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Temperature sensing in plants. | Functionality and mechanisms of Hsp70 chaperone system | In vitro reconstitution of human Hsp70 system and its implication in protein misfolding problems. | The effect of EMF and Enviromental stress on flatworms. | Functionality and Mechanism of the Stress-Responsive Molecular Chaperone Hsp70 and Heavy Metal Toxicity.

Temperature sensing in plants.


Land plants need to rapidly detect mild temperature increments at physiological temperatures to establish thermotolerance against the damages of an upcoming heat stress. The onset of plant thermotolerance requires the early perception of an upcoming heat stress by timely responding by accumulating heat shock proteins (Hsps), many of which, molecular chaperones that can protect proteins and membranes from heat-damage. Hence, land plants need precise molecular thermosensors, which have to be coupled to a specific signaling pathway to the cytoplasm and the nucleus to allow the upregulation of HSP genes. We found strong biochemical evidence for the existence of highly specific calcium channels in the plasma membrane of the moss Physcomitrella patens, which act as the most upstream heat-sensors of the plant cell (Saidi et al. 2009).
Our further aims are focused to establish the molecular identity of the Ca2+ channel serving as heat receptor(s) in moss P. patens and to characterize protein loss-of-function, especially with regards to the spectrum or intensity of chaperone expression and to the onset of acquired thermotolerance and correlate chaperone expression with Ca2+ transients. Using moss as a model system we would like to identify the equivalent heat receptor genes in higher plants and to characterize their phenotypes in terms of HSP expression and acquired thermotolerance.



Functionality and mechanisms of Hsp70 chaperone system


Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Molecular chaperones belong to several families of highly conserved proteins that favor the native folding of proteins, both as “holdases” that sequester hydrophobic regions in misfolding polypeptides and thus preventing protein aggregation, or as active unfoldases, that use the energy of ATP hydrolysis to unfold and disentangling misfolded and aggregated polypeptides. We try to understand central questions such as: By which mechanism may a single Hsp70 chaperone change the conformation of a stable misfolded polypeptide substrate? By which steps do Hsp70 and its co-chaperones use ATP to transforming inactive misfolded or alternatively folded polypeptides into active native proteins? Is chaperone-mediated protein refolding to the native state spontaneous or is it directed by the chaperone? Does Hsp70 behave as a true unfolding and/or folding catalyst capable of multiple turnovers to convert an excess of stable misfolded substrates into stable native products? What is the minimal ATP cost for transforming a single inactive misfolded polypeptide into an active, natively folded one? Chaperones are central cellular defenses against the formation of toxic protein conformers in protein-misfolding diseases, such as Alzheimer, Parkinson disease and aging in general.


In vitro reconstitution of human Hsp70 system and its implication in protein misfolding problems.

There is plethora of literature indicating that the molecular chaperones and chaperone controlled proteases, such as the proteasome, belong to a cellular network that can prevent and reduce the formation of toxic aggregates and possibly eliminate already formed toxic protein aggregates in neurodegenerative diseases. The respective analogues of Hsp70 system in bacteria are Dnak/DnaJ/GrpE. 70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins. My main objective is to develop efficient in vitro human Hsp70 chaperone system and its co-chaperones Hsp40/NEF. The in vitro bacterial Hsp70 machinery is already well established in the Goloubinoff Laboratory and this will serve as control for my work. I shall primarily study the kinetic parameters and energy cost of the human Hsp70 machinery. I will also focus on the possible implication of the Hsp70 machinery in protein conformation diseases.


The effect of EMF and Enviromental stress on flatworms.


The popular use of cellular phones and the proliferation of dedicated antenna have generated extensive exposure of living organisms to high frequency electromagnetic fields (HF-EMFs). This has raised concerns among health and environmental authorities and led to numerous epidemiological and biological studies. In addition, low frequency magnetic fields (LF-MFs) associated with overhead power lines, house appliances and railway systems represent a potential risk for health, which is an ongoing debate over the last decades. Despite continuous efforts, there are so far no clear answers to questions concerning the nature of the biological effects of HF-EMFs and LF-MFs.

Former studies carried out at the University of Lausanne have shown a possible mild EMF effect, resembling the effect of a mild heat shock. We started a project aimed at better understanding the impact of EMFs on cells. The project is carried out in the frame of the Swiss National Research Program (NRP57) "Non-Ionizing Radiation - Health and Environment" and is done in collaboration with Dr. Farhad Rachidi and Dr. Pierre Zweiacker from the Power Systems Laboratory of the Swiss Federal Institute of Technology of Lausanne (EPFL).

In our project, we use the flatworm Caenorhabditis elegans as a model organism to investigate possible cellular responses to EMFs. The experiments are conducted with genetically modified C. elegans expressing polyQ proteins (tagged polyglutamine-expansion proteins), which are associated with the formation of protein aggregates connected with various types of mammalian neurodegenerative diseases. In these flatworms, the mild heat shock-like EMF effect may be amplified by the dynamic accumulation of toxic protein aggregates and progressive paralysis of the animal. Possible EMF effects will be compared with effects induced by various environmental stresses, such as heat shock, oxidative stress and different chemical treatments.

The research has the potential to detect biological effects of EMFs and to characterize their nature. The results could provide a scientific basis for the development of EMF biosensing systems that may assist the authorities in defining safer exposure limits. The research will improve our understanding of fundamental aspects of cell biology in relation to cell sensing and responding to environmental stresses in general, and to EMF in particular.


Functionality and Mechanism of the Stress-Responsive Molecular Chaperone Hsp70 and Heavy Metal Toxicity.

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead may entail severe health hazards including prenatal and developmental damage.
We study the effect of heavy metals on protein folding, protein stability and chaperone assisted protein refolding. Perturbations of the native protein folding pathway by heavy metal ions that form stable complexes with misfolding intermediates may underlie an as yet unsuspected mechanism for the toxic effects by heavy metals in animals leading to protein folding and autoimmune diseases


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