D'hondt C.,Laboratory of Molecular and Cellular Signalling |
Iyyathurai J.,Laboratory of Molecular and Cellular Signalling |
Wang N.,Ghent University |
Gourdie R.G.,Medical University of South Carolina |
And 3 more authors.
Biochemical and Biophysical Research Communications | Year: 2013
Connexin 43 (Cx43)-hemichannel activity is controlled by intramolecular interactions between cytoplasmic loop and C-terminal tail. We previously identified the last 10 amino acids of the C-terminal tail of Cx43 as essential for Cx43-hemichannel activity. We developed a cell-permeable peptide covering this sequence (TAT-Cx43CT). In this study, we examined the critical molecular determinants in TAT-Cx43CT to restore Cx43-hemichannel activity. Using amino acid substitutions in TAT-Cx43CT, we identified the two aspartate (Asp378 and Asp379) and two proline (Pro375 and Pro377) residues as critical for TAT-Cx43CT activity, since TAT-Cx43CTDD/AA and TAT-Cx43CTPP/GG did not overcome the inhibition of Cx43-hemichannel activity induced by thrombin, micromolar cytoplasmic Ca2+ concentration or truncation of Cx43 at M239. Consistent with this, we found that biotin-Cx43CTDD/AA was much less efficient than biotin-Cx43CT to bind the purified CL domain of Cx43 in surface plasmon resonance experiments. In conclusion, we postulate that Asp378 and Asp379 in the C-terminal part of Cx43 are essential for loop/tail interactions in Cx43 hemichannels, while Pro375 and Pro377 may help to properly coordinate the critical Asp residues. © 2013 Elsevier Inc.
Zhong F.,Case Western Reserve University |
Harr M.W.,Case Western Reserve University |
Bultynck G.,Laboratory of Molecular and Cellular Signalling |
Monaco G.,Laboratory of Molecular and Cellular Signalling |
And 8 more authors.
Blood | Year: 2011
Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel, inhibiting IP3-dependent Ca2+ release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP 3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP 3R-dependent Ca2+ elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca2+ elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically. © 2011 by The American Society of Hematology.
Szatkowski C.,University dAmiens |
Parys J.B.,Laboratory of Molecular and Cellular Signalling |
Ouadid-Ahidouch H.,University dAmiens |
Matifat F.,University dAmiens
Molecular Cancer | Year: 2010
Background: Ca2+is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP3-induced Ca2+signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).Results: Our results show that the IP3receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+imaging experiments showed that serum and E2were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+release process and acted to counteract the proliferative effect of 10 nM E2.Conclusions: Altogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP3R3 expression by E2is involved in this effect. © 2010 Szatkowski et al; licensee BioMed Central Ltd.
PubMed | Laboratory of Molecular and Cellular Signalling
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2011
The amount of Ca(2+) taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca(2+) ions mainly originate from the inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores of the endoplasmic reticulum (ER). The uptake of these Ca(2+) ions in the mitochondria depends on the functional properties and the subcellular localization of the IP(3) receptor (IP(3)R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca(2+) ions from the ER to the mitochondria, structural interactions between IP(3)Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.