ho-lab @ Duke-NUS
SEP Functionalization
Our lab is dedicated to discovering and characterizing microproteins—small open reading frame–encoded peptides that have long evaded genomic and proteomic annotation—and to doing so through both computational and experimental lenses with a strong emphasis on mitochondrial biology. We have pioneered an integrated approach that begins with smORF-pro, our in-house computational toolkit for prioritizing candidate microproteins based on sequence-based features such as predicted signal peptides, transmembrane domains, and mitochondrial targeting sequences, alongside conservation metrics, predicted structural features, expression patterns, and ribosome profiling data. This allows us to distinguish potentially functional microproteins from spurious translational noise. Leveraging this pipeline, we curated a high-confidence set of over 2,500 candidate smORFs, which we then subjected to a custom-designed CRISPR-Cas9 loss-of-function screen under metabolic stress—specifically in galactose media, which forces cells to rely on mitochondrial oxidative phosphorylation. This screen, conducted in three distinct cell lines, identified 112 smORFs with functional roles in mitochondrial metabolism. From these, we prioritized 25 candidates for biochemical validation based on predicted stability, expression specificity, and mitochondrial localization signals. Experimental analyses, including mitochondrial fractionation, immunofluorescence, and expression assays, revealed that several of these microproteins localized robustly to mitochondria despite lacking classical import sequences. Functional assays such as Seahorse respirometry further demonstrated that the loss or overexpression of a subset of these microproteins significantly impacted mitochondrial respiration, ATP production, and membrane potential. Through proteomic analysis of immunoprecipitates, we also uncovered potential roles for these microproteins in respiratory complex assembly, protein quality control, and membrane organization. These findings not only reinforce the concept that mitochondria are a hotspot for microprotein activity but also establish a scalable discovery pipeline for functionally annotating the previously hidden microproteome. Our approach—combining in silico prioritization, metabolic stress-based CRISPR screening, and targeted biochemical validation—provides a generalizable framework for identifying novel bioactive microproteins, with particular relevance to mitochondrial and metabolic biology.
Cell Biology of SEPs
While the field of microproteomics has increasingly cataloged putative translated smORFs, our lab has focused on the next frontier: understanding how these microproteins are regulated, localized, and integrated into cellular pathways, particularly within the mitochondria. Many of the microproteins we have validated as mitochondrial residents defy canonical targeting paradigms—they lack predictable N-terminal mitochondrial targeting sequences, yet still localize efficiently to mitochondrial compartments, suggesting the existence of alternative import mechanisms or co-translational tethering. To explore this, we have established experimental platforms including split-GFP-based import assays, fractionation studies, and genome-wide CRISPR suppressor screens, which have identified non-classical factors required for the import of specific mitochondrial microproteins. In parallel, we are investigating whether microprotein translation is spatially regulated (eg. near mitochondria) and responsive to nutrient stress. So far, our data suggest that mitochondrial microproteins are embedded in a highly dynamic regulatory landscape that controls when, where, and how they are produced and imported. Understanding these processes not only illuminates basic principles of mitochondrial biology but also uncovers potential therapeutic levers for modulating mitochondrial function through targeted regulation of microprotein translation or localization.
Clinical Application of SEPs
A part of our lab is also committed to translating microprotein biology into clinical and therapeutic contexts, particularly for metabolic, inflammatory, and pregnancy-related disorders. One of our most well-characterized examples is ELABELA, a secreted microprotein originally identified as a hormone essential for cardiovascular development, which we and others have now shown to be dynamically regulated during pregnancy. ELABELA levels correlate with placental development and fetal growth, and our ongoing clinical collaborations are investigating its potential as a circulating biomarker for conditions such as preeclampsia and fetal growth restriction. In parallel, we are exploring therapeutic microproteins that modulate immune responses, most notably MOCCI (C15ORF48), which our lab characterized as a bifunctional transcript encoding both a peptide that suppresses inflammation and a noncoding RNA that regulates gene expression. MOCCI’s dual functionality enables fine-tuned control over innate immune responses, and we are developing delivery strategies to test its efficacy in models of inflammatory diseases. Beyond individual examples, our lab has initiated a proprietary effort with industry partners—referred to internally as the 65Lab initiative—to identify and develop a pipeline of secreted and disease associated microproteins. While the specific candidates and indications remain confidential, the overarching concept is to leverage the natural accessibility and systemic signaling capabilities of microproteins to develop first-in-class therapeutics. These proteins are ideally suited for pharmacologic targeting, whether through peptide mimetics, RNA-based delivery, or antibody-mediated modulation. Our lab is also testing lipid nanoparticle (LNP)-delivered mRNAs encoding selected microproteins in preclinical disease models, exploring their effects on tissue metabolism, inflammation, and organ protection. Altogether, our focus on secreted and targetable microproteins—supported by robust pipelines in discovery, delivery, and mechanistic validation—positions us to make meaningful contributions to the development of microprotein-based diagnostics and therapies.
Respiratory Supercomplexes
Mitochondrial respiratory supercomplexes are critical organizational units of the electron transport chain (ETC) that ensure efficient electron flow, minimize reactive oxygen species, and support adaptive metabolic responses. Our recent work, published in Cell Metabolism (2025), has expanded the conceptual framework of ETC assembly by identifying a novel higher-order structure—the I₂+III₂ “SC-XL” supercomplex—which exhibits enhanced respiratory capacity and redox efficiency under stress conditions. Through a combination of cryo-electron microscopy, blue-native PAGE, and functional bioenergetic assays, we demonstrated that SC-XL assembles in response to specific metabolic challenges, such as Complex III dysfunction or nutrient limitation. Remarkably, mitochondria enriched for SC-XL showed elevated NADH oxidation, reduced ROS production, and preserved ATP output even under ETC perturbation, indicating a bioenergetic advantage conferred by this alternate structural arrangement. We traced the emergence of this supercomplex to the activity of specific mitochondrial microproteins, including UQCC5 and UQCC6, which appear to act as assembly regulators. These discoveries challenge the previously held notion that ETC supercomplexes are fixed structures and instead suggest that mitochondrial architecture is dynamically remodeled in response to cellular cues. Moreover, the presence of SC-XL in tissues such as heart, brain, and liver—and its upregulation during ischemic or oxidative stress—points to its physiological relevance in supporting tissue resilience. We are currently exploring strategies to therapeutically induce SC-XL formation using small molecules, peptide delivery, or gene therapy approaches that enhance the expression or activity of key microprotein regulators. In sum, our work on SC-XL illustrates how respiratory supercomplex remodeling serves as a fundamental mechanism of mitochondrial adaptation and offers new therapeutic entry points for diseases characterized by mitochondrial insufficiency or oxidative stress.