Jacques Haiech

Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins

SummaryThe maximum parsimony method was used to reconstruct the genealogical history of the family of intracellular calcium-binding proteins represented by six major present-day lineages, three of which - calcium dependent modulator protein, heart and skeletal muscle troponin Cs, and alkali light chains of myosin - were found to share a closer kinship with one another than with the other lineages. Similarly, parvalbumins and regulatory light chains of myosin were depicted as more closely related, whereas the branch of intestinal calcium-binding protein proved to have the most distant separation. The computer-generated amino acid sequence for the common ancestor of these six lineages described a four domain protein in which each domain of approximately 40 amino acid residues had a mid-region, 12 residue segment that bound calcium and had properties most resembling those of the calcium dependent modulator protein. It could then be deduced that parvalbumins evolved by deletion of domain I, inactivation of calcium-binding properties in domain II, and acquisition of increased affinity for Ca++ and Mg++ in domains III and IV. Regulatory light chains of myosin lost the cation binding property from three domains, retaining it in I, whereas alkali light chains of myosin lost this ability from each of the four domains. In skeletal muscle troponin C all domains retained their calcium-binding activity; however, like parvalbumins, domains III and IV acquired high affinity properties. Cardiac troponin C lost its binding activity from domain I but otherwise resembled the skeletal muscle form. Finally, intestinal calcium-binding protein evolved by deletion of domains III and IV.Positive selection could be implicated in these evolutionary changes in that the rate of fixation of mutations substantially increased in the mid portions of those domains which were loosing calcium-binding activity. Likewise, when the cation binding sites were changing from low to high affinity, an accelerated rate of fixed mutations was observed. Once this new functional parameter was selected these regions showed a remarkable conservatism, as did those binding sites which were maintaining the lower affinity. Moreover even in sequence regions not directly involved in cation binding, the lineage of troponin C became very conservative over the past 300 million years, perhaps because of the necessity for maintaining specific interfaces in order for the molecule to interact with troponin I and T in a functional thin myofilament. A similar phenomenon was observed in domain II of the regulatory light chains of the myosin lineage suggesting a possible binding site with the heavy chain of myosin.

Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.

The participation of parvalbumins in the activation—relaxation cycle of vertebrate fast skeletal-muscle

Although the physiological role of muscular parvalbumins [l-3] remains an open question, several studies [4,5] have restricted the range of possibilities and suggested that these proteins constitute a non-interacting regulatory system of the sarcoplasmic Ca*-level which operates essentially in fast contracting muscles so as to facilitate their cyclic relaxation. In addition, a scheme has been proposed [S] , in which the affinity of parvalbumins for Ca*’ (thus their possible competition with troponin C for this ion) could be modulated by changes in hydrogen ion-concentration associated with the contraction process. This allows undisturbed activation of the myofibrils at higher pH, while ensuring their complete relaxation at lower pH. Experiments reported recently in the literature [6,7] have substantiated the validity of several steps of the above scheme. Thus, they have shown that calcium-free parvalbumins indeed have the potential to block contraction or ATPase activity of Ca’+-activated myofibrils. Sarcoplasmic reticulum, in turn, is able to remove efficiently all the Ca*-ions which can be bound by parvalbumins. The first experiments reported in the present communication were designed independently in order

Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

Focal adhesion kinase (FAK) activity and Ca2+ signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca2+ sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca2+ variations at FA sites and FA dynamics. Discrete subcellular Ca2+ oscillators initiate both propagating and abortive Ca2+ waves in migrating U87 astrocytoma cells. Ca2+-dependent FA disassembly occurs when the Ca2+ wave reaches individual FAs, indicating that local but not global Ca2+ increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca2+ elevation. FAK-related non-kinase domain-Ycam had a faster, Ca2+-insensitive recovery half-time (11 s), which is consistent with the effect of Ca2+ on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca2+ and FAK autophosphorylation was correlated to intracellular Ca2+ levels, we propose that local Ca2+ elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

Small Neutralizing Molecules to Inhibit Actions of the Chemokine CXCL12

The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.

Calcium signalling in Bacillus subtilis

Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury.

Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

BackgroundTumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.MethodsWe screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.ResultsA long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.ConclusionsOur results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Fluorescent Derivatives of the GFP Chromophore Give a New Insight into the GFP Fluorescence Process

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.

Calcium : the molecular basis of calcium action in biology and medicine

List of Contributors. Editorial R. Pochet. Overview: From Genes to drugs J. Haiech, M. Hilbert. Part One: Basic Mechanisms of Action of Calcium. Calcium and apoptosis: M. Maki, S. Christakos, F. Barletta, M. Huening, J. Kohut, M. Raval-Pandya. Calcium and cellular aging: A. Verkhratsky, E. Toescu. Calcium and growth factor: A. Villalobo, M.J. Ruano, P.I. Palomo-Jimenez, H. Li, J. Martin-Nieto, J. Heemskerk. Calcium signalling in excitable cells: N. Macrez, J. Mironneau, S.J. Marsh, N. Wanaverbecq, A.A. Selyanko, D. Brown. Calcium signalling in extracellular matrix: A. Koch, J. Engel, P. Maurer, D.T. Wardm D. Riccardi. Calcium signalling in non-excitable cells: A. Guse, G.J. Barritt, K.-H. Braunewell, C. Reissner, E.D. Gundelfinger, L. Combettes, C. Clair, M. Claret, T. Tordjmann. Calcium storage and release: J. Parys, I. Sienaert, S. Vanlingen, G. Callewaert, P. De Smet, L. Missiaen, H. de Smedt, M. Michalak, K. Nakamura, S. Papp, M. Opas, D. Rossi, V. Barone, I. Simeoni, V. Sorrentino, H. Kawamichi, A. Nakamura, M. Kohama. Part Two: Calcium Related Diseases: R. Valenta, A. Twardosz, I. Swoboda, B. Hayek, S. Spitzauer, D. Kraft, H. Pollard, M. Srivastava, P. Eggleton, D. Llewellyn, P. van Zwieten, D. Pietrobon, S. Islam, F. Rodriguez de Fonseca, M. Angel Gorriti, M. Navarro. Calcium and Muscle disease: Y. Ono, S. Hata, H. Sorimachi, K. Suzuki. Cancer: R.L. Gee, L. Subramanian, T.S. Walker, P.R. van Ginekl, A.S. Polans, P. Paterlini-Brechot, M. Chami, D. Gozuachik, D.J. Weber, R.R. Rustandi, F. Carrier, D.B. Zimmer, K.Y. Chun, D. Sacks, E. Brown, N. Chattopadhyay,M. Bai. Inflammatory diseases: G. Hagens, G. Siegenthaler. Liver diseases: K. Kobayashi, M. Iijima, T. Yasuda, D.S. Sinasac, N. Yamaguchi, L.-C. Tsui, S. Scherer, T. Saheki. Neurodegeneration: M.E. Spira, N. Ziv, R. Oren, A. Dormann, D. Gitler. Neurodegenerative diseases: M. Monteiro, S. Stabler, B. Keller. Part Three: Methodological Chapters: J. Haiech, J.-L. Galzi, M. Hibert, V. Dupriez. Calcium imaging, fluorescence microscopy, confocal microscopy: A. Nadal, B. Soria, A. Pesty, J. Pocock, G. Evans, G. Monteith, V. Dedov, B. Roufogalis. Index.