A Targeted Proteomic Approach for Heat Shock Proteins Reveals DNAJB4 as a Suppressor for Melanoma Metastasis

Miao, Weili; Li, Lin; Wang, Yinsheng

doi:10.1021/acs.analchem.8b00986

Cited by 32 publications

(49 citation statements)

References 41 publications

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“…10,11 Due to the high mass-resolving power and mass accuracy afforded by a time-of-flight or Orbitrap mass analyzer, the PRM method offers better accuracy and specificity than MRM in quantifying analytes present in complex sample matrices; 10,11 hence, it has become increasingly used in bioanalysis. 12–14…”

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confidence: 99%

Identification of Helicase Proteins as Clients for HSP90

Miao

Wang

2018

Anal. Chem.

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The 90-kDa heat shock protein (HSP90) is a molecular chaperone that maintains the proper folding of its client proteins including protein kinases and steroid hormone receptors. Helicases are a group of nucleic acid-binding ATPases that can unwind DNA and/or RNA and function in almost every aspect of nucleic acid metabolism. Not much, however, is known about the interactions between HSP90 and helicase proteins. Herein, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method that allows for quantifying >80% of the human helicase proteome. By employing this method, we demonstrated that a large number of helicase proteins exhibited diminished expression in cultured human cells upon treatment with two small-molecule inhibitors of HSP90. We further introduced a tandem affinity tag to the C-terminus of endogenous HSP90β protein by using the CRISPR-Cas9 genome editing method. Affinity purification followed by LC-PRM analysis revealed an enrichment of 40 out of the 66 quantified helicases from the lysate of cells expressing tagged HSP90β. Together, we developed a high-throughput targeted proteomic method for assessing quantitatively the human helicase proteome, and our results support that helicases may constitute an important group of client proteins for HSP90.

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confidence: 99%

Identification of Helicase Proteins as Clients for HSP90

Miao

Wang

2018

Anal. Chem.

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“…We also ensured that the distributions of relative intensities of multiple transitions associated with the same precursor ion are correlated with the theoretical distribution in the kinome MS/ MS spectral library entry, where we imposed a dot product (dotp) value 23 of at least 0.7. The sum of peak areas from all transitions of light or heavy peptides was used for quantification [44][45][46] , and no other adjustments were made. The relative standard deviations (RSD) for kinase protein quantification were 17.2%, 16.9% and 16.4% for WM-115/WM-266-4, IGR-39/IGR-37 and WM-793/1205Lu pairs of cell lines, respectively (Table S1).…”

Section: Tryptic Digestion Of Whole-cell Protein Lysates and Lc-prm mentioning

confidence: 99%

A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis

Miao

Liu

et al. 2020

Sci Rep

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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.

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“…In recent years, microarrays and high-throughput sequencing technologies that detect the expression levels of tens of millions of genes in humans have been widely used to predict potential targets for melanoma treatment (14,15).…”

Section: Introductionmentioning

confidence: 99%

Bioinformatic Analysis Identifies Potential Key Genes in the Pathogenesis of Melanoma

Han¹,

Li²,

Yan³

et al. 2020

Front. Oncol.

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Melanoma is the deadliest skin tumor and is prone to distant metastases. The incidence of melanoma has increased rapidly in the past few decades, and current trends indicate that this growth is continuing. This study was aimed to explore the molecular mechanisms of melanoma pathogenesis and discover underlying pathways and genes associated with melanoma. We used high-throughput expression data to study differential expression profiles of related genes in melanoma. The differentially expressed genes (DEGs) of melanoma in GSE15605, GSE46517, GSE7553, and the Cancer Genome Atlas (TCGA) datasets were analyzed. Differentially expressed genes (DEGs) were identified by paired t-test. Then the DEGs were performed cluster and principal component analyses and protein-protein interaction (PPI) network construction. After that, we analyzed the differential genes through bioinformatics and got hub genes. Finally, the expression of hub genes was confirmed in the TCGA databases and collected patient tissue samples. Total 144 up-regulated DEGs and 16 down-regulated DEGs were identified. A total of 17 gene ontology analysis (GO) terms and 11 pathways were closely related to melanoma. Pathway of pathways in cancer was enriched in 8 DEGs, such as junction plakoglobin (JUP) and epidermal growth factor receptor (EGFR). In the PPI networks, 9 hub genes were obtained, such as loricrin (LOR), filaggrin (FLG), keratin 5 (KRT5), corneodesmosin (CDSN), desmoglein 1 (DSG1), desmoglein 3 (DSG3), keratin 1 (KRT1), involucrin (IVL), and EGFR. The pathway of pathways in cancer and its enriched DEGs may play important roles in the process of melanoma. The hub genes of DEGs may become promising melanoma candidate genes. Five key genes FLG, DSG1, DSG3, IVL, and EGFR were identified in the TCGA database and melanoma tissues. The results suggested that FLG, DSG1, DSG3, IVL, and EGFR might play important roles and potentially be valuable in the prognosis and treatment of melanoma. These hub genes might well have clinical significance as diagnostic markers.

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A Targeted Proteomic Approach for Heat Shock Proteins Reveals DNAJB4 as a Suppressor for Melanoma Metastasis

Cited by 32 publications

References 41 publications

Identification of Helicase Proteins as Clients for HSP90

Identification of Helicase Proteins as Clients for HSP90

A Targeted Quantitative Proteomic Method Revealed a Substantial Reprogramming of Kinome during Melanoma Metastasis

Bioinformatic Analysis Identifies Potential Key Genes in the Pathogenesis of Melanoma

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