Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo. Using these Ub probes combined with mass spectrometry, we have successfully identified endogenous deconjugating enzymes in live cells. We believe that these genetically encoded Ub/Ubl probes are valuable for investigating biological functions of deconjugating enzymes in physiological environments.
A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–host interactions is crucial to developing effective therapeutics and addressing the threat of emerging coronaviruses. The role of noncoding regions of viral RNA (ncrRNAs) has yet to be systematically scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry and designed a diverse set of bait ncrRNAs to systematically map the interactome of SARS-CoV-2 ncrRNA in Calu-3, Huh7, and HEK293T cells. Integration of the results defined the core ncrRNA–host protein interactomes among cell lines. The 5′ UTR interactome is enriched with proteins in the small nuclear ribonucleoproteins family and is a target for the regulation of viral replication and transcription. The 3′ UTR interactome is enriched with proteins involved in the stress granules and heterogeneous nuclear ribonucleoproteins family. Intriguingly, compared with the positive-sense ncrRNAs, the negative-sense ncrRNAs, especially the negative-sense of 3′ UTR, interacted with a large array of host proteins across all cell lines. These proteins are involved in the regulation of the viral production process, host cell apoptosis, and immune response. Taken together, our study depicts the comprehensive landscape of the SARS-CoV-2 ncrRNA–host protein interactome and unveils the potential regulatory role of the negative-sense ncrRNAs, providing a new perspective on virus–host interactions and the design of future therapeutics. Given the highly conserved nature of UTRs in positive-strand viruses, the regulatory role of negative-sense ncrRNAs should not be exclusive to SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic affecting millions of lives. During replication and transcription, noncoding regions of the viral RNA (ncrRNAs) may play an important role in the virus–host interactions. Understanding which and how these ncrRNAs interact with host proteins is crucial for understanding the mechanism of SARS-CoV-2 pathogenesis. We developed the MS2 affinity purification coupled with liquid chromatography-mass spectrometry method and designed a diverse set of ncrRNAs to identify the SARS-CoV-2 ncrRNA interactome comprehensively in different cell lines and found that the 5′ UTR binds to proteins involved in U1 small nuclear ribonucleoprotein, while the 3′ UTR interacts with proteins involved in stress granules and the heterogeneous nuclear ribonucleoprotein family. Interestingly, negative-sense ncrRNAs showed interactions with a large number of diverse host proteins, indicating a crucial role in infection. The results demonstrate that ncrRNAs could serve diverse regulatory functions.
Protein degradation in eukaryotic cells is mainly carried out by the 26S proteasome, a macromolecular complex not only present in the cytosol and nucleus but also associated with various membranes. How proteasomes are anchored to the membrane and the biological meaning thereof have been largely unknown in higher organisms. Here we show that N-myristoylation of the Rpt2 subunit is a general mechanism for proteasome-membrane interaction. Loss of this modification in the Rpt2-G2A mutant cells leads to profound changes in the membrane-associated proteome, perturbs the endomembrane system and undermines critical cellular processes such as cell adhesion, endoplasmic reticulum-associated degradation (ERAD) and membrane protein trafficking. Rpt2G2A/G2A homozygous mutation is embryonic lethal in mice and is sufficient to abolish tumor growth in a nude mice xenograft model. These findings have defined an evolutionarily conserved mechanism for maintaining membrane protein homeostasis and underscored the significance of compartmentalized protein degradation by myristoyl-anchored proteasomes (MAPs) in health and disease.
A deep understanding of SARS-CoV-2-host interactions is crucial to the development of effective therapeutics. The role of non-coding regions of viral RNA (ncrRNAs) has not been scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry (MAMS) to systematically map the interactome of SARS-CoV-2 ncrRNA in different human cell lines. Integration of the results defined the core and cell-type-specific ncrRNA-host protein interactomes. The majority of ncrRNA-binding proteins were involved in RNA biogenesis, protein translation, viral infection, and stress response. The 5′ UTR interactome is enriched with proteins in the snRNP family and is a target for the regulation of viral replication and transcription. The 3′ UTR interactome is enriched with proteins involved in the cytoplasmic RNP granule (stress granule) and translation regulation. We show that the ORF10 is likely to be a part of 3′ UTR. Intriguingly, the interactions between negative-sense ncrRNAs and host proteins, such as translation initiation factors and antiviral factors, suggest a pathological role of negative-sense ncrRNAs. Moreover, the cell-type-specific interactions between ncrRNAs and mitochondria may explain the differences of cell lines in viral susceptibility. Our study unveils a comprehensive landscape of the functional SARS-CoV-2 ncrRNA-host protein interactome, providing a new perspective on virus-host interactions and the design of future therapeutics.
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