Moncef M. Ladjimi

Effect of nucleotides, peptides, and unfolded proteins on the self-association of the molecular chaperone HSC70.

In a previous study, we showed that the molecular chaperone HSC70 self-associates in solution in a reversible and likely unlimited fashion. Here, we examine the influence of nucleotides, nucleotide analogs, peptides, and unfolded proteins on the self-association properties of this protein. Whereas in the presence of ADP, HSC70 exists as a slow, concentration- and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, the protein is essentially monomeric, indicating that ATP shifts this equilibrium toward the monomer by stabilizing the monomer. Dissociation of oligomers into monomers is also obtained with the slowly hydrolyzable ATP analogs, adenosine 5′-O-(thiotriphosphate) and 5′-adenylyl-β,γ-imidodiphosphate, or the complex between ADP and the phosphate analog, BeF3, indicating that binding but not hydrolysis of ATP is necessary and sufficient for the stabilization of HSC70 monomer. Furthermore, binding of short peptides or permanently unfolded proteins to the peptide binding site of HSC70 promotes the dissociation of oligomers into monomers, suggesting that protein substrates are able to compete with HSC70 for the same binding site. Because the release of peptides or unfolded proteins from HSC70 has also been shown to require ATP binding, these results indicate that dissociation of oligomers is controlled by a mechanism similar to that of release of protein substrates and suggest that binding of HSC70 to itself occurs via the peptide binding site and mimics binding of HSC70 to protein substrates.

The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70

We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 105 M−1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.

Heterotropic interactions in Escherichia coli aspartate transcarbamylase : subunit interfaces involved in CTP inhibition and ATP activation

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

Comparative mechanisms of zearalenone and ochratoxin A toxicities on cultured HepG2 cells: is oxidative stress a common process?

Zearalenone (ZEN) and Ochratoxin A (OTA) are structurally diverse fungal metabolites that can contaminate feed and foodstuff and can cause serious health problems for animals as well as for humans. In this study, we get further insight of the molecular aspects of ZEN and OTA toxicities in cultured human HepG2 hepatocytes. In this context, we have monitored the effects of ZEN and OTA on (i) cell viability, (ii) heat shock protein (Hsp) 70 and Hsp 27 gene expressions as a parameter of protective and adaptive response, (iii) oxidative damage, and (iv) cell death pathways. Our results clearly showed that both ZEN and OTA inhibit cell proliferation. For ZEN, a significant induction of Hsp 70 and Hsp 27 was observed. In the same conditions, ZEN generated an important amount of reactive oxygen species (ROS). Antioxidant supplements restored the major part of cell mortality induced by ZEN. However, OTA treatment downregulated Hsp 70 and Hsp 27 protein and mRNA levels and did not induce ROS generation. Antioxidant supplements did not have a significant effect on OTA‐induced cell mortality. Using another cell system (Vero monkey kidney cells), we demonstrated that OTA downregulates three members of HSP 70 family: Hsp 70, Hsp 75, and Hsp 78. Our findings showed that oxidative damage seemed to be the predominant toxic effect for ZEN, while OTA toxicity seemed to be rather because of the absence of Hsps protective response. Furthermore, the two mycotoxins induced an apoptotic cell death.

Complementation of an Escherichia coli DnaK Defect by Hsc70-DnaK Chimeric Proteins

Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.

Identification of proteins related to early changes observed in Human hepatocellular carcinoma cells after treatment with the mycotoxin Zearalenone.

Zearalenone (ZEA) is a mycotoxin produced by some Fusarium species. ZEA often occur as a contaminant in cereal grains and animal feeds. Human exposure occurs by ingestion of mycotoxin-contaminated products and can cause serious health problems. It was established that this mycotoxin have an hepato, haemato, immuno and genotoxic properties (Maaroufi et al., 1996; Lioi et al., 2004). While most ZEA toxic effects have been quite well investigated, more studies are required to elucidate its mechanisms of toxicity. In order to better understand the molecular mechanisms involved in ZEA toxicity, we used a proteomic approach, to assess the early changes in protein expression initiated by ZEA in HepG2 cells. Our results showed that, after 8h of exposure, cells were still viable and showed a significant change in a number of proteins involved in diverse cellular processes. These changes may provide the early affected functions and yield further insight into mechanisms underlying the involvement of mycotoxin-induced diseases.

3‐D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.

iTRAQ: a method to elucidate cellular responses to mycotoxin zearalenone.

Mycotoxin zearalenone (ZEN) is a secondary metabolite produced by some Fusarium species that contaminate a large variety of grains and feedstuffs worldwide. ZEN has been associated with a wide variety of adverse health effects including hepatotoxic, hematologic, immunotoxic and genotoxic. In order to better understand the mechanism of ZEN toxicity, a proteomic approach was applied to characterize cellular responses of hepatocarcinoma cells (HepG2) to ZEN exposure. Protein extracts from cultured HepG2 cells treated with 100 µm ZEN for 8 h, as well as extracts from control cells. The screening method applied to compare the proteome was based on the stable isotope approach of isobaric tagging for relative and absolute quantification (iTRAQ). This study identified 982 proteins, among which peptides and their corresponding proteins were identified and quantified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Ingenuity pathways analysis software was then used to determine the biological functions and canonical pathways associated with the ZEN‐responsive proteins. Copyright

Engineering aspartate transcarbamylase

Aspartate transcarbamylase from Escherichia coli is one of the most extensively studied regulatory enzymes as a model of cooperativity and allostery. Numerous methods are used to engineer variants of this molecule: random and site-directed mutagenesis, dissociation and reassociation of the catalytic and regulatory subunits and chains, construction of hybrids made from normal and modified subunits or chains, interspecific hybrids and construction of chimeric enzymes. These methods provide detailed information on the regions, domains, interfaces and aminoacid residues which are involved in the mechanism of co-operativity between the catalytic sites, and of regulation by the antagonistic effectors CTP and ATP. These effectors induce the transmission of intramolecular signals whose pathways begin to be delineated.

Analysis of Monomeric Mutants of HSC70: A Possible Relationship Between Oligomerization and Functional Properties

Data of this study showed that alphaD-alphaE helices and the conserved interdomain linker are two interfaces essential not only for the self-association but also for the functional properties of rat HSC70. Self-association which is a conserved property of HSP70 seems to be important for the activity of these proteins.