UC San Diego

Lipids and proteins are essential natural small molecules that serve as the building blocks of eukaryotic cells. They play vital roles in regulating nearly all biological processes, including stress adaptation, development and growth, metabolism, and regeneration. An intricate interplay among these molecules facilitates proper functioning of cells and their machinery critical to human health. A disruption in their homeostasis would likely alter pathways that mediate physiology, leading to deleterious human diseases.For decades, a lack of available detection and experimental tools hindered our understanding of natural small molecules and their biology. It was not until recent technologies that led to groundbreaking discoveries of beneficial lipids called FAHFAs and protein-coding small open reading frames (smORFs). The identifications of these previously uncharacterized small natural molecules highlight that the current capacities of mammalian lipidome and transcriptome have been underestimated. Thus, this dissertation will focus on our efforts to functionally characterize these newly identified molecules and their biological roles. Recent discovery of a new bioactive lipid class called branched fatty acid esters of hydroxy fatty acids (FAHFAs) demonstrated great structural diversity, with some, such as palmitic acid 9-hydroxy stearic acid (9-PAHSA), exhibiting inflammation-reducing effects. While over 80 distinct FAHFA isomers have been reported, the biological activities among these lipid species are poorly defined. Because the PAHSAs were previously demonstrated to mediate inflammation in murine and human disease models, we investigated if other endogenous FAHFAs with more potent biological activities than PAHSAs exist in nature. We present that oat oil is a natural source rich of FAHFAs. Analysis of human serum following ingestion of liposomes consisted of fractionated oat oil revealed linoleic acid esters of hydroxy linoleic acids (LAHLAs) as a new FAHFA family, with 13-LAHLA being the most abundant LAHLA isomer. Comparison of 13-LAHLA and 9-PAHSA’s ability to attenuate LPS-stimulated cytokine release under acute inflammation in RAW264.7 macrophages demonstrates that 13-LAHLA was more active than 9-PAHSA, implying a structural-activity relationship (SAR) exists within the lipid class. Characterization of FAHFAs can be difficult due to their susceptibility to endogenous metabolism. FAHFAs are not only subjected to hydrolysis by FAHFA-specific lipases, such as CEL, AIG1 and ADTRP, they can also be incorporated to form FAHFA-containing triglycerols (FAHFA-TGs). While these pathways reflect that FAHFAs are dynamically regulated, metabolism of FAHFAs could diminish their beneficial effects in animal physiology. Therefore, we hypothesized that synthetic FAHFAs with greater stability could protect these derivates from endogenous degradation, thus prolonging their systemic levels and favorable effects. Syntheses and activity screening of a library of synthetic FAHFA derivatives nominated a number of selective potent regulators of inflammation. Subsequent testing of the lead compound in a mouse model of colitis confirms its anti-inflammatory activity and pharmacokinetics profile, potentiating a new non-steroidal therapeutic option against inflammation for this small molecule. In addition to FAHFAs, smORFs belong to a group of underexplored natural small molecules with translational potential. For decades, translation of eukaryotic genes strictly followed a monocistronic model such that translation would only occur once per transcript and only at the longest ORF found within the transcript. This prevented annotation of smaller transcripts like smORF. Recently, the advent ribosome profiling has transformed the understanding of eukaryotic transcriptomes by revealing that many transcripts are polycistronic and smORFs can encode functional microproteins critical to biology. Among smORFs, upstream ORFs (uORFs) are found in at least half of the human transcriptome, representing the largest type of smORFs. Relative to their prevalence, evidence supporting uORF-encoded microproteins remains extremely limited. uORFs are primarily known as cis-regulating elements in the transcriptome, where they mediate the translation of their downstream canonical transcript. However, the notable example of MIEF, a microprotein encoded by the uORF of MIEF1 gene and a regulator of mitochondrial translation, exemplifies that microproteins with independent functions can also arise from uORFs. This raises the possibility that the eukaryotic transcriptome is an abundant repository for many more unique uORF-encoded microproteins. Therefore, we selected the SLC35A4 uORF with previously demonstrated cis-regulating roles and functionally characterized its microprotein product (SLC35A4-MP). Here, we report that SLC35A4-MP is a bona fide member of the mitochondrial contact site and cristae organizing system (MICOS) complex, a protein complex essential for mitochondrial crista formation and mitochondrial respiration. Loss of SLC35A4-MP disrupts mitochondrial crista and crista junction integrity and impaired the optimal oxidative capacities of cells. This finding demonstrates that new functional members of vital protein complexes can be extracted from the abundant reservoir of uncharacterized uORF-encoded microprotein. As additional discoveries of natural small molecules are underway, we believe that our data provides an incentive for their further evaluation. Through development of systematic approaches to interrogate the fundamental biology of lipids and proteins, we aim to provide a template on how one can functionally characterize natural small molecules to counteract the molecular basis of human diseases. Our goal is to gain deeper knowledge of the mammalian lipidome and transcriptome, ultimately leading our path to designing better treatments.