Argonaute (AGO) proteins are core components of RNA interference (RNAi) but the mechanisms of their regulation, especially at the post-translational level, remain unclear. Among the ten AGOs in ...Arabidopsis, only AGO2 is induced by bacterial infection and is known to positively regulate immunity. Here we show that the N-terminal domain of AGO2 is enriched with arginine-glycine RG/GR repeats, which are methylated by protein arginine methyltransferase5 (PRMT5). Arginine methylation has dual functions in AGO2 regulation. Methylated arginine residues can promote AGO2 protein degradation and are also bound by Tudor-domain proteins (TSNs), which can degrade AGO2-associated small RNAs (sRNAs). PRMT5 is down-regulated during infection and the prmt5 mutant is more resistant to bacteria. We speculate that reduced PRMT5 expression during infection may lead to reduced arginine methylation of AGO2, resulting in accumulation of both AGO2 and, via reduced interaction with TSNs, accumulation of AGO2-associated sRNAs, to promote plant immunity. These results reveal that both the arginine methylation writer (PRMT5) and readers (TSNs) can regulate AGO2-mediated RNAi.
Heat shock proteins are molecular chaperones that are involved in protein folding. In this study, we developed a targeted proteomic method, relying on LC-MS/MS in the parallel-reaction monitoring ...(PRM) mode, for assessing quantitatively the human heat shock proteome. The method facilitated the coverage of approximately 70% of the human heat shock proteome and displayed much better throughput and sensitivity than the shotgun proteomic approach. We also applied the PRM method for assessing the differential expression of heat shock proteins in three matched primary/metastatic pairs of melanoma cell lines. We were able to quantify ∼45 heat shock proteins in each pair of cell lines, and the quantification results revealed that DNAJB4 is down-regulated in the three lines of metastatic melanoma cells relative to the corresponding primary melanoma cells. Interrogation of The Cancer Genome Atlas data showed that lower levels of DNAJB4 expression conferred poorer prognosis in melanoma patients. Moreover, we found that DNAJB4 suppresses the invasion of cultured melanoma cells through diminished expression and activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). Together, we established, for the first time, a high-throughput targeted proteomics method for profiling quantitatively the human heat shock proteome and discovered DNAJB4 as a suppressor for melanoma metastasis.
The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang ...1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.
Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking ...immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
Small-molecule inhibitors for the 90-kDa heat shock protein (HSP90) have been extensively exploited in preclinical studies for the therapeutic interventions of human diseases accompanied with ...proteotoxic stress. By using an unbiased quantitative proteomic method, we uncover that treatment with three HSP90 inhibitors results in elevated expression of a large number of heat shock proteins. We also demonstrate that the HSP90 inhibitor-mediated increase in expression of DNAJB4 protein occurs partly through an epitranscriptomic mechanism, and is substantially modulated by the writer, eraser, and reader proteins of N
-methyladenosine (m
A). Furthermore, exposure to ganetespib leads to elevated modification levels at m
A motif sites in the 5'-UTR of DNAJB4 mRNA, and the methylation at adenosine 114 site in the 5'-UTR promotes the translation of the reporter gene mRNA. This m
A-mediated mechanism is also at play upon heat shock treatment. Cumulatively, we unveil that HSP90 inhibitors stimulate the translation of DNAJB4 through an epitranscriptomic mechanism.
Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. ...However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs). Enhanced CPT1A/CPT2 expression was detected in the recurrent human breast cancers and associated with a worse prognosis in breast cancer patients. Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.
Kinases are involved in numerous critical cell signaling processes, and dysregulation in kinase signaling is implicated in many types of human cancers. In this study, we applied a parallel-reaction ...monitoring (PRM)-based targeted proteomic method to assess kinome reprogramming during melanoma metastasis in three pairs of matched primary/metastatic human melanoma cell lines. Around 300 kinases were detected in each pair of cell lines, and the results showed that Janus kinase 3 (JAK3) was with reduced expression in the metastatic lines of all three pairs of melanoma cells. Interrogation of The Cancer Genome Atlas (TCGA) data showed that reduced expression of JAK3 is correlated with poorer prognosis in melanoma patients. Additionally, metastatic human melanoma cells/tissues exhibited diminished levels of JAK3 mRNA relative to primary melanoma cells/tissues. Moreover, JAK3 suppresses the migration and invasion of cultured melanoma cells by modulating the activities of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). In summary, our targeted kinome profiling method provided by far the most comprehensive dataset for kinome reprogramming associated with melanoma progression, which builds a solid foundation for examining the functions of other kinases in melanoma metastasis. Moreover, our results reveal a role of JAK3 as a potential suppressor for melanoma metastasis.
Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 ...SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.
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