ABSTRACT
Mutations of the X-linked methyl-CpG-binding protein 2 (MECP2) gene in humans are responsible for most cases of Rett syndrome (RTT), an X-linked progressive neurological disorder. While genome-wide screens in clinical trials have revealed several putative RTT-associated mutations in MECP2, their causal relevance regarding the functional regulation of MeCP2 at the etiologic sites at the protein level requires more evidence. In this study, we demonstrated that MeCP2 was dynamically modified by O-linked-β-N-acetylglucosamine (O-GlcNAc) at threonine 203 (T203), an etiologic site in RTT patients. Disruption of the O-GlcNAcylation of MeCP2 specifically at T203 impaired dendrite development and spine maturation in cultured hippocampal neurons, and disrupted neuronal migration, dendritic spine morphogenesis, and caused dysfunction of synaptic transmission in the developing and juvenile mouse cerebral cortex. Mechanistically, genetic disruption of O-GlcNAcylation at T203 on MeCP2 decreased the neuronal activity-induced induction of Bdnf transcription. Our study highlights the critical role of MeCP2 T203 O-GlcNAcylation in neural development and synaptic transmission potentially via brain-derived neurotrophic factor.
Subject(s)
Animals , Humans , Mice , Methyl-CpG-Binding Protein 2/metabolism , Neurodevelopmental Disorders/genetics , Rett Syndrome/genetics , Synaptic Transmission , ThreonineABSTRACT
@#The aim of this study was to investigate the effect of transmembrane 9 superfamily protein member 2 (TM9SF2) in proliferation and migration of triple negative breast cancer cell line MDA-MB-231.The expression of TM9SF2 in triple negative breast cancer cell line MDA-MB-231 and nontumorigenic mammary epithelial cell line MCF-10A were measured by Western blot. MDA-MB-231 cells were treated with siRNA-TM9SF2 to knock-down the expression of TM9SF2. The effect of silencing TM9SF2 was measured with Western blot.The proliferation of cells was tested by MTS,and the migration was measured with Transwell and wound-healing assay.Proteins related to proliferation (PI3K,AKT,SRC and ERK) and migration (Snail,Slug and N-cadherin) were measured with Western blot.Protein expressions of TM9SF2 was better improved in triple negative breast cancer MDA-MB-231 cell line than MCF-10A.Compared with the control group, the siRNA-TM9SF2 infected group had lower expressions of PI3K, Snail, Slug and N-cadherin, and at the same time phosphorylation of AKT was decreased. The results suggest TM9SF2 can promote the proliferation and metastasis of triple negative breast cancer MDA-MB-231 cell line.
ABSTRACT
Objective To establish a sensitive,accurate and reliable LC-MS method to quantify the monoclonal antibody in rat blood and preclinical pharmacokinetics study. Methods The method of selective reaction monitoring(SRM)was established by using on-the-fly orthogonal array optimization(OAO)with anti-chemotherapeutic antibody-06(ACA-06)as a model drug,and the pharmacokinetics of ACA-06 in rats was studied. The optimal signature peptide selection and mass spectrometry conditions were opti?mized by single LC-MS run. Two highly specific and sensitive peptides,FSGVPDR and VNSAAFPAPIEK,were identified as the sig?nature peptides of quantification based on the OAO optimization result,which were located in the ACA-06 light chain and heavy chain complementary determining region(CDR)respectively. Results The method had good linearity in the range of 12.9~32250 ng/ml (r20.9819-0.9946). The lower limit of quantification was 12.9 ng/ml. The intra-day precision(RSD)was less than 11.5%,the inter-day RSD was less than 6%,and the accuracy was between-4.0%and 4.3%. Conclusion In this study,LC/SRM-MS method is es?tablished for the determination of ACA-06 in rat plasma. The method is specific,accurate and sensitive and enables an accurate phar?macokinetics investigation of ACA-06 in rat plasma.