A new study from MIT offers an explanation for how collagen can survive for so much longer than expected. The research team found that a special atomic-level interaction defends collagen from attack by water molecules. This barricade prevents water from breaking the peptide bonds through a process called hydrolysis.

“We provide evidence that that interaction prevents water from attacking the peptide bonds and cleaving them. That just flies in the face of what happens with a normal peptide bond, which has a half-life of only 500 years,” says Ron Raines, the Firmenich Professor of Chemistry at MIT.

Raines is the senior author of the new study, which will appear in ACS Central Science. MIT postdoc Jinyi Yang PhD ’24 is the lead author of the paper. MIT postdoc Volga Kojasoy and graduate student Gerard Porter are also authors of the study.

Water-resistant

Collagen is the most abundant protein in animals, and it is found in not only bones but also skin, muscles, and ligaments. It’s made from long strands of protein that intertwine to form a tough triple helix.

“Collagen is the scaffold that holds us together,” Raines says. “What makes the collagen protein so stable, and such a good choice for this scaffold, is that unlike most proteins, it’s fibrous.”

In the past decade, paleobiologists have found evidence of collagen preserved in dinosaur fossils, including an 80-million-year-old Tyrannosaurus rex fossil, and a sauropodomorph fossil that is nearly 200 million years old.

Over the past 25 years, Raines’ lab has been studying collagen and how its structure enables its function. In the new study, they revealed why the peptide bonds that hold collagen together are so resistant to being broken down by water.

Peptide bonds are formed between a carbon atom from one amino acid and a nitrogen atom of the adjacent amino acid. The carbon atom also forms a double bond with an oxygen atom, forming a molecular structure called a carbonyl group. This carbonyl oxygen has a pair of electrons that don’t form bonds with any other atoms. Those electrons, the researchers found, can be shared with the carbonyl group of a neighboring peptide bond.

Because this pair of electrons is being inserted into those peptide bonds, water molecules can’t also get into the structure to disrupt the bond.

To demonstrate this, Raines and his colleagues created two interconverting mimics of collagen — the one that usually forms a triple helix, which is known as trans, and another in which the angles of the peptide bonds are rotated into a different form, known as cis. They found that the trans form of collagen did not allow water to attack and hydrolyze the bond. In the cis form, water got in and the bonds were broken.

“A peptide bond is either cis or trans, and we can change the cis to trans ratio. By doing that, we can mimic the natural state of collagen or create an unprotected peptide bond. And we saw that when it was unprotected, it was not long for the world,” Raines says.

“No weak link”

This sharing of electrons has also been seen in protein structures known as alpha helices, which are found in many proteins. These helices may also be protected from water, but the helices are always connected by protein sequences that are more exposed, which are still susceptible to hydrolysis.

“Collagen is all triple helices, from one end to the other,” Raines says. “There’s no weak link, and that’s why I think it has survived.”

Previously, some scientists have suggested other explanations for why collagen might be preserved for millions of years, including the possibility that the bones were so dehydrated that no water could reach the peptide bonds.

“I can’t discount the contributions from other factors, but 200 million years is a long time, and I think you need something at the molecular level, at the atomic level in order to explain it,” Raines says.

The research was funded by the National Institutes of Health and the National Science Foundation.

Many different types of cells work together in the brain. In humans, nerve cells (neurons) make up less than half of the cells. The rest are called “glia.” The most common glial cells are astrocytes. They supply the neurons with nutrients, form part of the blood-brain barrier, regulate the synapses and support the immune cells.

However, a small proportion of astrocytes are able to produce nerve cells and other types of brain cells. These special astrocytes are therefore also known as brain stem cells. Brain stem cells and ordinary astrocytes hardly differ in their gene expression, i.e. in the activity of their genes. “How they can perform such different functions and what makes up the stem cell properties was previously completely unclear,” explains Ana Martin-Villalba, stem cell researcher at the DKFZ.

Methylation is the key

To solve this puzzle, the teams led by Martin-Villalba and Simon Anders (University of Heidelberg) isolated both ordinary astrocytes and brain stem cells from one of the regions of the brain where young neurons still develop in adult mice, the “ventricular-subventricular zone” (vSVZ). The researchers analyzed gene expression at the level of individual cells using mRNA sequencing as well as the patterns of methylation (“methylome”) in the entire genome. They used a specially developed tool to analyze the methylation data*.

DNA methylation refers to chemical “markers” with which the cell can switch off unused parts of its DNA. Methylation is therefore crucial for the identity of the cells.

During this study, the stem cell experts noticed that brain stem cells have a special DNA methylation pattern that distinguishes them from other astrocytes. “Unlike normal astrocytes, certain genes are demethylated in brain stem cells that are otherwise only used by nerve precursor cells. This allows the brain stem cells to activate these genes in order to produce nerve cells themselves,” explains Lukas Kremer, first author of the current publication. Co-first author Santiago Cerrizuela adds: “This pathway is denied to ordinary astrocytes, as the required genes are blocked by DNA methylation.”

Lack of blood supply triggers reprogramming of astrocytes to stem cells and increases new nerve formation

Could methylation also be used to convert astrocytes into brain stem cells in other regions of the brain, outside the vSVZ? “This would be an important step for regenerative medicine to repair damaged areas of the brain,” says Ana Martin-Villalba.

Earlier studies had already shown that a lack of blood supply, such as occurs in brain injuries or stroke, increases the number of newborn nerve cells. Do altered methylation profiles play a role in this process?

To investigate this, the researchers interrupted the blood supply to the brain of mice for a short time. As a result, astrocytes with the typical stem cell methylation profile could be detected even outside the vSVZ, as well as an increased number of nerve progenitor cells.

“Our theory is that normal astrocytes in the healthy brain do not form nerve cells because their methylation pattern prevents them from doing so,” explains study head Martin-Villalba. “Techniques to specifically alter the methylation profile could represent a new therapeutic approach to generate new neurons and treat nerve diseases.”

“The lack of blood supply apparently causes astrocytes in certain areas of the brain to redistribute the methyl marks on their DNA in such a way that their stem cell program becomes accessible. The reprogrammed cells then begin to divide and form precursors for new neurons,” summarizes Simon Anders and adds: “If we understand these processes better, we may be able to specifically stimulate the formation of new neurons in the future. For example, after a stroke, we could strengthen the brain’s self-healing powers, so that the damage can be repaired.”

Why studies on mice are necessary for this research

Strokes or accidents can lead to damage to the brain that is generally irreparable at present and often has dramatic consequences for those affected. As of today, there is no way to replace lost nerve cells. The aim of this work is to find ways to stimulate the regeneration of nerves in the adult brain.

This requires a profound understanding of how and under what circumstances brain stem cells can be induced to provide a supply of young nerve cells. To do this, the researchers need to study developmental processes that only take place in the brains of highly developed mammals. Epigenetic reprogramming cannot be observed in living animals using imaging techniques, but requires studies at the level of individual cells. The investigations cannot be carried out on cells from the culture dish, as the methylation profile of the astrocytes changes as soon as they are taken into culture, so that the epigenetic reprogramming can no longer be traced.