| Author: | Yang, Ying |
| Title: | Unveiling roles of sORF-encoded microproteins in liver development and cancer resistance by proteogenomic approaches |
| Advisors: | Zhao, Qian (ABCT) |
| Degree: | Ph.D. |
| Year: | 2025 |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xxii, 174 pages : color illustrations |
| Language: | English |
| Abstract: | Recent advancements in computational, genomic, and proteomic techniques have revealed the potential of unannotated small open reading frames (sORFs) that are capable of encoding peptides. These small open reading frame-encoded microproteins (SEPs), also known as alternative proteins (AltProts) or microproteins, are directly translated from sORFs and exhibit no similarity to the canonical reference proteins (RefProts) of the same gene. Microproteins have been demonstrated to contribute to the progression of various diseases by affecting cellular signaling and disease progression. Despite the growing recognition of their biological significance, microproteins have historically been overlooked because of their short length and relatively low abundance, which complicates their identification through mass spectrometry (MS). The liver, a vital organ during embryonic development, plays a crucial role in hepatic organogenesis and hematopoiesis. It is essential for cell proliferation, immune function, and the synthesis and transport of proteins and nucleic acids. Despite its significance, many biological processes involved in liver development remain poorly understood. Understanding the molecular mechanisms that govern liver development can facilitate the development of regenerative medical strategies to treat liver injuries and diseases. Hepatocellular carcinoma (HCC), a major focus in hepatology, ranks among the most prevalent cancers worldwide, facing challenges due to limited treatment options and a high rate of drug resistance. Therefore, it is essential to develop therapeutic strategies that can effectively address drug resistance to improve patient outcomes and increase survival rates in patients with advanced HCC. Microproteins play vital roles as biological regulators and are involved in numerous biological processes. However, their roles in liver development and drug resistance have not been fully explored. In this study, we focused on the role of microproteins in the field of hepatology, aiming to provide a robust foundation for the development of novel therapeutic strategies and diagnostic tools. We accomplished two key objectives: (i) to develop and refine a systematic methodological approach for the proteogenomic discovery of microproteins in liver tissue across various developmental stages, resulting in a comprehensive proteomic dataset for future functional studies, and (ii) to optimize both DIA and DDA MS-based proteomics methodologies to identify novel microproteins in lenvatinib-resistant HCC cells. This study allowed us to explore the microproteins associated with drug resistance in liver cancer, thereby enhancing our understanding of their mechanisms of action, particularly in relation to drug resistance. This has the potential to facilitate the development of novel strategies for future clinical interventions. First, we reported an approach utilizing size-exclusion chromatography (SEC) for the simultaneous enrichment and fractionation of microproteins from complex proteomes. This method greatly simplified the variance of microprotein discovery by enriching proteins smaller than 40 kDa. In a systematic comparison between the ten methods, our approach facilitated the discovery of more microproteins with overall higher intensities, while requiring less time and effort compared to other workflows. By applying this approach, we successfully identified 89 novel microproteins in the mouse liver, with 39 showing differential expression between the embryonic and adult stages. During embryonic development, upregulated microproteins were mainly involved in biological pathways related to RNA splicing and processing, whereas microproteins involved in metabolism were more active in adult livers. Our study not only presents an effective approach for identifying microproteins but also highlights novel microproteins that are potentially important in developmental biology. We also presented a novel approach combining Ribo-seq and multiple MS methods, identifying and quantifying 815 microproteins from human HCC cells. Notably, we found one microprotein PPGlue was downregulated in resistant cells. Functionally, PPGlue sensitized HCC to lenvatinib treatment both in vitro and in vivo, with enhanced apoptosis, suppressed proliferation and less cancer stemness. Mechanistic studies showed that PPGlue acted as a molecular glue to facilitate the assembly of the protein phosphatase complex PPP2R3C/PP5, reducing a drug exporter P-glycoprotein and subsequently increasing intracellular drug accumulation. Synthetic PPGlue also displayed a synergistic effect with P-glycoprotein's substrate such as lenvatinib, pazopanib and doxorubicin, highlighting its potential therapeutic value. Our study not only provides a practical proteogenomic methodology to identify microproteins in large scale, but also underscores the potential of microproteins as promising modalities in cancer treatment with PPGlue as a representative. In summary, we presented a comprehensive methodological optimization of the microprotein discovery workflow, which includes both sample preparation and MS-based analytical identification. As a result, we identified new molecular participants that are crucial for liver development and cancer biology. Our work offers a comprehensive framework for exploring microproteins, highlighting their identification methodologies and functional characteristics within the context of hepatopathy, thereby paving the way for future research and therapeutic development. |
| Rights: | All rights reserved |
| Access: | open access |
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