FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.

TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

LC-MS/MS CHARACTERIZATION OF FORCED DEGRADATION PRODUCTS OF TUCATINIB, A NOVEL TYROSINE KINASE INHIBITOR: DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD

Objective: The current study focused on the development, validation, and characterization of forced degradation products using LC-MS/MS. Methods: A simple, selective, validated and well-defined isocratic HPLC methodology for the quantitative determination of Tucatinib at a wavelength of 239 nm. An isocratic elution of samples was performed on an Inertsil ODS (250x4.6 mm, 5m) column with a mobile phase of 70:30v/v Acetonitrile and formic acid (0.1%) delivered at a flow rate of 1.0 ml/min. MS/MS was used to characterize degradation products formed in the forced degradation study. The validation and characterization of forced degradation products were performed in accordance with ICH guidelines. Results: Over the concentration range of 5-100μg/ml, a good linear response was obtained. Tucatinib's LOD and LOQ were determined to be 0.05 and 0.5, respectively. According to standard guidelines, the method was quantitatively evaluated in terms of system suitability, linearity, precision, accuracy, and robustness, and the results were found to be within acceptable limits. The drug was degraded under acidic, alkaline, and reduction conditions in forced degradation studies. Conclusion: The method was found to be applicable for routine tucatinib analysis. Because no LC-MS/MS method for estimating tucatinib and its degradation products has been reported in the literature. There is a need to develop a method for studying the entire tucatinib degradation pathway.

Phenylalanine Suppresses Cell Death Caused by Loss of Fumarylacetoacetate Hydrolase in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up‑regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-β-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up‑regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.