The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.     

Transcriptional Regulation of Programmed Hypertension by Melatonin: An Epigenetic Perspective

Melatonin is an endogenously produced indoleamine and secreted by the pineal gland. Melatonin has pleiotropic bioactivities and is involved in epigenetic regulation. Suboptimal conditions during maternal and perinatal phases can elicit epigenetic regulation of genes for nephrogenesis and reset physiological responses to develop programmed hypertension. This review discusses the early utility of melatonin to prevent programmed hypertension in later life by epigenetic regulation in the kidney, with an emphasis on: (1) the role of melatonin in epigenetic regulation; (2) the beneficial effects of melatonin on programmed hypertension; (3) epigenetic regulation of maternal melatonin therapy in different developmental windows of offspring kidneys analyzed by whole-genome RNA next-generation sequencing; and (4) current blocks in the application of melatonin in preventing programmed hypertension.     

Epigenetic Deregulation of Protein Tyrosine Kinase 6 Promotes Carcinogenesis of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and causes considerable morbidity and mortality. Epigenetic deregulation is a common mechanism underlying carcinogenesis. DNA methylation deregulation is the epigenetic change observed during the transformation of normal cells to precancerous and eventually cancer cells. This study investigated the DNA methylation patterns of PTK6 during the development of OSCC. Bisulfite genomic DNA sequencing was performed to determine the PTK6 methylation level. OSCC animal models were established to examine changes in PTK6 expression in the different stages of OSCC development. The DNA methylation of PTK6 was decreased during the development of OSCC. The mRNA and protein expression of PTK6 was increased in OSCC cell lines compared with human normal oral keratinocytes. In mice, the methylation level of PTK6 decreased after treatment with 4-nitroquinoline 1-oxide and arecoline, and the mRNA and protein expression of PTK6 was increased. PTK6 hypomethylation can be a diagnostic marker of OSCC. Upregulation of PTK6 promoted the proliferation, migration, and invasion of OSCC cells. PTK6 promoted carcinogenesis and metastasis by increasing STAT3 phosphorylation and ZEB1 expression. The epigenetic deregulation of PTK6 can serve as a biomarker for the early detection of OSCC and as a treatment target.     

UBL5/Hub1: An Atypical Ubiquitin-Like Protein with a Typical Role as a Stress-Responsive Regulator

Members of the ubiquitin-like protein family are known for their ability to modify substrates by covalent conjugation. The highly conserved ubiquitin relative UBL5/Hub1, however, is atypical because it lacks a carboxy-terminal di-glycine motif required for conjugation, and the whole E1-E2-E3 enzyme cascade is likely absent. Though the conjugation-mediated role of UBL5/Hub1 is controversial, it undoubtedly functions by interacting non-covalently with its partners. Several interactors of UBL5/Hub1 identified to date have suggested broad stress-responsive functions of the protein, for example, stress-induced control of pre-mRNA splicing, Fanconi anemia pathway of DNA damage repair, and mitochondrial unfolded protein response. While having an atypical mode of function, UBL5/Hub1 is still a stress protein that regulates feedback to various stimuli in a similar manner to other ubiquitin-like proteins. In this review, I discuss recent progress in understanding the functions of UBL5/Hub1 and the fundamental questions which remain to be answered.     

Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3'',4,4'',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2''-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.     

Zona Pellucida like Domain Protein 1 (ZPLD1) Polymerization Is Regulated by Two Distinguished Hydrophobic Motifs

Zona Pellucida Like Domain 1 Protein (ZPLD1) is a main component of the cupula, a gelatinous structure located in the labyrinth organ of the inner ear and involved in vestibular function. The N-glycosylated protein is likely able to organize high-molecular-weight polymers via its zona pellucida (ZP) module, which is common for many extracellular proteins that self-assemble into matrices. In this work, we confirmed that ZPLD1 can form multimers while setting up a cellular model leveraging Madin–Darby canine kidney (MDCK) cells to study protein polymerization. We identified two motifs within ZPLD1 which regulate its polymerization and follow previously published conserved regions, identified across ZP proteins. Mutational depletion of either one of these modules led to diminished or abnormal polymer formation outside of the cells, likely due to altered processing at the plasma membrane. Further, intracellular polymer formation was observed. Proteolytic cleavage during secretion, separating the regulatory motif located distinct of the ZP module from the mature monomer, seems to be necessary to enable polymerization. While the molecular interactions of the identified motifs remain to be proven, our findings suggest that ZPLD1 is a polymer forming ZP protein following an orchestrated mechanism of protein polymerization to finally build up a gelatinous hydrogel.     

Zebrafish Cyclin-Dependent Protein Kinase-Like 1 (zcdkl1): Identification and Functional Characterization

The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.     

TaNBR1, a Novel Wheat NBR1-like Domain Gene Negatively Regulates Drought Stress Tolerance in Transgenic Arabidopsis

Drought stress is an important factor that severely affects crop yield and quality. Autophagy has a crucial role in the responses to abiotic stresses. In this study, we explore TaNBR1 in response to drought stress. Expression of the TaNBR1 gene was strongly induced by NaCl, PEG, and abscisic acid treatments. The TaNBR1 protein is localized in the Golgi apparatus and autophagosome. Transgenic Arabidopsis plants overexpressing TaNBR1 exhibited reduced drought tolerance. When subjected to drought stress, compared to the wild-type (WT) lines, the transgenic overexpressing TaNBR1 plants had a lower seed germination rate, relative water content, proline content, and reduced accumulation of antioxidant enzymes, i.e., superoxide dismutase, peroxidase, and catalase, as well as higher chlorophyll losses, malondialdehyde contents, and water loss. The transgenic plants overexpressing TaNBR1 produced much shorter roots in response to mannitol stress, in comparison to the WT plants, and they exhibited greater sensitivity to abscisic acid treatment. The expression levels of the genes related to stress in the transgenic plants were affected in response to drought stress. Our results indicate that TaNBR1 negatively regulates drought stress responses by affecting the expression of stress-related genes in Arabidopsis.     

蛋白酪氨酸磷酸酶1B(PTP1B)抑制剂的研究进展

2型糖尿病是一种代谢综合症,其特点是胰岛素抵抗、高胰岛素血症、高血糖。研究表明,蛋白酪氨酸磷酸酶1B(PTP1B)是胰岛素转导信号的负调节因子,已经是治疗2型糖尿病和肥胖症的一个新的靶点。PTP1B抑制剂能够有效地治疗2型糖尿病和肥胖症。本文综述了近年来PTP1B抑制剂的研究进展。     

应用毕赤酵母表达中国株HIV-1核心蛋白Gag

目的 构建含HIV-1Gag全长基因的重组酵母表达质粒pPICGAG，并在毕赤酵母中进行表达。方法用Not I和XhoI将含HIV－1 Gag全长基因的质粒pKSGAG双酶切后，克隆到酵母表达载体pPIC9中，构建了重组表达质粒 pPICGAG。pPICGAG用 SacI线性化后，电转化毕赤酵母 GS115，PCR鉴定阳性酵母转化子，并分别将PCR阳性的酵母转化子在BMGY和BMMY培养基中进行诱导表达，表达产物进行SDS-PAGE电泳分析。结果 酵母转化子的整合率为72．7％。SDS-PAGE结果显示表达蛋白的相对分子质量为55000左右，与预计计算的值相同。Western blot结果显示表达蛋白能与单克隆抗体发生特异性反应。结论 在毕赤酵母中成功地表达了HIV-1核心蛋白Gag，且表达的蛋白具有良好的反应原性和特异性。     

Towards Dissecting the Mechanism of Protein Phosphatase-1 Inhibition by Its C-Terminal Phosphorylation

Phosphoprotein phosphatase-1 (PP1) is a key player in the regulation of phospho-serine (pSer) and phospho-threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well-established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C-terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell-cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.