Docking study of ligands into the colchicine binding site of tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of alpha and beta-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography

Background  

R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365~366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.     

HEAT repeats mediate plasma membrane localization of Tor2p in yeast

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.     

TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes TOR1 and TOR2 encode phosphatidylinositol kinase homologs. TOR2 has two essential functions. One function overlaps with TOR1 and mediates protein synthesis and cell cycle progression. The second essential function of TOR2 is unique to TOR2 and mediates the cell-cycle-dependent organization of the actin cytoskeleton. We have isolated temperature-sensitive mutants that are defective for either one or both of the two TOR2 functions. The three classes of mutants were as follows. Class A mutants, lacking only the TOR2-unique function, are defective in actin cytoskeleton organization and arrest within two to three generations as small-budded cells in the G2/M phase of the cell cycle. Class B mutants, lacking only the TOR-shared function, and class C mutants, lacking both functions, exhibit a rapid loss of protein synthesis and a G1 arrest within one generation. To define further the two functions of TOR2, we isolated multicopy suppressors that rescue the class A or B mutants. Overexpression of MSS4, PKC1, PLC1, RHO2, ROM2, or SUR1 suppressed the growth defect of a class A mutant. Surprisingly, overexpression of PLC1 and MSS4 also suppressed the growth defect of a class B mutant. These genes encode proteins that are involved in phosphoinositide metabolism and signaling. Thus, the two functions (readouts) of TOR2 appear to involve two related signaling pathways controlling cell growth.     

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuc lease for s s DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.     

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.     

β1A integrin is a master regulator of invadosome organization and function

Invadosomes are adhesion structures involved in tissue invasion that are characterized by an intense actin polymerization–depolymerization associated with β1 and β3 integrins and coupled to extracellular matrix (ECM) degradation activity. We induced the formation of invadosomes by expressing the constitutive active form of Src, SrcYF, in different cell types. Use of ECM surfaces micropatterned at the subcellular scale clearly showed that in mesenchymal cells, integrin signaling controls invadosome activity. Using β1^−/− or β3^−/− cells, it seemed that β1A but not β3 integrins are essential for initiation of invadosome formation. Protein kinase C activity was shown to regulate autoassembly of invadosomes into a ring-like metastructure (rosette), probably by phosphorylation of Ser785 on the β1A tail. Moreover, our study clearly showed that β1A links actin dynamics and ECM degradation in invadosomes. Finally, a new strategy based on fusion of the photosensitizer KillerRed to the β1A cytoplasmic domain allowed specific and immediate loss of function of β1A, resulting in disorganization and disassembly of invadosomes and formation of focal adhesions.