Post-translational Protein Stability Research Articles

S100A1-SUMO interaction via SUMO interaction motif (SIM) of the S100A1 C-terminus domain is critical for S100A1 post-translational protein stability

S100A1-SUMO interaction via SUMO interaction motif (SIM) of the S100A1 C-terminus domain is critical for S100A1 post-translational protein stability

Berberine enhances posttranslational protein stability of p21/cip1 in breast cancer cells via down-regulation of Akt.

Berberine has shown anticancer properties and has potential for a chemopreventive and/or chemotherapeutic agent for breast cancer. Berberine showed cytotoxicity to breast cancer cells, with an increase in the levels of p21/cip1 and p27/kip1, cyclin-dependent kinase inhibitors (CDKI), but mechanisms involved in up-regulating these molecules are largely unknown. Herein, we studied the key regulatory mechanisms involved in berberine-mediated up-regulation of p21/cip1 and p27/kip1. Berberine treatment for 24 and 48h decreased the number of cells by 44-84% (P < 0.0001) and 38-78% (P < 0.0001), and increased cell death by 12-17% (P < 0.005) and 38-78% (P < 0.0001) in MCF-7 and MDA-MB-231 cells, respectively. Cells were arrested in G1 phase by berberine which was accompanied with up-regulation of mRNA and protein level of both p21/cip1 and p27/kip1. Berberine decreased the expression of protein levels of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 to cause G1 phase arrest. Berberine caused nuclear localization of p21/cip1 in both the cell lines. Our data for the first time showed that the post-translational stability of both the proteins was strongly increased by berberine as examined by cycloheximide chase assay. Inhibition of Akt was associated with berberine-mediated up-regulation of p21/cip1 and also led to a decrease in cell viability accompanied with significant G1 phase cell cycle arrest. Our study revealed that berberine not only up-regulates mRNA and protein levels of p21/cip1 and p27/kip1 but also increases their nuclear localization and post-translational protein stability. Further, Akt inhibition was found to mediate berberine-mediated up-regulation of p21/cip1 but not the p27/kip1.

Abstract SY38-03: Targeting post-translational activation of MYC for the treatment of cancer

Abstract The MYC oncoprotein is considered to be one of the most important targets in cancer; however, successful targeting of MYC has not been achieved to date. MYC is a transcription factor, which binds to DNA in target genes as a heterodimer with its partner protein, MAX. While MAX is constitutively expressed, MYC protein expression is tightly regulated at multiple levels, including gene transcription, mRNA stability, protein translation, and post-translational protein stability. In cancer, MYC expression is upregulated through deregulation at many of these levels, resulting in an increase in MYC:MAX heterodimers and MYC-regulated gene expression. MYC regulated genes are involved in nearly all cellular processes, including cell intrinsic properties such as cell cycle control, cell growth and metabolism, self-renewal, migration and invasion, as well as cell extrinsic programs including angiogenesis, stromal cell expansion, and alterations in immune cell surveillance. MYC is thus a master regulator of cellular phenotype, and its deregulation contributes to tumorigenesis through many mechanisms. Importantly, elegant studies have recently demonstrated the tractability of targeting MYC for cancer therapeutics. Specifically, loss of MYC in adult tissues caused very little toxicity and metronomic expression of a MYC dominant negative protein caused no observable toxicity, but eliminated KRAS-driven pancreatic and lung tumors. Since targeting MYC directly has proven difficult, we are developing new strategies to target the post-translational activation of MYC. MYC is both stabilized and transcriptionally activated following cell stimulation via receptor tyrosine kinase signaling pathways leading to ERK mediated phosphorylation of Serine 62. We have demonstrated that Serine 62 phosphorylated MYC is upregulated in human cancer cells and that this facilitates its regulation of pro-oncogenic target genes by increasing its DNA binding activity and co-activator recruitment, as well as increasing its protein stability. We have identified Protein Phosphatase 2A (PP2A) as the enzyme responsible for dephosphorylating Serine 62. PP2A is a critical tumor suppressor that is known to inactivate multiple oncogenic signaling pathways, as well as cell cycle drivers and survival factors. Furthermore, inactivation of PP2A has been shown to be critical for the transformation of human cells, and PP2A activity is suppressed in most tested human tumors. We are utilizing a recently developed small molecule, orally available, allosteric PP2A activator drug. This drug (DTx) is in clinical development by Dual Therapeutics. We have tested this drug, as well as a peptide mimetic PP2A activator, OP449, in cell lines and in mouse models of tumorigenesis. We have observed cytotoxic activity in both breast and pancreas cancer cell lines associated with dephosphorylation of MYC at Serine 62. We have also observed reduced MYC DNA target gene binding and target gene expression with PP2A activation therapy. In order to test novel therapeutic strategies targeting MYC activity, we have developed new mouse models of Myc-driven tumorigenesis using our ROSA26-LSL-Myc mice that we generated to conditionally express transcriptionally deregulated, physiological levels of Myc in response to Cre recombinase. By crossing these mice with mice expressing other organ specific oncogenic drivers, we have developed mouse mammary tumor models representing HER2+ and Triple Negative breast cancer. We have also developed a novel mouse model of pancreatic tumorigenesis using the same strategy. All of these models appear to molecularly recapitulate the corresponding human disease, and we are using them as pre-clinical testing platforms for our PP2A activation therapy trials. So far, we have seen dramatic tumor growth inhibition and even tumor shrinkage with DTx treatment in all three of these models. This is associated with loss of Serine 62 phosphorylation in the treated tumors. Importantly, we see no signs of toxicity in vivo with this novel compound and pharmacokinetic studies look very promising for bringing this compound to the clinic. In summary, we believe that targeting pathways that post-translationally activate MYC's oncogenic potential is a promising strategy for targeting this important oncoprotein. Citation Format: Xiaoyan Wang, Amy Farrell, Mahnaz Janghorban, Brittany Allen-Petersen, Juan Liang, Tyler Risom, Michael Ohlmeyer, Goutham Narla, Rosalie C. Sears. Targeting post-translational activation of MYC for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY38-03. doi:10.1158/1538-7445.AM2015-SY38-03

A dual luciferase system for analysis of post-transcriptional regulation of gene expression in Leishmania

Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.

Preferential Retention of Circadian Clock Genes during Diploidization following Whole Genome Triplication in Brassica rapa

Much has been learned about the architecture and function of the circadian clock of Arabidopsis thaliana, a model for plant circadian rhythms. Circadian rhythms contribute to evolutionary fitness, suggesting that circadian rhythmicity may also contribute to agricultural productivity. Therefore, we extend our study of the plant circadian clock to Brassica rapa, an agricultural crop. Since its separation from Arabidopsis, B. rapa has undergone whole genome triplication and subsequent diploidization that has involved considerable gene loss. We find that circadian clock genes are preferentially retained relative to comparison groups of their neighboring genes, a set of randomly chosen genes, and a set of housekeeping genes broadly conserved in eukaryotes. The preferential retention of clock genes is consistent with the gene dosage hypothesis, which predicts preferential retention of highly networked or dose-sensitive genes. Two gene families encoding transcription factors that play important roles in the plant core oscillator--the PSEUDO-RESPONSE REGULATORS, including TIMING OF CAB EXPRESSION1, and the REVEILLE family, including CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL--exhibit preferential retention consistent with the gene dosage hypothesis, but a third gene family, including ZEITLUPE, that encodes F-Box proteins that regulate posttranslational protein stability offers an exception.

Centrosomal Protein 55 (Cep55) Stability Is Negatively Regulated by p53 Protein through Polo-like Kinase 1 (Plk1)

Centrosomal protein 55 (Cep55), which is localized to the centrosome in interphase cells and recruited to the midbody during cytokinesis, is a regulator required for the completion of cell abscission. Up-regulation of Cep55 and inactivation of p53 occur in the majority of human cancers, raising the possibility of a link between these two genes. In this study we evaluated the role of p53 in Cep55 regulation. We demonstrated that Cep55 expression levels are well correlated with cancer cell growth rate and that p53 is able to negatively regulate Cep55 protein and promoter activity. Down-regulation of expression of Cep55 was accompanied by repression of polo-like kinase 1 (Plk1) levels due to p53 induction. Overexpression of Plk1 and knockdown of p53 expression both enhanced the post-translational protein stability of Cep55. BI 2356, a selective Plk1 inhibitor, however, prevented Cep55 accumulation in p53 knockdown cells while persistently keeping Plk1 levels elevated. Our results, therefore, indicate the existence of a p53-Plk1-Cep55 axis in which p53 negatively regulates expression of Cep55, through Plk1 which, in turn, is a positive regulator of Cep55 protein stability.

U-box-type ubiquitin E4 ligase, UFD2a attenuates cisplatin mediated degradation of ΔNp63α

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, including cisplatin. But little is understood about the post-translational protein stability of ΔNp63α. In this present study we demonstrate for the first time that ΔNp63α physically interacts with U-box-type E4 ubiquitin ligase UFD2a. UFD2a stabilizes ΔNp63α, and ubiquitylation of ΔNp63α is attenuated by UFD2a both in the presence and absence of cisplatin. Ectopic expression of UFD2a increased the half-life of ΔNp63α in association with a significant enhancement of the repressive transcriptional activity of ΔNp63α. Downregulation of endogenous UFD2a by RNAi resulted in degradation of ΔNp63α. Taken together, our current study provides an insight onto the regulation of ΔNp63α protein levels in response to cisplatin and also suggests that UFD2a might play an important role in the regulation of cisplatin mediated cell death mediated by p63.

The oncogenic fusion protein Pax3–FKHR has a greater post-translational stability relative to Pax3 during early myogenesis

The childhood solid muscle tumor Alveolar Rhabdomyosarcoma (ARMS) is characterized by the t(2;13)(q35;q14) chromosomal translocation, which results in the fusion of two transcription factors important for myogenesis, Pax3 and FKHR (FOX01a). The effects of myogenic differentiation on the stability of FKHR have been well characterized. However, similar studies have yet to be performed on Pax3 or the oncogenic fusion protein Pax3–FKHR. Therefore, we demonstrate in the physiologically relevant mouse primary myoblast system that the expression of Pax3 decreases nearly 95% during the first 24 h of myogenic differentiation. In contrast, there is an aberrant persistence of expression of Pax3–FKHR during this same time period. These differences in protein expression levels do not result from changes on the transcriptional nor the translational level since we observed no concomitant decrease in the levels of Pax3 or Pax3–FKHR mRNA or in the ability of both proteins to be translated. Instead, a pulse-chase analysis determined that Pax3–FKHR has a half-life significantly greater than\\ the half-life of wild type Pax3 demonstrating for the first time that Pax3–FKHR has greater post-translational protein stability relative to wild type Pax3 during early myogenic differentiation. Finally, the persistence of expression of Pax3–FKHR prevents the terminal differentiation of primary myoblasts demonstrating a biological consequence of its aberrant expression.