New Experimental Mouse Model Enables Direct Investigation of Protein's Mechanical Function

A new study published in the journal Nature Communications outlines the creation of the first experimental mouse model allowing for direct analysis of the mechanical function of proteins in living organisms.

The relationship between force and biological molecules

Animal models are integral to scientific research, helping scientists to understand and manipulate the molecular mechanisms behind a variety of physiological states in a laboratory context. As such, a wide variety of animal models exist, with mouse models being the most commonly used.

Researchers from the Cardiovascular System group at the Centro Nacional de Investigaciones Cardiovasculares (CNIC), in collaboration with an international team, have created a novel experimental mouse model, one that permits analysis of the mechanical function of proteins in living organisms.

An array of single-molecule techniques utilizing recombinant proteins have enabled scientists to formulate hypotheses on how mechanical forces are both generated and sensed within biological tissues. This has advanced our understanding of the relationship between force and biological molecules.

"This relationship between cells and the mechanical components of their environment is extremely important, and explains many phenomena related to disease, such as cancer metastasis and atherosclerosis, which underlies several cardiovascular conditions," says Jorge Alegre-Cebollada, leader of the research team.

Despite this, the testing of such hypotheses in a native environment has proven challenging – until now. Alegre-Cebollada told Technology Networks: "So far, studying protein mechanics has relied on applying biophysical tools to purified proteins, so the context in which mechanical proteins work was lost in the process."

In the study, the scientists utilized a HaloTag-TEV genetic cassette to achieve the new mouse model, inserting it into titin, a protein responsible for elasticity in skeletal and cardiac muscle.

"Using the HaloTag-TEV cassette, we can play around with protein mechanics without resorting to protein purification – and if we want to purify it anyways, the resulting protein is more representative of reality. The model allows us to put to experimental test many hypotheses that have been generated in the biophysics field for the last two decades," Alegre-Cebollada adds.

Triggering a tug-of-war game gone wrong at the biological tissue level

HaloTag is a self-labeling protein tag that covalently binds to a synthetic ligand and fuses to a researcher's protein of interest. I ask Alegre-Cebollada how, exactly, the HaloTag-TEV cassette works in the context of this study. He tells me: "It combines two elements well known in the protein engineering field, which for the first time, are introduced in to the genome of a vertebrate model. On one side, there is the HaloTag domain, which allows for controlled chemical reactions to both visualize the targeted protein and for purification purposes."

"The second element is a recognition site for the protease TEV, a well-known biotechnological tool that cleaves TEV sites very efficiently. So, the tagged protein can be cleaved when we add TEV protease… and this is a very efficient trigger of a fast-mechanical response. We are triggering a tug-of-war game gone wrong at the biological tissue level!" he adds.

The mechanical function of the target protein (in this instance, titin) can be disrupted at any specific moment, enabling the scientists to study the physiological impact of disrupting the protein.

"In love with titin"

Why exactly did the researchers focus on the protein titin?

"We are in love with titin. It is the classical example of a protein with mechanical function and, as such, it has led to many milestone contributions in the field of protein mechanics. We understand quite well the mechanical properties of titin using highly simplified, biophysical approaches. However, we are still missing some pieces in the puzzle of how titin mechanics and biochemistry work together in muscle cells to provide mechanical power and elasticity. The HaloTag-TEV titin gives us unprecedented opportunities to look into the contribution of titin mechanics to biological function," Alegre-Cebollada adds.

But the applications of the HaloTag-TEV cassette do extend beyond titin; it can be inserted into a variety of other proteins that are implicated in mechanical function and human disease.

"There are many mechanical proteins that I can think of in which the HaloTag-TEV cassette may be useful. For example, talin, which is fundamental for the cells to probe their mechanical environment, or filamin, which senses cytoskeletal forces. We are currently pursuing the application of the HaloTag-TEV cassette to understand triggers of pathogenicity in dilated cardiomyopathy, a devastating disease of the heart that is most commonly caused by mutations in titin," Alegre-Cebollada concludes.

Jorge Alegre-Cebollada was speaking to Molly Campbell, Science Writer, Technology Networks.

Reference: Rivas-Pardo et al. (2020). A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues. Nature Communications. DOI: https://doi.org/10.1038/s41467-020-15465-9.