New insight in disease-causing mechanisms of genetic component LINE-1

26 January 2024
Billions of years ago, as primitive lifeforms were becoming more complex, a selfish genetic component became a sort of genome colonizer. Using a copy-and-paste mechanism, this tenacious bit of code replicated and inserted itself again and again into a variety of genomes. Over time, all eukaryotic organisms inherited the code—including us. In fact, this ancient genetic element wrote about one-third of the human genome—and was considered junk DNA until relatively recently.

This genetic component is known as LINE-1 and its mobility within our genomes can lead to disease-causing mutations. A key protein, encoded by LINE-1 genes, is called ORF2p; this protein enables LINE-1’s success at replicating itself. Therefore, understanding ORF2p’s structure and mechanics could enable targeting it therapeutically for diseases involving LINE-1’s activity.

Now, in a large international collaboration with multiple academic and industry research groups, UMCG scientists have experimentally rendered the protein’s structure for the first time, revealing a host of new insights about LINE-1’s key disease-causing mechanisms. The results were published in Nature.

“The work will facilitate rational drug design targeting LINE-1 and may lead to novel therapies and strategies to combat cancer, autoimmune disease, neurodegeneration, and other diseases of aging,” says senior author John LaCava, group leader of ERIBA’s Laboratory of Macromolecules and Interactomes. One of the contributions of his team was to use a technique called crosslinking-mass spectrometry to reveal which parts of the ORF2p protein were near to one another – providing a basis to understand its shape. The experiments were carried out by Luciano Di Stefano, a former post-doc in the lab. This information was combined with that obtained from other techniques (e.g., crystallography, electron microscopy, computational modeling) used in the tour de force study to calculate of the structure of the full length ORF2p.

Evolutionary mates

LINE-1 is a retrotransposon, a kind of mobile genetic code that transfers its RNA back into DNA – replicating its sequence into different places throughout an organism’s genome. While there are different kinds of retrotransposons, LINE-1 is thought to be the only autonomous retrotransposons in the genomes of modern-day humans – this autonomy is provided by the activity of its ORF2 protein.

LINE-1’s precise origin is unclear, but it has an evolutionary connection to group II introns, a class of ancient mobile elements dating back about 2.5 billion years. Retrotransposons like LINE-1 have been co-evolving with their host organisms during all this time.

Millions of genetic fragments derived from ORF2p activity are found in our cells. The vast majority are inactive evolutionary relics, evidence of the ancient molecular arms race between the host and retrotransposons. Yet, about 100 LINE-1 genes are still operational—and usually not helpful. LaCava and senior author Martin Taylor, of Massachusetts General Hospital and Harvard Medical School, have collaboratively studied LINE-1 and its proteins for more than a decade, but because ORF2p expresses so lowly and infrequently, it has remained poorly understood. Another protein produced by LINE-1, known as ORF1p, is churned out by cancer cells, as a recent study by LaCava and their collaborators described.

Nucleus and cytoplasm

Using a combination of structural and biochemical methods, the researchers discovered two novel folded domains within ORF2p’s core that contribute to LINE-1’s ability to make copies of itself.

“ORF2p has structural adaptations uniquely suited for these endeavors. It’s a sort of jack-of-all-trades protein, capable of handling everything from replication to insertion” says co-lead author Trevor van Eeuwen of The Rockefeller University.

Although most attention has been paid to LINE-1 activity in the nucleus, where it makes new insertions in our genomes, LINE-1 can also become activated in the cytoplasm of cells. When LINE-1 is activated in the cytoplasm, “it acts like a viral mimic. It creates RNA:DNA hybrids that look like a viral infection when they’re sensed,” van Eeuwen notes. This viral mimicry, a topic LaCava’s lab is studying, suggests a possible solution to the puzzle of how ORF2p activates the innate immune system, contributing to autoimmune disease and other conditions. Their research found that interactions with LINE-1-derived genetic material in the cytoplasm activates the cGAS/STING antiviral pathway. In turn, that pathway causes cells to produce interferons, stimulating the immune system and leading to inflammation, in a manner analogous to what happens during an infection by a virus.

The path ahead

In the future, the researchers will seek to explore the potential clinical applications of their findings. Because there is a kinship between retrotransposons and retroviruses, in the current study they tested treatments for the retroviruses HIV and HBV to see if they would inhibit LINE-1. They did not, suggesting that the design of therapeutics will have to be tailored to LINE-1’s unique characteristics. “The work opens the door to rational drug design of better LINE-1 inhibitors, and we hope these will lead to clinical trials soon,” LaCava says.

This research project was an international collaboration. In addition to scientists from the UMCG, crucial contributions were made by ROME Therapeutics, The Rockefeller University (M. Rout and J LaCava lab), MSKCC (B. Greenbaum lab), Rutgers (E. Arnold lab), University of Alberta (M. Götte lab), Dana Farber (K. Burns lab), UCSF (A. Sali lab), MPI Tübingen (O. Weichenreider lab), and UT Arlington (S. Christensen).

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