The largest protein in the human body, titin, has a molecular weight of three million Daltons and is composed of 27,000 amino acids. But in biology, the smallest things can often have the biggest impact, as a new discovery proves.
With lengths of less than 100 amino acids, small open reading frame-encoded peptides (SEPs) are tiny in comparison. Their small size also makes them hard to detect with conventional methods and few studies have attempted to validate their functionality and biological relevance in a systematic manner.
In particular, nuclear-encoded SEPs that are localized to the mitochondria, called mitochondria-targeted SEPs (mito-SEPs), represent an unexplored repository of new gene functions and therapeutic targets.
“The major challenge is that there is no definitive way of predicting if a protein is part of the mitochondrial proteome, which consists of about 1,500 proteins,” said study corresponding author Lena Ho, an Assistant Professor at Duke-NUS Medical School and a Joint Principal Investigator at the A*STAR Institute of Medical Biology (IMB). The study also included A*STAR-affiliated researchers from the Institute of Molecular and Cell Biology (IMCB) and the Skin Research Institute of Singapore (SRIS).
“To come up with a plausible candidate list, we decided to combine independent methods of proteomic and transcriptomic-based analyses to search for these proteins and tailor our approach to small proteins.”
Through a combination of proteomics, metabolomics and metabolic flux modeling, the researchers screened the SEP peptidome, which led them to identify 16 mito-SEPs, including one with significant implications in energy metabolism: BRAWNIN.
In knockout studies, zebrafish lacking BRAWNIN were growth-stunted and displayed a range of features characteristic of mitochondrial diseases in humans—a dangerous build-up of lactic acid and early death. BRAWNIN was shown to be a central regulator for the assembly of vertebrate respiratory complex III (CIII), which is crucial for survival.
“CIII is essential for life, thus humans with mutations in components of CIII are rare,” explained Ho. “Figuring out the assembly of mammalian CIII and how to manipulate the process to exert metabolic control are very fundamental questions.”
“In parallel, we are also trying to understand how the electron transport chain—the machinery that produces ATP —responds to external metabolic cues,” she added. According to Ho, this is an important question because the failure of the electron transport chain to keep up with cellular energetic demands is a feature of most degenerative diseases, including aging. Small proteins like BRAWNIN could be a link in this mechanism, she said.
“The progressive failure caused by mitochondrial decline and dysfunction underlies all degenerative diseases, and has also been widely implicated in cancer,” said Ho. “The proteins we’ve discovered, including BRAWNIN, represent potential targets in reversing this.”
The A*STAR-affiliated researchers contributing to this research are from the Institute of Medical Biology (IMB), Institute of Molecular and Cell Biology (IMCB) and Skin Research Institute of Singapore (SRIS).