Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.

Arginylation is an emerging posttranslational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1 knockout embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell-cell contacts, and chromosomal aberrations. Ate1 knockout fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild type fibroblasts. Furthermore, Ate1 knockout cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is down-regulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1 knockout results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth.

Most prostate cancer cases remain indolent for long periods of time, but metastatic progression quickly worsens the prognosis and leads to mortality. However, little is known about what promotes the metastasis of prostate cancer and there is a lack of effective prognostic indicators, making it immensely difficult to manage options for treatment or surveillance. Arginyltransferase 1 (Ate1) is the enzyme mediating post-translational protein arginylation, which has recently been identified as a master regulator affecting many cancer-relevant pathways including stress response, cell cycle checkpoints, and cell migration/adhesion. However, the precise role of Ate1 in cancer remains unknown. In this study, we found the occurrence of metastasis of prostate cancer is inversely correlated with the levels of Ate1 protein and mRNA in the primary tumor. We also found that metastatic prostate cancer cell lines have a reduced level of Ate1 protein compared to non-metastatic cell lines, and that a depletion of Ate1 drives prostate cancer cells towards more aggressive pro-metastatic phenotypes without affecting proliferation rates. Furthermore, we demonstrated that a reduction of Ate1 can result from chronic stress, and that shRNA-reduced Ate1 increases cellular resistance to stress, and drives spontaneous and stress-induced genomic mutations. Finally, by using a prostate orthotropic xenograft mouse model, we found that a reduction of Ate1 was sufficient to enhance the metastatic phenotypes of prostate cancer cell line PC-3 in vivo. Our study revealed a novel role of Ate1 in suppressing prostate cancer metastasis, which has a profound significance for establishing metastatic indicators for prostate cancer, and for finding potential treatments to prevent its metastasis.

Mutagenesis reporters are critical for quantifying genome stability. However, current methods rely on cell survival/death to report mutation, which takes weeks and prevents evaluation of acute or time-dependent changes. Existing methods also have other limitations, such as cell type restrictions. Using our discovery that mCherryFP fluorescence depends on residue Trp98, we replaced this codon with a stop codon to generate a mutation biosensor (termed CherryOFF), with a green fluorescence protein (GFP) as an internal control. We found that the red fluorescence of this biosensor is activated by a specific A/T-G/C nucleotide transition. Compared with the established hypoxanthine phosphoribosyl transferase assay, our reporter has similar or better ability to detect changes of mutation frequency induced by physical/chemical mutagens or manipulation of mutation-related genes. Furthermore, CherryOFF-GFP can report mutagenesis independently of cell-death events, can be adapted to many cell types, and can generate readouts within 1 day for the measurement of acute or time-dependent events.

Circular dichroism (CD) is a sensitive method that enables accurate detection of protein conformation, as well as structural changes in response to environmental factors such as processing and handling. Whey protein is generally considered the most nutritionally available protein powder source, but variance may exist even among whey proteins. Taking into account a variety of processing, storage and transport conditions, the purpose of this study is to explore whether those variations could result in detectable differences in CD assessed protein structure among different whey protein products available on the market. Secondary and tertiary CD as well as HT spectra of 10 various commercial dry whey products are presented, illustrating marked structural variations among the materials. This might point to the fact that external factors such as processing and/or handling techniques of whey products might have an impact on product characteristics and possibly their nutritional effects.

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In the originally published version of this article, lentivirus and retrovirus were both used for stable gene delivery. However, several vectors were accidentally mislabeled. The vectors CherryOFF-GFP and mCherryFP-GFP are retroviral vectors in the pQC backbone but not lentiviral vectors. Therefore, they should be packaged with retroviral helper plasmids including Gag-Pol and VSV.G (Addgene #14887 and #8454, respectively).