How stressed out cells remain stable

U. PENNSYLVANIA (US) — Studying how proteins respond to physical stress is helping scientists understand how normal and mutated red blood cells remain stable.

In stark contrast with much of the architecture people interact with every day, the internal architecture of the human body is predominantly soft. Other than bones, all of the organs, tissues and structures in the body are pliable and flexible and need to be that way in order to work.

To understand what keeps flexible structures stable, Dennis Discher, professor of chemical and biomolecular engineering and bioengineering at the University of Pennsylvania, used red blood cells because they survive for months, despite making a complete lap of the circulatory system every few minutes.

“Red blood cells are disks, and they have proteins right below the membrane that give it resilience, like a car tire,” Discher says. “The cells are filled with hemoglobin like the tires are filled with air, but where the rubber meets the road is the exterior.”

To measure stress in that membrane on an atomic level, researchers needed a way to track changes to the shape of those supporting proteins. Amino acid cysteine proved to be the perfect proxy.

Proteins are long chain of amino acids that are tightly folded in on themselves. The order and chemical properties of the acids determine the locations of the folds, which in turn determine the function of the protein.

Cysteine is hydrophobic, meaning it interacts poorly with water and so is usually on the inside of a protein. And because stress changes a folded protein’s shape, measuring the degree to which cysteine is exposed would in effect measure how stressed the protein and cells containing it are.

Discher simulated the shear forces originating from the beating heart, which forcefully pumps blood and ultimately pulls apart the folds that keep cysteine on the inside of proteins at the red blood cell membrane, allowing it to bind with a fluorescent marker dye.

He was able to visually confirm that the more stressed cells were more fluorescent under the microscope but then tested the levels of marked cysteine using mass spectrometry.

The research is reported in the journal Proceedings of the National Academy of Sciences.

“Just like a polymer engineer designing a tire, we’re looking at the relationship between the chemical makeup and the physical stability of the structure and how it performs,” Discher says.  “We can use this technique to look at the relationship between structure, flexibility and function.”

Investigating the structural elements of blood cells could pave the way to breakthroughs for human health, Discher says.

“How long can blood be stored? Why are there no good blood substitutes? There are a lot of things we don’t understand about the forces cells can sustain before fragmenting and falling apart, especially when we consider age and mutations.”

Discher studied the mutated blood cells that result in disorders known as elliptocytosis; cells that are elliptical, rather than round, and therefore have shorter functional lifespans.

Elliptical cells are often missing a chemical rivet that anchors the support proteins to the outer membrane, which means that stress causes them to disconnect, rather than unfold.

That kind of structural change is crippling to the function of anatomical structures like blood cells. The flexibility provided by unfolding is therefore key to their overall stability.

“At least for this cell, the first mechanism of response is to unfold proteins and keep the interactions between proteins the same,” Discher says. “That constant back and forth with unfolding within these cells as the cells flow and distort while in the blood stream, allows their architecture to be maintained.”

The next step is to use the cysteine-mass-spectometry technique to investigate the role of softness and flexibility in responding to stress in other biological systems, particularly stem cells.

The research was supported by the National Institutes of Health.

More news from University of Pennsylvania news: