The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Aha1 is a co-chaperone of heat shock protein 90 (HSP90), and it stimulates the ATPase activity of HSP90 to promote the folding of its client proteins. By employing ascorbate peroxidase (APEX)-based proximity labeling and proteomic analysis, we identified over 30 proteins exhibiting diminished abundances in the proximity proteome of HSP90 in HEK293T cells upon genetic depletion of Aha1. Dicer1 is a top-ranked protein, and we confirmed its interactions with HSP90 and Aha1 by immunoprecipitation followed by western blot analysis. Genetic depletion of Aha1 and pharmacological inhibition of HSP90 both led to reduced levels of Dicer1 protein. Additionally, HSP90 and Aha1 bind preferentially to newly translated Dicer1. Reconstitution of Aha1-depleted cells with wild-type Aha1 substantially rescued Dicer1 protein level, and a lower level of restoration was observed for complementation with the HSP90-binding-defective Aha1-E67K, whereas an Aha1 mutant lacking the first 20 amino acids-which abolishes its chaperone activity-failed to rescue Dicer1 protein level. Moreover, knockdown of Aha1 and inhibition of HSP90 led to diminished levels of mature microRNAs (miRNAs), but not their corresponding primary miRNAs. Together, we uncovered a novel mechanism of HSP90 and Aha1 in regulating the miRNA pathway through promoting the folding of Dicer1 protein, and we also demonstrated that Aha1 modulates this process by acting as an autonomous chaperone and a co-chaperone for HSP90.

ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N6 -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein.

Tamoxifen has been clinically used in treating estrogen receptor (ER)-positive breast cancer for over 30 years. The most challenging aspect associated with tamoxifen therapy is the development of resistance in initially responsive breast tumors. We applied a parallel-reaction monitoring (PRM)-based quantitative proteomic method to examine the differential expression of kinase proteins in MCF-7 and the isogenic tamoxifen-resistant (TamR) cells. We were able to quantify the relative protein expression levels of 315 kinases, among which hexokinase 2 (HK2) and mTOR were up-regulated in TamR MCF-7 cells. We also observed that the TamR MCF-7 cells exhibited elevated rate of glycolysis than the parental MCF-7 cells. In addition, we found that phosphorylation of S6K - a target of mTOR - was much lower in TamR MCF-7 cells, and this phosphorylation level could be restored upon genetic depletion or pharmacological inhibition of HK2. Reciprocally, the level of S6K phosphorylation was diminished upon overexpression of HK2 in MCF-7 cells. Moreover, we observed that HK2 interacts with mTOR, and this interaction inhibits mTOR activity. Lower mTOR activity led to augmented autophagy, which conferred resistance of MCF-7 cells toward tamoxifen. Together, our study demonstrates that elevated expression of HK2 promotes autophagy through inhibiting the mTOR-S6K signaling pathway and results in resistance of MCF-7 breast cancer cells toward tamoxifen; thus, our results uncovered, for the first time, HK2 as a potential therapeutic target for overcoming tamoxifen resistance.

N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m6A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of previously published m6A mapping results revealed the presence of m6A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.

Tamoxifen has been used for years for treating estrogen receptor-positive breast cancer; drug resistance, however, constitutes one of the main challenges for this therapy. We found that the protein expression level of ATF3 is significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than the corresponding parental cancer cells. In addition, ATF3 protein expression is positively correlated with the resistance of TamR MCF-7 cells to 4-hydroxytamoxifen (4-OHT). Mechanistically, elevated ATF3 protein expression in TamR MCF-7 cells results from a lower level of expression of YTHDF2, an m6A reader protein, and the ensuing stabilization and increased translational efficiency of ATF3 mRNA. Additionally, TamR MCF-7 cells exhibited decreased methylation at A131, a consensus motif site for m6A, in the 5-untranslated region (5-UTR) of ATF3 mRNA. Moreover, augmented ATF3 stimulates the expression of ABCB1, an efflux pump that confers drug resistance in breast cancer cells, and ATF3 itself is also positively regulated by adenylate kinase 4. Together, our results uncovered a novel molecular target for m6A modification (i.e., ATF3 mRNA) and the epitranscriptomic regulator for this target (i.e., YTHDF2). We also illustrated the role of ATF3 in drug resistance, revealed its downstream target (i.e., ABCB1), and suggested ATF3 as a candidate therapeutic target for overcoming drug resistance in cancer cells.

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.

Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.

Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ζ or Pol η. Genetic ablation of Pol ι, Pol κ, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G → A transition and G → T transversion mutations for these lesions. Moreover, further depletion of Pol ζ in Pol κ- or Pol ι-deficient cells gives rise to elevated rates of G → A and G → T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.

Metastasis is a major obstacle in the therapeutic intervention of melanoma, and several GTP-binding proteins were found to play important roles in regulating cancer metastasis. To assess systematically the regulatory roles of these proteins in melanoma metastasis, we employed a targeted chemoproteomic method, which relies on the application of stable isotope-labeled desthiobiotin-GTP acyl phosphate probes in conjunction with scheduled multiple-reaction monitoring (MRM), for profiling quantitatively the GTP-binding proteins. Following probe labeling, tryptic digestion, and affinity pull-down of desthiobiotin-conjugated peptides, differences in expression levels of GTP-binding proteins in two matched pairs of primary/metastatic melanoma cell lines were measured using liquid chromatography-MRM analysis. We also showed that among the top upregulated proteins in metastatic melanoma cells, AK4 promotes the migration and invasion of melanoma cells; overexpression of AK4 in primary melanoma cells leads to augmented migration and invasion, and reciprocally, knockdown of AK4 in metastatic melanoma cells results in repressed invasiveness. In summary, we examined the relative expression levels of GTP-binding proteins in two pairs of primary/metastatic melanoma cell lines. Our results confirmed some previously reported regulators of melanoma metastasis and revealed a potential role of AK4 in promoting melanoma metastasis.