso1p and the homologous Sso2p. The amino-terminal peptides of several syntaxins do not refract well in crystals. This suggests that even when present in the analyzed protein the peptide is unstructured in monomeric syntaxins.

shRNA expression reduced the proliferation of cells and prevented colony formation on soft agar plates, indicative of a defect in anchorage independent growth. These cells lines enabled us to fine-tune PHB1 and PHB2 silencing under standardized conditions and to investigate the mechanism underlying the strong phenotype associated with prohibitin depletion. We first analyzed the effect of prohibitin depletion on the levels of mitochondrial fusion protein OPA1 as previous publications have shown that prohibitins are necessary for stable expression of OPA1. We could show that OPA1 expression is dependent on the levels of prohibitins. A slight reduction of prohibitins, seen early after induction of shRNA production, led to a partial fragmentation of the fusion competent OPA1 fragments a and b. At later time points of induction and consequently stronger depletion of prohibitins, OPA1 fragmentation increased and mitochondria appeared fragmented. An incomplete reduction of prohibitin proteins, as seen with expression of shRNAs shPHB1-0 and shPHB2-0, only resulted in mild OPA1 fragmentation and no fragmentation of the mitochondrial network. Further analysis revealed that even strong and long-lasting depletion of PHB1 e.g. in cells producing shPHB1-3, did not cause the dissipation of MMP. To date, the effects of prohibitin loss in mammalian cells are still unclear: Schleicher et al. and Ross et al. show a reduction in MMP upon PHB1 and PHB1/2 depletion via RNAi in primary endothelial cells and a T-cell line, respectively; in contrast, Merkwirth and colleagues demonstrated that although PHB2 depletion in MEFs causes OPA1 fragmentation, respiratory activity and MMP are not affected. Treatment of cells with the ionophore CCCP resulted in the rapid loss of MMP accompanied by a gradual and consecutive fragmentation of the OPA1 fusion-competent protein fragment and the mitochondrial network. Different patterns of OPA1 fragmentation were observed in CCCP treated cells and prohibitin-depleted cells: All fusion incompetent fragments were upregulated upon CCCP treatment, but not as a consequence of prohibitin reduction. In addition, fragment d was downregulated when prohibitin levels were reduced, but upregulated upon CCCP treatment. Thus, our data confirm the findings of Merkwirth at al., showing downregulation of fragment d in PHB2-depleted MEFs. Studies in yeast have shown prohibitins interact with mAAA protease and are important for the VE-822 proper integration of respiratory chain complex proteins. Furthermore, rhomboid like protease PARL is thought to be involved in OPA1 processing. Considering the fragmentation pattern of OPA1 in prohibitin knockdown cells, it seems most likely that multiple proteases are involved in OPA1 processing and not all of them are affected by prohibitin loss. This would be in accordance with our observation that both mitochondrial cristae morphology and ATP synthesis were unaffected by a loss of prohibitins. Mitochondrial fragmentation only occurred in cell with a strong depletion of prohibtins as we saw it upon shPHB1-3 expression.