Researchers have mapped thousands of small proteins in the human frontal cortex that are missing from standard protein databases. The atlas, published in Nature Aging, finds that many of these microproteins have altered levels in Alzheimer’s disease and points to roles in gene regulation and cellular energy production.
Microproteins are typically made up of fewer than 150 amino acids. Because they are short and can be encoded within or near larger genes, standard methods and databases may overlook them or attribute their activity to a better-known protein.
A team led by Brendan Miller and Alan Saghatelian of the Salk Institute for Biological Studies built a custom reference database using RNA sequencing from tissue samples from 12 postmortem brains. They then used it to examine mass spectrometry data from 610 brain samples, initially identifying 4,321 microproteins. More than 3,000 had not been catalogued in standard, manually reviewed protein databases.
The researchers checked these findings against ribosome-profiling data and used a deep-learning tool to assess the mass spectrometry evidence. This process yielded 1,067 previously unreviewed microproteins with strong evidence that they are present in the brain.
Comparing samples from people with and without symptomatic Alzheimer’s, the team found dozens of microproteins with altered levels. A microprotein derived from the BCL3 gene was less abundant in Alzheimer’s samples, while one associated with RAB3C was more abundant. In laboratory-grown microglia, the two proteins remained stable and occupied specific locations within the cells.
About half of the microproteins appeared to be regulated independently of the larger genes near which they are encoded. In some cases, microprotein RNA levels changed in Alzheimer’s disease even when the RNA levels of the related main gene did not. The researchers linked this separation to alternative splicing, in which cells assemble RNA segments in different ways, and identified patterns near splice sites that may help regulate the process.
The team also investigated a microprotein encoded by MKKS. Although standard databases list a larger protein as the gene’s main product, the researchers found that the microprotein was the dominant MKKS product in the frontal cortex. Its levels were lower in brains from people with symptomatic Alzheimer’s, and its activity was associated with genes involved in mitochondria, which produce energy inside cells.
In laboratory microglia, the researchers used CRISPR to remove the DNA sequence that produces the MKKS microprotein without altering the main gene. The modified cells consumed less oxygen during a stress test, indicating that the microprotein contributes to cellular respiration.
The atlas also points to microproteins produced from pseudogenes, genetic sequences often considered inactive. One candidate, derived from the ACTBP7 pseudogene, altered the distribution of structural fibers when introduced into laboratory microglia.
The findings do not show that altered microprotein levels cause Alzheimer’s disease. The changes could instead reflect cellular responses to damage or shifts in regulation. The researchers also note that some proteins may be present below current mass spectrometry detection limits, and that the production pathways for many microproteins remain to be mapped.
Sources: PsyPost.