Single-protein mechanochemical regulation of cell function

Descripción del proyecto

Estudio de los efectos mecánicos en proteínas individuales y su translocación a través de poros

Las proteínas participan en la mayoría de los procesos celulares. Su función está regulada por cambios en su estructura tridimensional, que, a su vez, puede ser determinada por fuerzas mecánicas. Los estudios de los efectos mecánicos sobre las proteínas se han limitado a experimentos de molécula única «in vitro», que no permiten estudiar los efectos sobre la función celular y tisular. El proyecto ProtMechCell, financiado por el Consejo Europeo de Investigación, tiene por objeto analizar si la estabilidad mecánica y la estructura local de las proteínas son un regulador maestro de la cinética de translocación de proteínas a través de poros de todo tipo. La elasticidad de las proteínas, el movimiento de los factores de transcripción y las proteínas a través de los poros, así como la estabilidad intracelular de las proteínas individuales sometidas a tracción, se investigarán empleando técnicas punteras de espectrometría de fuerza mecánica, optogenética e intracelular.

Objetivo

Mechanical stimuli regulate an increasingly large number of cellular and tissue functions. However, the effect that those macroscopic forces have on the underpinning proteins is poorly understood.

Most of our knowledge of protein dynamics under force is still restricted to in vitro single-molecule experiments. Collectively, these molecular measurements have revealed that, when denatured with force, proteins unfold and stretch along their end-to-end length, following a completely different pathway from that sampled in biochemical denaturation. Yet, we are still lacking fundamental understanding of how mechanical unfolding of proteins occurs in the cell, and how it impacts cellular function.

Here, we propose to develop and apply a combination of state-of-the-art mechanical techniques across scales aiming to correlate the nanomechanical properties of proteins measured in vitro with their behaviour in their physiological cellular context.

We will first study how the regulation of protein elasticity –through dynamic force-induced unfolding and refolding, protein binding and post-translational modifications in cryptic sites of key mechanosensors– affects cellular mechanotransduction. We will also examine, using single-cell optogenetic experiments, how the mechanical unfolding of mechanosensitive transcription factors regulate their import rate into the cell nucleus across the nuclear pore complex, and the potential physiological implications.

We will then test the hypothesis that the mechanical stability and local structure of proteins emerge as a general master regulator of their translocation kinetics across pores of varying sizes and functions, including those present in proteasomes, peroxisomes and lysosomes.

Finally, we will harness the natural specificity of the distinct cellular pores to inspire an intracellular force spectrometer to measure changes in the mechanical stability of a single protein under its functional pulling velocity inside the cell.