Scientists have created an atlas of microproteins to study Alzheimer's disease
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- Scientists have created an atlas of microproteins to study Alzheimer's disease
Researchers at the Salk Institute have created an atlas of human brain microproteins that can help study aging and neurodegenerative diseases. After analyzing more than 600 samples of postmortem frontal cortex tissue, the scientists identified 1,067 microproteins missing from the main proven protein database. One of them turned out to be related to energy metabolism in microglia, the immune cells of the brain involved in the development of Alzheimer's disease. About what microproteins are and why scientists have not noticed them for decades, see the "Izvestia" material.
What are microproteins?
Virtually every cell in the human body contains the same genetic code. However, different cells use different parts of it to produce proteins that determine their functions and features. That is why a neuron is different from a heart cell, and an immune cell is different from a muscle cell.
Proteins are made up of chains of amino acids. Many well-studied proteins include hundreds or thousands of such elements. Microproteins are usually referred to as significantly shorter molecules — in the new study, they included proteins with a length of no more than 150 amino acids.
However, it is much more difficult to detect microproteins than large proteins. During standard proteomic analysis, proteins are broken down into fragments called peptides, which are then recorded by a mass spectrometer. To reliably confirm the existence of a protein, it is usually necessary to find at least two peptides characteristic of it. A short molecule may simply not have such suitable fragments.
As a result, many microproteins could remain outside scientific databases for decades, even if cells were actively producing them.
"We still don't fully understand the molecular mechanisms of healthy aging. This is especially true for microproteins, which have been unintentionally ignored for decades," said Alan Saghatelyan, senior author of the study and professor at the Salk Institute.
According to him, the new atlas will allow systematic study of microproteins in aging and neurodegeneration, including Alzheimer's and Parkinson's diseases.
How scientists compiled the atlas
The researchers combined data from transcriptomics, ribosomal profiling, and mass spectrometry. The first method allows us to determine which parts of the genome are read in cells, the second — which sequences are used for protein synthesis, and the third confirms the physical presence of protein fragments in the tissue.
The work was based on samples of the dorsolateral prefrontal cortex, an area of the frontal lobe of the brain associated with memory, planning, attention, and decision—making. The study included materials from several postmortem collections of the human brain. 610 samples passed through the proteogenomic conveyor, 608 of them met the quality criteria. For a comparative analysis of the microprotein content, scientists selected 480 samples from the Religious Orders Study and Memory and Aging Project.
Among them, 111 belonged to people without Alzheimer's disease, 182 belonged to people with characteristic pathological brain changes but without lifelong symptoms of dementia, and another 187 belonged to patients with pathological changes and clinical manifestations of the disease. The average age of donors in this part of the study was about 88 years.
The scientists re-processed the existing data using their own computer programs. In particular, they used the ShortStop machine learning tool created by Saghatelyan's laboratory. It analyzes small open reading frames — sections of the genetic code that are potentially capable of producing microproteins.
The algorithm takes into account the sequence of amino acids and the physico-chemical properties of the proposed molecules. He was trained to distinguish known short proteins from artificially created sequences with similar characteristics.
Calculations alone were not enough. To confirm the existence of the found molecules, the scientists compared the predictions with the results of mass spectrometry. In fact, the researchers compared experimentally recorded protein fragments with what their spectrum should look like according to a computer model. This comprehensive approach has helped to reduce the likelihood that a random signal will be mistaken for a microprotein.
How many microproteins were found
In total, the researchers analyzed 3,001 microproteins missing from the peer-reviewed section of the UniProtKB database. Spectral data of a high level of reliability was obtained for 1067 molecules. Another 1,433 microproteins have received an average confidence score and will require additional verification.
Many of the 1,067 most reliably confirmed microproteins had not previously been found in human brain tissue. However, the researchers do not claim that all the found molecules necessarily perform independent biological functions. The atlas primarily confirms their presence and provides a basis for subsequent experiments.
The difficulty was that 814 out of 1067 microproteins could be confirmed with only one unique peptide. Using a standard rule requiring the detection of two peptides, most of these molecules would have gone unnoticed. Therefore, the authors additionally checked whether the corresponding regions of the genome were read by ribosomes, and evaluated the coincidence of the registered and predicted spectra.
Almost three quarters of the detected microproteins were encoded by regions of the genome that were already considered responsible for the production of larger proteins. This means that the same genetic site can create several different products, some of which were not taken into account in traditional models.
In some cases, the amount of microprotein varied regardless of the content of the main protein encoded by the same gene. Therefore, analysis only at the level of genes or large proteins may not show all the molecular changes occurring in a neurodegenerative disease.
How did microproteins differ in Alzheimer's disease
The authors compared samples of people without the disease and patients with symptomatic Alzheimer's disease. In the most rigorous model, where the microprotein had to be detected in at least half of the samples, the scientists identified 22 molecules that had not previously been included in the verified database, the content of which differed statistically significantly between the groups.
As the criteria expanded, the number of potentially disease-related microproteins increased. A less rigorous search analysis allowed us to identify up to 258 candidates. However, such results require additional confirmation, since when working with thousands of short molecules, the risk of accidental coincidences increases.
The researchers also found 99 already known short proteins, the content of which differed between the brain tissues of people with and without Alzheimer's disease. Some of the changes coincided with the results of previous studies, which provided additional verification of the accuracy of the developed system.
However, the differences were not always large. The authors emphasized that postmortem brain tissue consists of many types of cells, so changes specific only to a specific cell population may be less noticeable when analyzing a common sample.
The scientists paid special attention to microglia, the immune cells of the central nervous system. They monitor the condition of the brain tissue, remove damaged cells and participate in the inflammatory response. With age and neurodegenerative diseases, the functions of microglia may be impaired.
What scientists have found in microglia
Among the potentially important molecules, the researchers identified a 63 amino acid-long microprotein called micro-MKKS63. It is encoded by a short reading frame in the MKKS gene.
Previously, this region of the genome was known mainly due to a larger protein with a length of 570 amino acids. However, the researchers found almost no evidence of large protein production in the frontal cortex samples. Micro-MKKS63, on the contrary, turned out to be the predominant protein product of this genetic region.
In patients with symptomatic Alzheimer's disease, the content of micro-MKKS63 was lower than in people without the disease. At the same time, the level of transcripts encoding the large MKKS protein did not change significantly. This confirmed that the microprotein can be regulated separately from the main product of the same gene.
Additional analysis showed that micro-MKKS63 is associated with the work of mitochondria— intracellular structures that provide energy production. The researchers found a microprotein in the mitochondrial region of human microglial cells.
Then, using the CRISPR–Cas9 genome editing system, the scientists disrupted only the short reading frame responsible for micro-MKKS63, while preserving the region encoding the large MKKS protein.
After removal of the microprotein, the baseline oxygen consumption, maximum respiratory intensity, and ATP-related energy production decreased in two independently created cell lines. The data obtained indicate that micro-MKKS63 is necessary for normal oxidative phosphorylation— one of the main mechanisms of energy production in the cell.
Thus, a decrease in the content of micro-MKKS63 may be associated with energy disorders in microglia in Alzheimer's disease. However, it is not yet known whether the lack of microprotein contributes to the development of the disease or becomes a consequence of already occurring pathological processes.
Why is the discovery important for genome research?
The work shows that the current map of the human proteome remains incomplete. Scientists have studied many genes and large proteins well, but this does not mean that all the molecules that cells are capable of producing are known.
"Sometimes it is assumed that we know everything about our genome and all the proteins that our cells can create. This is simply not true," Saghatelyan stressed.
The discovery of micro-MKKS63 also demonstrates that the traditionally isolated "core" protein of the gene is not necessarily its main product in all tissues. In brain cells, a shorter and previously little-studied molecule can be produced much more actively than a large protein that has already been listed in reference books.
This is important for the search for biomarkers and therapeutic targets. If scientists study only the canonical protein, they may not notice another product of the same gene that is more strongly associated with the work of a certain type of cell or disease.
The atlas allows researchers to check which microproteins are present in brain cells, how their content changes with aging and neurodegeneration, and which molecules deserve a separate functional study. The authors made available data on expression, estimates of the reliability of spectra, and information processing programs.
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