Knockout of the Hmt1p Arginine Methyltransferase in Saccharomyces cerevisiae Leads to the Dysregulation of Phosphate-associated Genes and Processes.

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1Δ, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3 Analysis of the hmt1Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1Δ cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

MethylQuant: A Tool for Sensitive Validation of Enzyme-Mediated Protein Methylation Sites from Heavy-Methyl SILAC Data.

The study of post-translational methylation is hampered by the fact that large-scale LC-MS/MS experiments produce high methylpeptide false discovery rates (FDRs). The use of heavy-methyl stable isotope labeling by amino acids in cell culture (heavy-methyl SILAC) can drastically reduce these FDRs; however, this approach is limited by a lack of heavy-methyl SILAC compatible software. To fill this gap, we recently developed MethylQuant. Here, using an updated version of MethylQuant, we demonstrate its methylpeptide validation and quantification capabilities and provide guidelines for its best use. Using reference heavy-methyl SILAC data sets, we show that MethylQuant predicts with statistical significance the true or false positive status of methylpeptides in samples of varying complexity, degree of methylpeptide enrichment, and heavy to light mixing ratios. We introduce methylpeptide confidence indicators, MethylQuant Confidence and MethylQuant Score, and demonstrate their strong performance in complex samples characterized by a lack of methylpeptide enrichment. For these challenging data sets, MethylQuant identifies 882 of 1165 true positive methylpeptide spectrum matches (i.e., >75% sensitivity) at high specificity (<2% FDR) and achieves near-perfect specificity at 41% sensitivity. We also demonstrate that MethylQuant produces high accuracy relative quantification data that are tolerant of interference from coeluting peptide ions. Together MethylQuant's capabilities provide a path toward routine, accurate characterizations of the methylproteome using heavy-methyl SILAC.

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates.

All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through (13)CD3-methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-ß-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling. Data are available via ProteomeXchange with the data identifier PXD002857.

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human.

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys(79) in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys(79) methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.

Yeast proteins Gar1p, Nop1p, Npl3p, Nsr1p, and Rps2p are natively methylated and are substrates of the arginine methyltransferase Hmt1p.

The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five.

Proteogenomic Discovery of a Small, Novel Protein in Yeast Reveals a Strategy for the Detection of Unannotated Short Open Reading Frames.

In recent years, proteomic data have contributed to genome annotation efforts, most notably in humans and mice, and spawned a field termed "proteogenomics". Yeast, in contrast with higher eukaryotes, has a small genome, which has lent itself to simpler ORF prediction. Despite this, continual advances in mass spectrometry suggest that proteomics should be able to improve genome annotation even in this well-characterized species. Here we applied a proteogenomics workflow to yeast to identify novel protein-coding genes. Specific databases were generated, from intergenic regions of the genome, which were then queried with MS/MS data. This suggested the existence of several putative novel ORFs of <100 codons, one of which we chose to validate. Synthetic peptides, RNA-Seq analysis, and evidence of evolutionary conservation allowed for the unequivocal definition of a new protein of 78 amino acids encoded on chromosome X, which we dub YJR107C-A. It encodes a new type of domain, which ab initio modeling suggests as predominantly α-helical. We show that this gene is nonessential for growth; however, deletion increases sensitivity to osmotic stress. Finally, from the above discovery process, we discuss a generalizable strategy for the identification of short ORFs and small proteins, many of which are likely to be undiscovered.

Annexin/S100A Protein Family Regulation through p14ARF-p53 Activation: A Role in Cell Survival and Predicting Treatment Outcomes in Breast Cancer.

The annexin family and S100A associated proteins are important regulators of diverse calcium-dependent cellular processes including cell division, growth regulation and apoptosis. Dysfunction of individual annexin and S100A proteins is associated with cancer progression, metastasis and cancer drug resistance. This manuscript describes the novel finding of differential regulation of the annexin and S100A family of proteins by activation of p53 in breast cancer cells. Additionally, the observed differential regulation is found to be beneficial to the survival of breast cancer cells and to influence treatment efficacy. We have used unbiased, quantitative proteomics to determine the proteomic changes occurring post p14ARF-p53 activation in estrogen receptor (ER) breast cancer cells. In this report we identified differential regulation of the annexin/S100A family, through unique peptide recognition at the N-terminal regions, demonstrating p14ARF-p53 is a central orchestrator of the annexin/S100A family of calcium regulators in favor of pro-survival functions in the breast cancer cell. This regulation was found to be cell-type specific. Retrospective human breast cancer studies have demonstrated that tumors with functional wild type p53 (p53wt) respond poorly to some chemotherapy agents compared to tumors with a non-functional p53. Given that modulation of calcium signaling has been demonstrated to change sensitivity of chemotherapeutic agents to apoptotic signals, in principle, we explored the paradigm of how p53 modulation of calcium regulators in ER+ breast cancer patients impacts and influences therapeutic outcomes.

Sphingosine kinase 1 isoform-specific interactions in breast cancer.

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor ß-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-ß-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.