Scientists are building atomic-level models of enzymes linked to lupus and Alzheimer's

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When nucleic acids such as DNA or RNA build up in a cell's cytoplasm, it sets off a warning call to the immune system. Enzymes normally destroy these nucleic acids before they cause a problem, but when these enzymes are called upon by the immune system to malfunction, it can lead to autoimmune and inflammatory diseases.

In a new study published in the journal March 26, 2024 structure, Scripps Research scientists present the previously undescribed structures of these two nucleic acid-degrading enzymes – PLD3 and PLD4. Understanding the structures and molecular details of these enzymes is an important step in designing treatments for dysfunction in a variety of diseases, including lupus erythematosus, rheumatoid arthritis and Alzheimer's disease.

„These enzymes are critical for cleaning the cellular environment and also set the threshold for whether or not they are considered infectious,” says the senior author. David Nemazy, PhD, professor in the Department of Immunology and Microbiology at Scripps Research. „I hope that one day we can help patients based on this information.”

Enzymes are proteins that speed up chemical reactions by binding and reacting with specific molecules called substrates. In the case of PLD3 and PLD4, the substrate is a strand of RNA or DNA that enzymes cleave nucleotide by nucleotide.

The team used X-ray crystallography to create atomic-scale models of PLD3 and PLD4 in multiple states or conditions, allowing them to examine how their conformations changed during the catalytic reaction. This includes when enzymes are resting or when they are actively bound to a substrate.

„These models allow us to visualize PLD3 and PLD4 very clearly and with high resolution, so we know how each atom interacts, which means we can learn how the enzymes work,” says Meng Yuan, staff scientist at the Integrated Department. Structural and Computational Biology at Scripps Research.

Structural analyzes revealed that PLD3 and PLD4 are structurally identical and that although PLD4 is a larger protein, they degrade DNA and RNA in a similar fashion. Both enzymes degrade nucleic acids through a two-step process.

„We call this process two-step catalysis: bite and release,” says Yuan. “In the first step, the enzyme bites the DNA strand and separates a single 'brick' or nucleotide from the other strand, and in the second step, it opens its 'mouth' and releases the brick for recycling. „

Because the enzymatic reaction happens so quickly—within milliseconds—researchers must use an alternative substrate to visualize the structure of the enzymes during catalysis. To do this, they incubate the enzymes with a molecule that looks like DNA that the enzyme would normally degrade, but the enzymes degrade much more slowly.

This method uncovered a previously unknown function of one of the enzymes: in addition to cleaving nucleotides from single-stranded RNA and DNA, PLT4 also showed phosphatase activity, meaning it may also be involved in breaking down the phosphate backbone of DNA.

„I think it's amazing what the crystal structure tells us about this phosphatase activity,” says Nemasi. „Finding a new enzyme activity is unheard of in structural biology. It's because Meng was able to solve such an amazingly accurate and detailed structure that he was able to tell us about this additional enzyme activity that we didn't know about.

After they elucidated the typical structure of PLD3 and PLD4, the researchers studied the structure of variants associated with diseases including Alzheimer's and spinocerebellar ataxia. The analyzes revealed that some of these variants had reduced enzyme capacity, while others – including a mutation associated with late-onset Alzheimer's disease – appeared to be more active.

„Some of our data suggests that one of these Alzheimer's-related enzyme types might be working better, which I was surprised by, but it might be less stable and more easily aggregated,” says Nemesee.

The researchers plan to continue studying the structure and function of these enzymes. Their next steps include exploring possible ways to block the enzymes in situations where they are overactive, and they also plan to explore the possibility of replacing the enzymes in people with inactive (or non-working) versions.

Note: Yuan M, Peng L, Huang D, et al. Structural and mechanistic insights into the disease-associated endolysosomal exonucleases PLD3 and PLD4. structure. 2024:S0969212624000790. doi: 10.1016/j.str.2024.02.019

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