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Published on: January 12, 2024
Nascent alt-protein chemoproteomics reveals a pre-60S assembly checkpoint inhibitor
Xiongwen Cao1,2, Alexandra Khitun1,2, Cecelia M Harold3
1Department of Chemistry, Yale University, New Haven, CT, USA.
Abstract:
Many unannotated microproteins and alternative proteins (alt-proteins) are coencoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing regulated synthesis could play important cellular roles, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated alt-proteins or N-terminal extensions, one of which is post-transcriptionally upregulated by DNA damage stress. We further defined a nucleolar, cell-cycle-regulated alt-protein that negatively regulates assembly of the pre-60S ribosomal subunit (MINAS-60). Depletion of MINAS-60 increases the amount of cytoplasmic 60S ribosomal subunit, upregulating global protein synthesis and cell proliferation. Mechanistically, MINAS-60 represses the rate of late-stage pre-60S assembly and export to the cytoplasm. Together, these results implicate MINAS-60 as a potential checkpoint inhibitor of pre-60S assembly and demonstrate that chemoproteomics enables hypothesis generation for uncharacterized alt-proteins.
Insights
Researchers discovered novel alternative proteins (alt-proteins) using chemoproteomics. One identified alt-protein, MINAS-60, regulates ribosome assembly, impacting protein synthesis and cell growth.
Area of Science:
- Molecular Biology
- Proteomics
- Cell Biology
Background:
- Many microproteins and alternative proteins (alt-proteins) are co-encoded with canonical proteins but lack functional characterization.
- Understanding the roles of these unannotated proteins is crucial for comprehending cellular complexity.
Purpose of the Study:
- To develop a chemoproteomic pipeline for identifying nascent alt-proteins in human cells.
- To investigate the cellular roles and regulation of newly identified alt-proteins.
Main Methods:
- Development of a chemoproteomic pipeline to detect actively translated alt-proteins.
- Identification of 22 actively translated alt-proteins or N-terminal extensions.
- Characterization of a specific alt-protein, MINAS-60, involved in ribosome biogenesis.
Main Results:
- Identified 22 actively translated alt-proteins, including one upregulated by DNA damage.
- Defined MINAS-60, a nucleolar, cell-cycle-regulated alt-protein that inhibits pre-60S ribosomal subunit assembly.
- Demonstrated that MINAS-60 depletion enhances 60S subunit availability, increasing global protein synthesis and cell proliferation.
Conclusions:
- MINAS-60 acts as a checkpoint inhibitor for pre-60S assembly and cytoplasmic export.
- Chemoproteomics is a powerful tool for generating hypotheses about uncharacterized alt-proteins.
- Regulated alt-proteins can play significant roles in cellular processes like protein synthesis and cell cycle control.
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