Beyond Protein Synthesis; The Multifaceted Roles of Tuberin in Cell Cycle Regulation

The ability of cells to sense diverse environmental signals, including nutrient availability and conditions of stress, is critical for both prokaryotes and eukaryotes to mount an appropriate physiological response. While there is a great deal known about the different biochemical pathways that can detect and relay information from the environment, how these signals are integrated to control progression through the cell cycle is still an expanding area of research. Over the past three decades the proteins Tuberin, Hamartin and TBC1D7 have emerged as a large protein complex called the Tuberous Sclerosis Complex. This complex can integrate a wide variety of environmental signals to control a host of cell biology events including protein synthesis, cell cycle, protein transport, cell adhesion, autophagy, and cell growth. Worldwide efforts have revealed many molecular pathways which alter Tuberin post-translationally to convey messages to these important pathways, with most of the focus being on the regulation over protein synthesis. Herein we review the literature supporting that the Tuberous Sclerosis Complex plays a critical role in integrating environmental signals with the core cell cycle machinery.

RNA-Binding Profiles of CKAP4 as an RNA-Binding Protein in Myocardial Tissues

Background: Pathological tissue remodeling such as fibrosis is developed in various cardiac diseases. As one of cardiac activated-myofibroblast protein markers, CKAP4 may be involved in this process and the mechanisms have not been explored.Methods: We assumed that CKAP4 held a role in the regulation of cardiac fibrotic remodeling as an RNA-binding protein. Using improved RNA immunoprecipitation and sequencing (iRIP-seq), we sought to analyze the RNAs bound by CKAP4 in normal atrial muscle (IP1 group) and remodeling fibrotic atrial muscle (IP2 group) from patients with cardiac valvular disease. Quantitative PCR and Western blotting were applied to identify CKAP4 mRNA and protein expression levels in human right atrium samples.Results: iRIP-seq was successfully performed, CKAP4-bound RNAs were characterized. By statistically analyzing the distribution of binding peaks in various regions on the reference human genome, we found that the reads of IP samples were mainly distributed in the intergenic and intron regions implying that CKAP4 is more inclined to combine non-coding RNAs. There were 913 overlapping binding peaks between the IP1 and IP2 groups. The top five binding motifs were obtained by HOMER, in which GGGAU was the binding sequence that appeared simultaneously in both IP groups. Binding peak-related gene cluster enrichment analysis demonstrated these genes were mainly involved in biological processes such as signal transduction, protein phosphorylation, axonal guidance, and cell connection. The signal pathways ranking most varied in the IP2 group compared to the IP1 group were relating to mitotic cell cycle, protein ubiquitination and nerve growth factor receptors. More impressively, peak analysis revealed the lncRNA-binding features of CKAP4 in both IP groups. Furthermore, qPCR verified CKAP4 differentially bound lncRNAs including LINC00504, FLJ22447, RP11-326N17.2, and HELLPAR in remodeling myocardial tissues when compared with normal myocardial tissues. Finally, the expression of CKAP4 is down-regulated in human remodeling fibrotic atrium.Conclusions: We reveal certain RNA-binding features of CKAP4 suggesting a relevant role as an unconventional RNA-binding protein in cardiac remodeling process. Deeper structural and functional analysis will be helpful to enrich the regulatory network of cardiac remodeling and to identify potential therapeutic targets.

Non-histone methylation of SET7/9 and its biological functions

Background: (su(var)-3-9,enhancer-of-zeste,trithorax) domain-containing protein 7/9 (SET7/9) is a member of the protein lysine methyltransferases (PLMTs or PKMTs) family. It contains a SET domain. Recent studies demonstrate that SET7/9 methylates both lysine 4 of histone 3 (H3-K4) and lysine(s) of non-histone proteins, including transcription factors, tumor suppressors, and membrane-associated receptors. Objective: This article mainly reviews the non-histone methylation effects of SET7/9 and its functions in tumorigenesis and development. Methods: PubMed was screened for this information. Results: SET7/9 plays a key regulatory role in various biological processes such as cell proliferation, transcription regulation, cell cycle, protein stability, cardiac morphogenesis, and development. In addition, SET7/9 is involved in the pathogenesis of hair loss, breast cancer progression, human carotid plaque atherosclerosis, chronic kidney disease, diabetes, obesity, ovarian cancer, prostate cancer, hepatocellular carcinoma, and pulmonary fibrosis. Conclusion: SET7/9 is an important methyltransferase, which can catalyze the methylation of a variety of proteins. Its substrates are closely related to the occurrence and development of tumors.

Pharmacological activity and biochemical interaction of zingerone: a flavour additive in spice food

Zingerone (4-(4-Hydroxy-3-methoxyphenyl)-2-butanone) is one of the non-volatile and nontoxic compounds of ginger. It is also called vanillylacetone with a crystalline solid form which is sparingly soluble in water and more soluble in ether. The contribution of this compound in ginger is about 9.25%. The chemical structure is made of a phenolic ring with methoxy group attached to benzene ring. Gingerol can be heated to form zingerone by retroaldol reaction. It has been reported that zingerone has multiple pharmacological activities. It is effective against diarrhoea causing enterotoxigenic bacteria that leads to infant death. It is also used against intestinal gastric, oxidative stress, weak immunity, obesity. During its activity against cancer, it governs the expression of different cell cycle protein and TGF-?1 expression. Antioxidant response is controlled by inducing the activity of ROS neutralising enzymes like superoxide dismutase, catalase and glutathione reductase. It can also reduce various inflammations by restricting the activity of interleukins. This review summarizes the multiple pharmacology activities of zingerone against various important diseases like cancers, tumors, inflammations, oxidative conditions, microbial infections, biofilm formations, thrombosis and other diseases. In addition, the molecular regulation of these pharmacological responses by zingerone is also critically discussed.

PM2.5 Upregulates the Expression of MUC5AC via the EGFR-PI3K Pathway in Human Sinonasal Epithelial Cells

<b><i>Background:</i></b> Fine particulate matter (PM) (PM with an aerodynamic diameter &#x3c;2.5 μm, PM2.5) exposure contributes to respiratory disease development and exacerbation. <b><i>Objective:</i></b> We sought to investigate the effect of PM2.5 exposure on mucociliary function in primary human nasal epithelial cells (HNECs) and the underlying mechanism. <b><i>Methods:</i></b> HNECs derived from control subjects and patients with chronic rhinosinusitis with nasal polyps were established as air-liquid interface cultures. Confluent cultures were exposed to 100 or 200 μg/mL PM2.5 for 24 h and assessed for expression of specific mucociliary-associated factors, the percentage of β-tubulin IV-positive and MUC5AC-positive cells, expression of epidermal growth factor receptor (EGFR) ligand and activation of phosphoinositide 3-kinase (PI3K)-AKT/ERK. In addition, cultures pretreated for 30 min with AG1478 (an EGFR inhibitor) or LY294002 (a PI3K inhibitor) following PM2.5 exposure were assessed for MUC5AC mRNA and protein expression. <b><i>Results:</i></b> PM2.5 exposure at 100 or 200 μg/mL for 24 h did not affect geminin coiled-coil domain containing, multiciliate differentiation and DNA synthesis associated cell cycle protein, FOXJ1, or DNAI2 mRNA expression or the percentage of β-tubulin IV-positive cells. However, 200 μg/mL PM2.5 exposure significantly increased mRNA expression of SAM-pointed domain-containing ETS transcription factor and MUC5AC and the percentage of MUC5AC-positive cells. PM2.5 also increased expression of EGFR ligands, including heparin-binding EGF-like growth factor and amphiregulin. Furthermore, PM2.5induced activation of PI3K, AKT, and ERK, and pretreatment of HNECs with AG1478 or LY294002 attenuated PM2.5-induced MUC5AC mRNA and protein expression. <b><i>Conclusions and Clinical Relevance:</i></b> This study demonstrates that short-term PM2.5 exposure increases MUC5AC expression in HNECs. Furthermore, this study shows that PM2.5-induced MUC5AC expression is likely mediated through the EGFR-PI3K pathway.

CSIG-28. REGULATION OF GEMC1 MULTICILIOGENESIS PROGRAM IN CHOROID PLEXUS CARCINOMA

Tumors of the choroid plexus (CP) are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP is more benign, CPC is a highly lethal and little understood cancer with poor survival rate and a tendency for recurrence and metastasis. CP tumors are thought to arise from CP epithelial cells that secrets cerebral spinal fluid and generate multiple cilia on their apical surface. Here we show that aberrant NOTCH and Sonic Hedgehog signaling in mice drive tumors that resemble CPC in humans. In contrast to CP epithelial cells with clusters of multiple cilia, NOTCH-driven CP tumors were monociliated, and disruption of the NOTCH complex restored multiciliation and decreased tumor growth. NOTCH suppressed multiciliation in tumor cells by inhibiting the expression of Geminin Coiled-Coil Domain Containing 1 (GEMC1), and multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS), early transcriptional regulators of multiciliated cell (MCC) differentiation. Consistently, Gemc1-Mcidas deficiency led to a lack of MCCs in the CP, and impaired the correction of the multiciliation defect in tumor cells by a NOTCH inhibitor. Disturbances to the GEMC1 program are commonly observed in human CPCs characterized by solitary cilia and frequent somatic TP53 mutations. Accordingly, CPC driven by deletion of tumor suppressors Trp53 and Rb1 in mice exhibits a cilia deficit consequent to loss of Gemc1-Mcidas expression. Taken together, these findings reveal that the GEMC1-MCIDAS multiciliogenesis program in the CP is critical for inhibiting tumorigenesis, whereas a defective multiciliation program promotes CPC and may represent a therapeutic avenue for this cancer.

Deficiency of ZC3HC1 increases vascular smooth muscle cell migration, proliferation and neointima formation following injury

The ZC3HC1 gene is associated with various cardiovascular traits in that its common missense variant, rs11556924-T (p.Arg363His), lowers risk of coronary artery disease (CAD) and blood pressure, but increases carotid intima-media thickness (IMT). This study was designed to determine the mechanisms by which ZC3HC1 modulates IMT using in vitro and in vivo models.We assessed the effect of the rs11556924-T allele on ZC3HC1 expression in vascular smooth muscle cells (SMCs) from 151 multi-ethnic heart transplant donors and found that rs11556924-T was significantly associated with lower ZC3HC1 expression and faster SMC migration. These results were supported by in vitro silencing experiments. At the protein level, ZC3HC1 deficiency resulted in the accumulation of cyclin B1, a key cell cycle protein. Further, transcriptome analysis revealed changes in the regulation of canonical SMC marker genes, including ACTA2, CNN1, LMOD1, and TAGLN. Pathway analysis of differentially expressed genes in SMCs secondary to ZC3HC1 knockdown showed decreased expression of genes in the cell division and cytoskeleton organization pathways.In line, primary SMCs isolated from the aortas of Zc3hc1-/- mice migrated faster and proliferated more compared to SMCs isolated from wild-type littermates, with the former also showing accumulation of cyclin B1. Neointima formation was also enhanced in Zc3hc1-/- mice in response to arterial injury mimicking restenosis.Taken together, these findings demonstrate that genetic modulation or deficiency of ZC3HC1 leads to the accumulation of cyclin B1 in SMCs and increased migration, proliferation, and injury-induced neointima formation. We further discuss and propose that a genetic variant regulating SMC proliferation may enhance IMT and early atherosclerosis progression but may be beneficial for plaque stability in advanced lesions.

Estrogen Receptor Variant ER-α36 Facilitates Estrogen Signaling via EGFR Signaling in Glioblastoma

Background: Glioblastoma (GBM) is a deadly and common primary brain tumor. Poor prognosis is linked to high proliferation and cell heterogenity. Sex differences may play a role in patient outcome. Previous studies showed that ER-α36, a variant of the estrogen receptor, mediated non-genomic estrogen signaling and is highly expressed in many estrogen receptors (ER)-negative malignant tumors. ER-α36 also associates with the epidermal growth factor receptor (EGFR).Aim: The primary purpose of this study is to investigate the cross-talk between ER-α36 and EGFR in estrogen (E2) induced glioblastoma cell proliferation.Methods: The expression of estrogen receptors were tested by immunofluorescence in the specimens. The expression of ER-α36, EGFR and cell cycle protein were measured by qPCR and Western blot. The numbers of cells were tested through cell counting, and cell signaling pathway activation also determined by Western blot.Results: Here, we showed that ER-α36 was highly expressed and confirmed that ER-α36 co-labels with EGFR in human glioma specimens using immunohistochemical techniques. We also investigated the mechanisms of estrogen induced proliferation in ER-a-negative cell lines U87 and U251. We found that glioblastoma cell lines U87 and U251 showed varying responsive to mitogenic estrogen signaling which correlated with ER-α36 expression, and knockdown of ER-α36 diminished the response. Exposure to estrogen also caused up-regulation of cyclin protein expression in vitro. We also found that low concentrations of estrogen promoted SRC-Y-416 and inhibited SRC-Y-527 phosphorylation, corresponding with activated SRC signaling. Inhibiting SRC or EGFR abolished estrogen-induced mitogenic signaling, including cyclin expression and MAPK phosphorylation.Conclusion: Cumulatively, our results demonstrate that ER-α36 promotes non-genomic estrogen signaling via the EGFR/SRC/MAPK pathway in glioblastoma. This may be important for the treatment of ER-a-negative glioblastomas that retain high level of ER-α36, since estrogen may be a viable therapeutic target for these patients.

Novel roles of small extracellular vesicles in regulating the quiescence and proliferation of neural stem cells

Neural stem cells (NSCs) quiescence plays pivotal roles in securing sustainable neurogenesis and avoiding stemness exhaustion in the adult brain. The maintenance of quiescence and transition between proliferation and quiescence are complex processes associated with multiple niche signals, and environmental stimuli. Though the mechanisms of the transitions between NSC states have been extensively investigated, they remain to be fully elucidated. Exosomes are small extracellular vesicles (sEVs) containing functional units such as proteins, microRNAs, and mRNAs. It has already been demonstrated that sEVs actively participate in cancer cell proliferation and metastasis. However, the role of sEVs in NSC quiescence has not been investigated. Here, we applied proteomics to analyze the protein cargos of sEVs derived from proliferating, quiescent, and reactivating NSCs. Our findings revealed expression level fluctuations of NSCs sEV protein cargo at different proliferative conditions. We also identified functional clusters of gene ontology annotations from differentially expressed proteins in three sources of exosomes. Moreover, the use of exosome inhibitors revealed the contribution of exosomes to NSC quiescence at the entrance into quiescence, as well as in quiescence maintenance. Exosome inhibition delayed the entrance into quiescence by proliferating NSCs and allowed quiescent NSCs to exit from the G0 phase of the cell cycle. Protein translation was significantly upregulated in both quiescent NSCs and quiescent-induced NSCs via the exosome inhibition. Our results demonstrated that NSC exosomes are involved in regulating the quiescence of NSCs and provide a functional prediction of NSCs exosome protein cargos in terms of cell-cycle regulation and protein synthesis.