Unit: Protein Folding and Modeling
Director: GOLDBERG Michel
The unit combines genetics, biochemistry and physical-chemistry to study various problems related to protein structure, function and integration in a variety of cellular processes such as the acquisition of their functional conformation or the mechanisms of molecular transmital of the abnormal conformation of a non-conventional pathogen (the "prion" protein).
One group of the unit deals with the conformation of the PrP protein inside the prion. A second group has a long term interest in the molecular mechanisms involved in the cellular detection of misfolded bacterial proteins and, besides these fundamental studies, develops cloning strategies for recombinant protein production.
1- Immunochemical approach of the PrP protein conformation inside the "Prion""(M.-C. Blom-Potar, P. Bittoun, M. Cardona, P. Falanga, M. Goldberg and M. Hontebeyrie)
The prion is the infectious agent responsible for spongiform encephalopathies such as the sheep scrappie, the Mad Cow Disease, or the human Creutzfeldt-Jakob disease. The prion is made of a protein, the PrP, that exists in healthy as well as diseased individuals. However, the conformation (shape) of the PrP of healthy individuals differs from that of PrP in the prion. The "Prion" group aims at characterizing this conformation difference by means of specific antibodies. Besides its purely scientific interest, this research aims at improving the sensitivity of prion detection in potentialy infected individuals or animals.
This year, the group has brought together all the technical tools and biological material required for its research: transgenic mice expressing sheep PrP (provided by H. Laude- INRA), PrP specific antibodies (provided by J. Grassi-CEA), prion infectivity tests, culture of prion producing cell lines, ...
After having isolated, last year, a cell line expressing high levels of PrP, we succeeded this year in infecting these cells and in showing that they produce high levels of prions. The determination of the amount of infectious PrP they produce is underway.
We aim at investigating the reactivity to monoclonal antibodies of the structured part (the "core") of the pathogenic PrP. This study can not be achieved using "natural" prions. Indeed, to the purely proteinic moiety of natural PrP are linked two large molecular structures made of complex sugars which, in the prion, build up a shell that covers the protein and prevent it from reacting with antibodies. We are therefore attempting to remove this shell so as to render possible an investigation of the PrP conformation in the prion. To this effect we have grown cells in culture medium containing a glycosylation inhibitor (i.e. a molecule preventing the attachment of sugars to proteins). The optimal conditions for use of such an inhibitor have been defined. This enabled us to obtain a preparation of purified, "non-glycosylated" prions, the immunochemical characterization of which has been undertaken.For that purpose, we first have developped a miniaturized, high sensitivity immunochemical test (spot test - see Figure 1) to check the immunoreactivity of a monoclonal antibody towards normal structured or unstructured PrP, normal structured non-glycosylated PrP, structured or unstructured glycosylated prion, structured non-glycosylated prion.
We thus could verify that, among the many antibodies analyzed that bind to the core of PrP, none could react with structured glycosylated prions. However, two of these antibodies reacted well with structured, unglycosylated prions. This confirms that the sugars effectively hide some antigenic determinants of PrP in the prion, thus preventing them from reacting with the antibodies. Moreover, the reactivity of these two antibodies to unglycosylated prions led us to identify two regions of the PrP that, in its pathogenic conformation (the prion) are located on the surface of the protein. One of these two antibodies was provided to us by the CEA. The second originates from a new hybridome prepared, within the frame of our project, by F. Nato and J. Gregoire (Institut Pasteur Technical Platform for Protein Expression and Purification headed by P. Beguin). This "anti-PrP" hybridoma is one among 33 cell lines thus far obtained after immunizing PrP knock-out mice with synthetic peptides corresponding to different regions of the PrP core. The array of antibodies we expect to accumulate in this way should lead to an immunochemical mapping of the surface of PrPwithin the prion. We also hope to obtain "conformational" monoclonal antibodies able to bond to the unglycosylated prion and not to normal PrP. Such an antibody would be a precious reagent for developping more sensitive tests to detect the presence of prions in non-symptomatic potentialy infected animals.
2- Bacterial periplasm (N. Sassoon-Clavier, M. Miot and J.-M. Betton)
The compartimentalization of Gram-negative bacteria implies the existence of specific mechanisms monitoring protein folding in the envelope. To study protein folding in the bacterial envelope, we use a model system, MalE31, a defective folding variant of the maltose-binding protein, the periplasmic receptor for the high affinity transport of maltose in E. coli. The defective folding pathway of MalE31 leads, at high levels of production, to the aggregation of folding intermediates and to the formation of inclusion bodies. Using MalE31 with altered signal sequences, we showed that the quality control in the cytoplasm allows a more efficient degradation of misfolded proteins. Under these conditions, the increased level of cytoplasmic heat-shock proteins can partially suppress the misfolding pathway of MalE31. The crystal structure of this aggregation-prone protein, solved this year, supports a model in which the effect of the mutation is exerted at the level of folding intermediates, rather than at that of the final conformation. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. We have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. This variant displays defective transport behaviour, even though it retains maltose-binding affinity comparable with that of the wild-type protein. The cristallographic information on MalE31 gives no direct details on the mechanism of misfolding but indicates that the mutated residues are in a region on the surface of MalE, not previously identified, involved in the interactions with the membrane components of the maltose transport.
The temperature effect on inclusion body formation was examined. We showed that MalE31 aggregation did not affect bacterial growth at 30°C, but is lethal at 37°C. Suprisingly, under mild heat-shock conditions at 42°C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat-shock periplasmic protease DegP/HtrA via the CpxA/CpxR two-component signaling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31 aggregation. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathaway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system. We are currently using this toxicity to search extragenic suppressors from a multi-copy DNA library.
3- ATP-dependent Clp proteases from Mycobacterium tuberculosis (N. Benaroudj and M. Miot)
Clp proteins are ATP-dependent autocompartimentalized proteases which are widely recognized as essential factors for cell survival and involved in the virulence of many bacteria. In E. coli, Clp proteases are composed of the proteolytic subunit, ClpP, and the ATPase subunits, CpA or ClpX. The ClpP subunits are organized into two superimposed heptameric rings, which form a central chamber. In M. tuberculosis, these proteases have not been studied, but genomic sequencing revealed the presence of two genes coding for ClpP proteins (ClpP1 and ClpP2). We have produced in E. coli, purified and started the characterization of ClpP1 and ClpP2 of M. tuberculosis. Preliminary results indicate that both proteins, separately produced, are unable to hydrolyze substrate peptides, and form single heptameric rings under experimental conditions tested. We wish to determine whether the different ClpP peptidases work independently or in concert in protein breakdown and how they could be linked to the virulence of M. tuberculosis. A more long term goal will be to study new chaperones that do not belong to the DnaK or GroEL families and whose functions are still unknown.
4- Recombinant protein production (J. Rogé and J.-M. Betton)
With the recent improvements of cell-free transcription-translation systems, we have developped a new cloning strategy which is based on the possibility to express, in vitro, a gene from a PCR product. This strategy which allows a rapid optimisation of expression parameters, was succesfully applied for several proteins from M. tuberculosis and M. leprae selected inside targets of the structural genomic program at Pasteur Institute. Finally, several proteins or protein domains of SARS coronavirus were produced and purified in order to develop serologic diagnostic tests and a vaccine.
5- Production and study of a recombinant protein, candidate vaccine against malaria (A. Chaffotte and A.-G. Planson)
The C-terminal fragment F19 of MSP1 (Merozoïte Surface Protein) from Plasmodium falciparum is an anti-malaria vaccine candidate. We aim at producing it in large quantities and at low cost in the bacterium E. coli. Comparison of the structural characteristics by NMR of the F19 fragment, expressed either as an individual protein, or fused at the C-terminal end of MalE, revealed a remarkable property of MalE as an assistant to the oxidative folding of F19 within the fusion. To determine if this assisting effect is linked to the periplasmic expression context, we examined the properties of the in vitro refolding of the fusion protein MalE-F19. The refolding kinetics of MalE-F19 showed a large slowing down of the earliest observable phase of the MalE moiety, whatever the F19 region was or not reduced. Under oxidizing conditions, the refolding kinetics of the fusion protein leads to the reconstitution of épitopes of F19 as shown by the regain of the immunoreactivity towards monoclonal antibodies specific for the native fragment. The structural characterisation, using NMR (in collaboration with Inaki Guijarro, Unité de RMN des biomolécules) of the F19 fragment isolated (from proteolytic cleavage with Factor Xa) after oxidative refolding of the fusion showed that the fragment refolds in its native conformation: the TOCSY and NOESY spectra superimpose perfectly to the corresponding spectra of native (never denatured) F19. This result demonstrates that the assisting effect of MalE to the oxidative folding of the fragment is intrinsic and thus does not imply the expression context of the fusion MalE-F19.
6- Role of SecB in the secretion process of HasA (A. Chaffotte and C. Bodenreider)
Detailed kinetic studies using stopped-flow and conducted in collaboration with N. Wolff (Unit of NMR of Biomolecules) and P. Delepelaire (Unit of Bacterial Membranes) showed that the presence of the chaperone SecB dramatically slows down the refolding of HasA, an extracellular hemophore from S. marcescens. These studies evidence the role of SecB during the secretion process of HasA through the ABC transporter.
7- Contribution to the technical platform of Biological Macromolecular Biophysics (A. Chaffotte, M. Goldberg, R. Nageotte)
The unit is in charge of the technical aspects and the scientific supervision (help in the conception and interpretation) of experiments in circular dichroism, Fourier transform infrared spectroscopy and analyticla ultracentrifugation performed within the framework of the technical platform of Biological Macromolecular Biophysics. It houses and takes care of the maintenance of the corresponding equipments.
8- Teaching and education
The Unit is in charge of organizing the "Protein Biochemistry" laboratory course (co-Director: A. Chaffotte and J.M. Betton) of the Institut Pasteur, which is associated to the Master's program of the Paris 6, Paris 7 and Orsay Universities, the Ecole Normale Supérieure, the Ecole Polytechnique, and the CEA and members of this Unit have contributed to the laboratory sessions of the 2003 course.
Figure 1: Specificity test of anti-PrP antibodies
1 microliter of cell extract or of prion suspension was deposited on a nitrocellulose membrane according to the following disposition: top, cells lacking PrP; right, cells producing glycosylated PrP; bottom, cells producing non-glycosylated PrP; left, structured glycosylated prions; center, glycosylated prions destructured by heating at 95°C in 3M urea.
A black dot indicates strong reactivity; no dot indicates no, or weak reactivity.
Keywords: Prion, deglycosylation, molecular chaperone, folding, stress response