Proteins are the fundamental workhorses within all living cells, executing the vast majority of biological tasks, from catalyzing chemical reactions to providing structural support. For decades, scientific focus centered primarily on large, complex proteins that were easily identifiable using existing biochemical methods. This focus inadvertently caused researchers to overlook an entire class of much smaller, yet highly influential, molecules. The recent recognition of microproteins, typically defined as very small proteins containing fewer than 100 amino acids, has begun to reshape our understanding of cellular regulation. Their discovery suggests that many processes previously thought to be fully understood are actually fine-tuned by these overlooked regulators.
Defining the Hidden Players
Microproteins are defined as polypeptides containing 150 amino acids or fewer, with most studied examples falling below the 100 amino acid threshold. This minute size is why these molecules remained hidden, challenging traditional criteria used to identify protein-coding genes. Standard genome annotation pipelines filtered out genetic sequences that did not meet a minimum length requirement, typically 300 base pairs. Consequently, the genetic instructions for these smaller molecules were routinely dismissed as non-functional “junk” or transcriptional noise.
The structure of microproteins distinguishes them from other small peptides, such as hormones like insulin. Classic peptides are synthesized as large precursor proteins, which are then cleaved into active, smaller forms through post-translational modification. In contrast, microproteins are translated directly from their messenger RNA as fully mature, functional molecules that do not require subsequent processing. Advances in proteomics and bioinformatics have finally allowed scientists to look past the size filter and reveal the thousands of microproteins encoded within the human genome.
The Genetic Blueprint
The instructions for building microproteins are found within short Open Reading Frames (sORFs), which are small sequences of genetic code that instruct the cell’s machinery to start translation. These sORFs are often located in regions previously labeled as non-coding RNA (ncRNA) or untranslated regions (UTRs). For years, these ncRNA regions were considered transcriptional leftovers without the capacity to encode functional proteins. The discovery that these areas harbor sORFs capable of directing microprotein production challenges the traditional view of genetic organization.
Their discovery is complicated by the fact that many sORFs initiate protein synthesis using non-canonical start codons, rather than the standard AUG codon (methionine). While AUG is the typical signal to begin translating a protein, many microproteins begin with alternative codons like CUG, GUG, UUG, or ACG. This reliance on non-standard start signals made it difficult for early computational models to accurately predict their location. Identifying these small coding sequences has expanded the known protein library, revealing a new layer of regulatory complexity in the cell.
Key Roles in Health and Disease
The functional roles identified for microproteins are broad, suggesting they act as fine-tuning mechanisms that regulate the activity of larger cellular components. Many microproteins function as allosteric regulators, binding to a large protein in one location to modify its activity at a distant site. Others work independently as signaling molecules or effector proteins, participating in fundamental cellular processes like ion transport, stress signaling, and energy generation. This regulatory capacity is evident in tissues with high energy demands, such as the heart and skeletal muscle.
Muscle Contraction and Calcium Handling
Several microproteins modulate calcium handling, the process that dictates the contraction and relaxation of muscle cells. For instance, Phospholamban (PLN) and Sarcolipin (SLN) interact directly with the Sarcoplasmic Reticulum Calcium ATPase (SERCA) pump. By binding to SERCA, these molecules regulate the uptake of calcium ions into the cell’s storage units, controlling the strength and speed of muscle contraction. The microprotein DWORF works in opposition to these inhibitors by competitively binding to SERCA, displacing PLN and other regulators to enhance calcium cycling.
Metabolic Regulation
Microproteins also influence metabolic health, affecting processes like glucose homeostasis, fat storage, and energy expenditure. Mitochondria, the cell’s energy factories, are a frequent destination for newly synthesized microproteins. For example, Mitoregulin (MTLN) regulates the breakdown of triglycerides and influences mitochondrial beta-oxidation in fat cells and skeletal muscle. Another microprotein, Mitolamban (Mtlbn), localizes to the inner mitochondrial membrane in the heart. Mtlbn associates with Complex III of the electron transport chain, contributing to the assembly and function of this complex responsible for generating cellular energy. Recent screening technologies identifying microproteins involved in lipid accumulation are providing new insights into the regulation of obesity.
Unlocking Therapeutic Potential
The discovery of microproteins opens new avenues for developing treatments for a range of human diseases. Since these molecules are involved in fundamental regulatory pathways, manipulating their function could offer novel ways to treat conditions like heart failure, muscle wasting disorders, and metabolic diseases. For example, the microprotein DWORF, which enhances muscle calcium handling, is under investigation for treating cardiomyopathy. Identifying microproteins that regulate lipid metabolism also offers promising drug targets for obesity and type 2 diabetes.
Researchers are exploring two main therapeutic strategies: developing drugs that target microprotein pathways, or using the microproteins themselves as therapeutic agents. Miniproteins, a related group, are being studied as drug scaffolds because their small size allows for good tissue penetration, combining the advantages of traditional small molecules and large antibody-based biologics. The ability to rapidly identify and validate these molecules has been accelerated by modern molecular techniques, including advanced mass spectrometry and large-scale genetic screening methods like CRISPR/Cas9. These tools are uncovering the function of previously unknown microproteins, making them actionable targets for future medicine.