PCMT1 confirmed as a pan-cancer immune biomarker and a contributor to breast cancer metastasis.

Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT, gene name PCMT1) is an enzyme that repairs proteins with altered aspartate residues by methylation, restoring their normal structure and function. This study conducted a comprehensive analysis of PCMT1 in pan-cancer. The Cancer Genome Atlas, Human Protein Atlas website, and the Genotype-Tissue Expression were utilized in analysis of PCMT1 expression. We examined the association between PCMT1 expression and various factors, including gene modifications, DNA methylation, immune cell infiltration, immunological checkpoints, drug susceptibility, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analyses determined the potential biological roles and pathways involving PCMT1. Our focus then shifted to the role of PCMT1 in breast invasive carcinoma (BRCA). We found that PCMT1 expression was aberrant in many tumors and significantly influenced the prognosis across several cancer types. Gene alterations in PCMT1 predominantly involved deep deletions and amplifications. A negative correlation was observed between DNA methylation and PCMT1 expression across all studied cancer types except thyroid carcinoma PCMT1 exhibited positive correlations with common lymphoid progenitor and CD4(+) T helper 2 cells, whereas it was inversely correlated with central and effector memory T cells, memory CD8(+) T cells, and CD4(+) T helper 1 cells. In many cancer types, PCMT1 expression closely correlated with immunological checkpoint inhibitors, TMB, and MSI. It was also significantly linked to pathways involved in epithelial-mesenchymal transition (EMT), highlighting its role in cancer metastasis. PCMT1 emerged as a significant predictor of breast cancer progression. In vitro experiments demonstrated that reducing PCMT1 expression decreased BRCA cell migration and invasiveness. Additionally, animal studies confirmed that inhibition of PCMT1 slowed tumor growth.

PCMT1 as a prognostic marker in breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is one of the most aggressive cancers in women, therefore it is necessary to determine novel prognostic markers to estimate survival and advancement the treatment of the disease. Recently, PCMT1, a protein mediating TNBC immune infiltration, has gained attention as a potential therapeutic target. The aim of the study was to demonstrate the relationship between PCMT1 protein overexpression as a prognostic indicator for patients with TNBC cancer and patient survival. MATERIALS AND METHODS: The study included 64 samples collected from 64 TNBC patients. We used the ImageJ software with the IHC Profiler driver for image analysis. To improve the reliability of the results, we expanded the analysis by including The Cancer Genome Atlas cohort. RESULTS: We observed strong PCMT1 immunoreactivity in breast cancer samples and PCMT1 expression in TNBC was significantly higher than in the control group. Patients with high PCMT1 expression had a significantly lower overall survival rate (60.62% vs. 90.35%, respectively) than patients with low PCMT1 expression. In our study and TCGA groups, PCMT1 expression did not correlate with lymph node involvement and distant metastases but correlated with tumor stage. The results obtained in a larger TCGA group are consistent with those in our research group. Overexpression of PCMT1 was a prognostic marker of shorter survival in patients with TNBC. CONCLUSIONS: The overexpression of the PCMT1 protein in triple negative breast cancer significantly correlated with shorter overall survival. The confirmed association could be a potential prognostic biomarker for patients with TNBC.

Silencing PCMT1 enhances the sensitivity of breast cancer cells to paclitaxel through the PI3K/Akt/STMN1 pathway.

This study aimed to investigate whether silencing Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) expression can enhance the sensitivity of breast cancer cells to paclitaxel and its possible mechanism. Tumor tissues and adjacent histologically normal tissues were collected from patients with breast cancer admitted to our hospital. Human normal breast epithelial cells MCF10A, human breast cancer cells MCF-7, and paclitaxel-resistant breast cancer cells MCF-7/PR were purchased. MCF-7/PR cells were further grouped into negative control (NC) group, si-PCMT1 group (transfected with si-PCMT1), 740Y-P group (treated with 740Y-P, an activator of phosphatidylinositol 3-kinase (PI3K)/ v-Akt Murine Thymoma Viral Oncogene (AKT) signaling pathway), and si-PCMT1 + 740Y-P group (transfected with si-PCMT1 and then treated with 740Y-P). The expression level of PCMT1 in tissues and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot analysis was used to detect the protein expression level of PCMT1 in tissues and cells as well as the protein level of p-PI3K, PI3K, p-Akt, Akt, and Stathmin1 (STMN1) in cells. 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and colony formation assays were used to determine cell viability, scratch assay was used to assess the migration ability of cells, and Transwell assay was used to assess the invasion ability of cells. The expression of PCMT1 was remarkably up-regulated in breast cancer tissues and MCF-7/PR cells. Silencing PCMT1 expression significantly inhibited the proliferation, migration, and invasion of MCF-7/PR cells, and alleviated the resistance of cancer cells to paclitaxel. Additionally, silencing PCMT1 expression also inhibited the activation of PI3K/Akt/STMN1 pathway in MCF-7/PR cells, while activating PI3K/Akt/STMN1 pathway significantly reversed the effect of silencing PCMT1 expression on MCF-7/PR cells. PCMT1 is highly expressed in breast cancer tissues and MCF-7/PR cells, and silencing PCMT1 expression can not only inhibit the development of breast cancer but also enhance paclitaxel sensitivity. Its mechanism of action may be achieved by inhibiting PI3K/Akt/STMN1 signaling.

Immunotherapy targeting isoDGR-protein damage extends lifespan in a mouse model of protein deamidation.

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

PCMT1 knockdown attenuates malignant properties by globally regulating transcriptome profiles in triple-negative breast cancer cells.

Background: As the most frequently diagnosed cancer in women, Breast cancer has high mortality and metastasis rate, especially triple-negative breast cancer (TNBC). As an oncogene, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a prognostic biomarker in breast cancer and is highly expressed, while its underlying functions remain unknown. Methods: In this study, we silenced PCTM1 in TNBC MDA-MB-231 cells by short hairpin RNA (shPCMT1) to investigate its cellular functions using cell proliferation, apoptosis, migration, and invasion experiments. Following this, the transcriptome sequencing (RNA-seq) experiment was conducted to explore the molecular targets of PCMT1, including differentially expressed genes (DEGs) and regulated alternative splicing events (RASEs). Results: The results showed that shPCMT1 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. We obtained 1,084 DEGs and 2,287 RASEs between shPCMT1 and negative control (NC) groups through RNA-seq. The DEGs were significantly enriched in immune or inflammation response and cell adhesion-associated pathways, pathways associated with PCMT1 cellular function in cell migration. The RASE genes were enriched in cell cycle-associated pathways and were associated with the altered cell proliferation rate. We finally validated the changed expression and splicing levels of DEGs and RASEs. We found that 34 RNA binding protein (RBP) genes were dysregulated by shPCMT1, including NQO1, S100A4, EEF1A2, and RBMS2. The dysregulated RBP genes could partially explain how PCMT1 regulates the global transcriptome profiles. Conclusion: In conclusion, our study identified the molecular targets of PCMT1 in the TNBC cell line, expands our understanding of the regulatory mechanisms of PCMT1 in cancer progression, and provides novel insights into the progression of TNBC. The identified molecular targets are potential therapeutic targets for future TNBC treatment.

PCMT1 regulates the migration, invasion, and apoptosis of prostate cancer through modulating the PI3K/AKT/GSK-3ß pathway.

Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a repair enzyme that catalyzes the conversion of isomerized aspartic acid (iso-Asp) residues into their normal structure, thereby restoring the configuration and function of proteins. Studies have shown that PCMT1 is overexpressed in several tumors and affects patients' prognosis. However, there are few reports on the role of PCMT1 in prostate cancer (PCa). In the present research, with the assistance of The Cancer Genome Atlas Program (TCGA) database, we found that PCMT1 was overexpressed in PCa tissues. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry staining also showed that PCMT1 expression was significantly increased in PCa tissues and cell lines. In PCa clinical samples, PCMT1 expression was closely related to Gleason score, clinical stage, lymph node metastasis and bone metastasis. The experiments of overexpression and knockdown of PCMT1 in vitro or in vivo showed that PCMT1 can significantly promote the proliferation, migration and invasion of PCa cells, inhibit cell apoptosis, and promote the growth of PCa. We furthermore confirmed that PCMT1 regulated the migration, invasion and apoptosis of PCa cells by modulating the phosphatidylinositol 3-kinase/AKT kinase/glycogen-synthase kinase-3ß (PI3K/AKT/GSK-3ß) signaling pathway. Collectively, PCMT1 plays a cancer-facilitative role in PCa by promoting the proliferation, migration and invasion of PCa cells, and inhibiting apoptosis. Therefore, PCMT1 is considered to represent a novel target for treating PCa.

PCMT1 is a potential target related to tumor progression and immune infiltration in liver cancer.

BACKGROUND: Liver cancer is a prevalent and deadly form of cancer with high incidence and mortality rates. The PCMT1 protein has been linked to cell anti-apoptosis and tumor metastasis, but its significance in liver hepatocellular carcinoma (LIHC) remains largely unexplored. METHODS: We conducted a pan-cancer analysis to examine the expression differences of PCMT1. Kaplan-Meier curves were employed to assess the prognostic impact of PCMT1 on LIHC patients, and we investigated the association between PCMT1 and clinical features, which we validated using a GEO therapeutic dataset. Gene enrichment analysis helped identify signaling pathways associated with PCMT1 expression. Moreover, we evaluated the relationship between PCMT1 and immune cell infiltration, as well as the differences in gene mutations between high-expression and low-expression groups. In vitro and in vivo experiments were performed to assess the effect of PCMT1 on tumor cell lines and mouse tumor models, and potential pathways were explored through gene sequencing. RESULT: PCMT1 is highly expressed in most tumors and exhibits a significant association with prognosis in LIHC patients. Pathway enrichment analysis revealed that PCMT1 is involved in cell cycle regulation, immunity, and other processes. Further immune analysis demonstrated that high expression of PCMT1 could reduce tumor-killing immune cell infiltration. In vitro experiments indicated that PCMT1 knockdown could inhibit cancer cell proliferation and migration while promoting apoptosis. In vivo experiments showed that PCMT1 knockdown significantly reduced tumor growth rate, enhanced CD8+T cell infiltration, and increased caspase-3 expression in the tumor area. Gene sequencing suggested that PCMT1 may function through the PI3K-AKT pathway. CONCLUSION: Our findings suggest that PCMT1 acts as a promoter of liver cancer progression and may serve as a novel prognostic indicator and therapeutic target for patients with LIHC.

In vitro Anti-malignant Property of PCMT1 Silencing and Identification of the SNHG16/miR-195/PCMT1 Regulatory Axis in Breast Cancer Cells.

BACKGROUND: Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) is a highly conserved protein repair enzyme that participates in regulating the progression of human cancers. We therefore studied the function and the related mechanisms of PCMT1 in breast cancer cells. METHODS: Expression profile and prognostic analysis of PCMT1 in breast cancer patients were analyzed using online databases. PCMT1 expression in breast cancer cells was detected by western blot analysis. Cell proliferation was determined by CCK-8 and colony formation assays. Apoptosis was evaluated using flow cytometry analysis and caspase-3/7 activity assay. Cell invasion was assessed by Transwell invasion assay. The small nucleolar RNA host gene 16 (SNHG16)/miR-195/PCMT1 regulatory axis was identified using bioinformatics analysis. RESULTS: PCMT1 expression was increased in breast cancer tissues and cells. High PCMT1 expression was correlated with poor prognosis in breast cancer patients. PCMT1 knockdown suppressed cell proliferation and colony formation ability in breast cancer cells. Moreover, PCMT1 knockdown induced apoptosis and restrained the invasive ability in breast cancer cells. PCMT1 overexpression increased the proliferative and invasive abilities of breast cancer cells. miR-195 was identified as the unique upstream miRNA of PCMT1. SNHG16 was identified as the unique upstream lncRNA of miR-195. SNHG16 knockdown downregulated PCMT1 by increasing miR-195 expression. Breast cancer cell proliferation was regulated by the SNHG16/miR-195/PCMT1 axis. CONCLUSION: PCMT1 silencing inhibited cell proliferation and invasion and induced apoptosis in breast cancer cells and the SNHG16/miR-195/PCMT1 regulatory axis might serve as a potential therapeutic target for breast cancer.

[Prognostic Value of PCMT1 Expression in Gastric Cancer and Its Regulatory Effect on Spindle Assembly Checkpoints]. / 胃癌中PCMT1è¡¨è¾¾çš„é¢„åŽä»·å€¼åŠå ¶å¯¹çººé”¤ä½“ç»„è£ æ£€æŸ¥ç‚¹çš„è°ƒæŽ§ä½œç”¨.

Objective: The study was conducted to investigate the expression of protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) in gastric cancer and its effect on the prognosis, and to analyze its potential mechanism. Methods: UALCAN, a cancer data analysis platform, was used to conduct online analysis of the expression of PCMT1 in gastric cancer tissues. Through the Database for Annotation, Visualization and Integrated Discovery (DAVID), Gene Ontology (GO) annotation and signaling pathway enrichment by Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the possible functions and signaling pathways. A total of 120 patients who underwent radical gastrectomy for gastric cancer between January 2014 and December 2017 in our hospital were enrolled for the study. Immunohistochemical staining was performed to determine the expression of PCMT1 and Ki67 in gastric cancer tissues. Cox regression, Kaplan-Meier curve, and receiver operating characteristic (ROC) curves were used for prognostic analysis of 5-year survival in gastric cancer patients after surgery. Lentivirus was used to construct PCMT1-interfering or PCMT1-overexpressing vectors, which were then used to transfect human gastric cancer cell lines of MGC-803 and HGC-27 cells. The interfering empty vector (sh-NC) group, the interfering PCMT1 vector (sh-PCMT1) group, the overexpressing empty vector (LV-Vec) group, and the overexpressing PCMT1 vector (LV-PCMT1) group were set up. Western blot was performed to determine the protein expression levels of PCMT1, CyclinB1, and CDC20. CCK-8 assay was performed to measure the proliferation of gastric cancer cells. Flow cytometry was performed to determine the cell cycle. MGC-803 cells were injected in four groups of nude mice to construct a subcutaneous xenograft tumor model, with three nude mice in each group. The body mass of the nude mice was measured. The nude mice were sacrificed after 14 days and the tumor volume was monitored. The expression levels of CyclinB1 and CDC20 proteins in the tumor tissues were determined by Western blot assay. Results: Analysis with UALCAN showed that PCMT1 was highly expressed in gastric cancer tissues. Moreover, elevated expression was found in gastric tumor tissues of different pathological stages and grades and those with lymph node metastasis (P<0.05). GO and KEGG enrichment analyses showed that PCMT1 was mainly involved in the signal regulation of mitosis, spindle assembly checkpoints, and cell cycle. The immunohistochemical results showed that PCMT1 and Ki67 were highly expressed in gastric cancer tissues and that they were positively correlated with each other (P<0.05). Cox multivariate analysis showed that high PCMT1 expression (hazard ratio [HR]=2.921, 95% confidence interval [CI]:1.628-5.239) was one of the independent risk factors affecting the 5-year survival rate of gastric cancer patients after surgery. Kaplan-Meier curve showed that patients with high PCMT1 expression had a lower 5-year survival after surgery (16.7%, HR=4.651, 95% CI: 2.846-7.601) than patients with low PCMT1 expression (70.0%, HR=0.215, 95% CI: 0.132-0.351) did. The ROC curve showed that PCMT1 had an area under the curve (AUC) of 0.764 (95% CI: 0.674-0.854) for predicting 5-year patient survival after surgery. Western blot results showed that lentiviral interference or overexpression of PCMT1 cell lines was successfully constructed. The results of CCK-8 showed that the proliferative ability of MGC-803 and HGC-27 cells was weakened with the downregulation of PCMT1, and the overexpression of PCMT1 promoted cell proliferation (P<0.05). With the interference of PCMT1, the expression of CDC20 protein was decreased, the expression of CyclinB1 protein was increased, and the cell cycle was arrested in the G2/M phase. In contrast, the overexpression of PCMT1 led to the opposite trends (P<0.05). In the sh-PCMT1 group, the tumor volume and mass were decreased and the expression of CDC20 protein was decreased and the expression of CyclinB1 protein was increased in the tumor tissues of the nude mice (P<0.05, compared with those of the sh-NC group. In contrast, the LV-PCMT1 group showed the opposite trends (P<0.05, compared with those of the LV-Vec group). Conclusion: The high expression of PCMT1 in gastric cancer tissues is associated with poor prognosis in patients and may affect tumor cell malignant proliferation via regulating spindle checkpoints in the process of mitosis.

Elevated expression of protein-L-isoaspartate O-methyltransferase-1 (PCMT1) in cervical cancer.

Background: Protein-L-isoaspartate O-methyltransferase-1 (PCMT1) is a protein carboxyl methyltransferase enzyme, which has been found to play roles in cancers. However, no clinical information about the correlation between cervical cancer and PCMT1 expression has been reported. Methods: We used immunohistochemistry (IHC) to characterize the protein level of PCMT1 in human cervical intraepithelial neoplasia and cervical cancer specimens. The mRNA expression profile of PCMT1 in cervical cancer was also analyzed by using Gene Expression Omnibus (GEO) databases. The prognostic value of PCMT1 in patients with cervical cancer was evaluated by using the Kaplan-Meier plotter. Gene set enrichment analysis (GSEA) was conducted by using The Cancer Genome Atlas (TCGA) cervical cancer dataset. Results: The protein level of PCMT1 was increased in cervical high-grade squamous intraepithelial lesion (HSIL) (7.40±0.42) and cervical cancer tissues (10.70±0.54), compared to normal cervix (5.00±0.86) and low-grade squamous intraepithelial lesion (LSIL) (6.22±0.57) (P<0.05). the immunoreactivity score (IRS) of PCMT1 was also higher in cervical cancer tissues than in paired adjacent non-cancerous cervical tissues (9.03±0.52 vs. 6.32±0.46) (P<0.05). High expression of PCMT1 was associated with decreased overall survival (OS) of patients with cervical cancer (P=0.0022). GSEA demonstrated that cervical cancer patients with high expression of PCMT1 were enriched in the various cancer-related signaling pathways. Conclusions: These results suggest that PCMT1 might be a diagnostic and prognostic biomarker for cervical cancer, and further validation studies should be performed.

Genome-wide CRISPR/Cas9 library screen identifies PCMT1 as a critical driver of ovarian cancer metastasis.

BACKGROUND: The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance. METHODS: Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging. RESULTS: We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors. CONCLUSIONS: Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

LINC00511/miRNA-143-3p Modulates Apoptosis and Malignant Phenotype of Bladder Carcinoma Cells via PCMT1.

Bladder cancer has easy recurrence characteristics, but its occurrence and development mechanism are still unclear. Non-coding RNA is a kind of RNA that exists widely and cannot be translated into proteins, which has played a key role in the regulation of biological functions of tumor cells. However, the regulation mechanism of non-coding RNA on bladder tumors is not fully understood. By microarray analysis and database analysis, we found that LINC00511 was significantly highly expressed in bladder cancer. The expressions of LINC00511, miR-143-3p, and PCMT in bladder cancer tissues and cells were detected by quantitative reverse transcription-polymerase chain reaction. The relationship between the expressions of miR-143-3p and PCMT1 and the clinicopathological parameters of the tumor was analyzed. The proliferation and invasion of bladder cancer cells were detected by MTT assay and Transwell assay. The expression levels of E-cadherin and vimentin in bladder cancer cells were detected by Western blot. Cell apoptosis was detected by flow cytometry. In vivo, TCCSUP or SW780 cells were inoculated into BALB/c nude mice to detect tumor volume and weight. Bioinformatics and dual luciferase reporter gene were used to analyze the relationship between LINC00511 and miR-143-3p and its downstream target gene PCMT1. The results showed that LINC00511 could target miR-143-3p/PCMT1 to regulate the proliferation, migration, and apoptosis of bladder cancer TCCSUP or SW780 cells and promote the occurrence and development of bladder cancer.

Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

PCMT1 is an unfavorable predictor and functions as an oncogene in bladder cancer.

The role of protein l-isoaspartate (d-aspartate) O-methyltransferase (PCMT1) in human cancer was generally cognized. The clinical significance and biological function of PCMT1 in bladder cancer is still unknown. PCMT1 mRNA and protein expression levels in bladder cancer tissues and cell lines were detected by qRT-PCR, immunohistochemistry, or western blot. The correlation between PCMT1 expression and clinicopathological factors was analyzed through immunohistochemistry in 108 bladder cancer patients. Loss-of-function and gain-of-function studies were conducted to explore the biological function of PCMT1 in bladder cancer cell lines in regulating cell proliferation, migration, and invasion. In our results, we found that PCMT1 was overexpressed in bladder cancer tissues compared with normal urothelium tissues in microarray datasets (GSE3167). Then, we confirmed PCMT1 mRNA and protein expression were increased in bladder cancer tissues and cell lines compared with paired normal urothelium tissues and normal uroepithelial cell line. PCMT1 protein expression was obviously correlated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis. Survival analysis showed that PCMT1 protein high-expression was an independent unfavorable prognostic factor for bladder cancer patients. The in vitro experiments showed PCMT1 regulated bladder cancer cells migration and invasion through modulating epithelial-mesenchymal transition (EMT)-associated genes expression including E-cadherin, vimentin, Snail and Slug, but had no effect on proliferation. In conclusion, PCMT1 is an unfavorable prognostic biomarker and involves in cells migration and invasion through regulating EMT-associated genes. © 2018 IUBMB Life, 70(4):291-299, 2018.

PCMT1 Ameliorates Neuronal Apoptosis by Inhibiting the Activation of MST1 after Subarachnoid Hemorrhage in Rats.

Mammalian sterile 20-like kinase 1 (MST1) is found to promote neuronal apoptosis. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1), an anti-apoptosis factor, was recently identified as an MST1-interacting protein. This study aims to explore the potential role of PCMT1 in reducing MST1-induced neuronal apoptosis after subarachnoid hemorrhage (SAH) in rats. One hundred ninety-eight male Sprague-Dawley rats were used. An exogenous PCMT1 agonist, CGP 3466B, was injected subcutaneously 1 h after the SAH induced by endovascular perforation. Chelerythrine or calyculin A was given immediately via intracerebroventricular administration after SAH. The SAH grade, Garcia score, and brain water content were measured at 24 and 72 h after the SAH. Neuronal apoptosis was detected by an immunofluorescent assay. The expression levels of endogenous PCMT1, MST1, phospho-MST1 (p-MST1), cleaved MST1 (cl-MST1), and apoptosis-related proteins were studied by western blotting. The expression of PCMT1 and MST1 decreased, while the level of active caspase 3 increased in rats after SAH. CGP 3466B treatment improved neurobehavioral function, reduced brain water content, inhibited the activity of MST1, and relieved neuronal apoptosis. These neuroprotective effects were significantly weakened either through accelerating MST1 phosphorylation by calyculin A or increasing cl-MST1 by chelerythrine. PCMT1 inhibited neuronal apoptosis by reducing MST1 phosphorylation and the level of cl-MST1. PCMT1/MST1 pathway might be an alternative therapeutic target for alleviating early brain injury after SAH.

Dopamine down-regulation of protein L-isoaspartyl methyltransferase is dependent on reactive oxygen species in SH-SY5Y cells.

Parkinson's disease (PD) is a chronic and progressive neurological disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra. Dopamine, via the oxidative stress that it generates in the cytosol, could contribute to the selective loss of neurons observed in PD. Protein L-isoaspartyl methyltransferase (PIMT) is an enzyme that repairs L-isoaspartyl-containing proteins and possesses anti-apoptotic properties. PIMT expression has been shown to decrease with age. Together, these observations prompted us to investigate whether dopamine can regulate PIMT expression in SH-SY5Y neuroblastoma cells. Here, we report that dopamine down-regulated PIMT at both gene and protein levels. The same inhibition of PIMT protein level was caused by the electron transport chain inhibitor, rotenone, which was accompanied, in both cases, by an increase in cell death and reactive oxygen species (ROS) production. In fact, pre-treatment with the antioxidant N-acetyl cysteine blocked PIMT dopamine-associated down-regulation. PCMT1 promoter mapping experiments allowed the identification of two regions that showed different sensitivity to DA action. A first region localized between 61 and 94bp upstream of transcription start site was very sensitive to dopamine inhibition while a second region between 41 and 61bp appeared more resistant to dopamine inhibitory effect. The inhibition of PCMT1 promoter activity was mediated by dopamine-induced ROS since it was prevented by the hydroxyl radical scavenger N,N'-dimethylthiourea. Conversely, H2O2 inhibited in a dose-dependent manner the transcriptional activity of PCMT1 promoter. Therefore, our findings identified new molecular mechanisms, cytosolic dopamine and its resulting ROS, as inhibitors of PIMT expression. This suggests that ROS generated from cytosolic dopamine could reduce both the PCMT1 gene promoter activity and the PIMT protein level thus decreasing its capacity to repair proteins involved in apoptosis and could contribute to neuronal cell death observed in PD.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase protects cardiomyocytes against hypoxia induced apoptosis through inhibiting proapoptotic kinase Mst1.

BACKGROUND: Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known. METHODS AND RESULTS: To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis. CONCLUSIONS: These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.