Partial regulation of serotonin transporter function by γ-synuclein

Human α-synuclein (α-Syn) is instrumental in maintaining homeostasis of monoamine neurotransmitters in brain, through its trafficking, and regulation of the cell surface expression and, thereby, activity of dopamine, serotonin and norepinephrine transporters. Here we have investigated whether other members of the synuclein family of proteins, γ-synuclein (γ-Syn) and β-synuclein (β-Syn) can similarly modulate the serotonin transporter (SERT). In Ltk⁻ cells co-transfected with SERT and γ-Syn, γ-Syn reduced [³H]5-HT uptake, in a manner dependent on its expression levels. The decrease in SERT activity was via decreased V_max of the transporter, without change in K_m, compared to cells expressing only SERT. By contrast, β-Syn co-expression failed to alter SERT uptake activity, and neither the V_max nor the K_m was changed in the presence of β-Syn. γ-Syn modulation of SERT was only partial, with a maximal ∼27% decrease in SERT activity seen even at high expression levels of γ-Syn. By contrast, α-Syn attenuated SERT activity by ∼65% at identical expression levels as γ-Syn. Co-immunoprecipitation studies showed the presence of heteromeric protein:protein complexes between γ-Syn or α-Syn and SERT, while β-Syn failed to physically interact with SERT. Both α-Syn and γ-Syn colocalized with SERT in rat primary raphae nuclei neurons. These studies document a novel physiological role for γ-Syn in regulating 5-HT synaptic availability and homeostasis, and may be of relevance in depression and mood disorders, where SERT function is dysregulated.     

大鼠脂肪细胞中14-3-3蛋白与葡萄糖转运子4的相互作用

目的研究在大鼠脂肪细胞中,14-3-3蛋白与葡萄糖转运子4(GLUT4)之间是否存在相互作用。方法用胶原酶I消化雄性SD大鼠附睾上的脂肪垫,获得分离的脂肪细胞。在纯化的细胞中,采用低渗裂解和甘油梯度速率沉降技术获得3个含有GLUT4的组分:T、H和L。用交联了1F8(GLUT4特异性单抗)的琼脂糖微珠对脂肪细胞总提取物以及上述3个组分分别进行免疫吸附实验,经吸附后在上清液和微珠的洗脱液中分别进行14-3-3蛋白和GLUT4的免疫印迹分析。结果在脂肪细胞的总提取物以及上述GLUT4的3个组分T、H和L中,14-3-3蛋白都能够与GLUT4发生免疫共沉淀。共沉淀下来的14-3-3蛋白在免疫印迹分析时显示为2个条带,其相对分子质量分别约为29 ku和60 ku,提示14-3-3蛋白可能以二聚体的形式与GLUT4发生相互作用。结论在生理条件下,14-3-3蛋白与GLUT4在大鼠脂肪细胞中存在相互作用。     

HCV NS5A结合蛋白顺乌头酸酶1的验证研究

目的筛选人肝脏cDNA文库中与HCV NS5A的结合蛋白基因,验证其中顺乌头酸酶1与HCV NS5A的相互作用。方法应用酵母双杂交系统3筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,应用哺乳动物双杂交及免疫共沉淀技术验证其中顺乌头酸酶1蛋白与HCV NS5A之间的相互作用。结果成功筛选出人肝脏cD-NA文库中与HCV NS5A存在相互作用的蛋白基因,哺乳动物双杂交及免疫共沉淀实验结果证实HCV NS5A与顺乌头酸酶1蛋白在HepG2细胞内存在相互作用。结论本实验成功筛选人肝脏cDNA文库中的HCV NS5A结合蛋白基因,并且在体外水平即细胞内证实HCV NS5A与其中的顺乌头酸酶1蛋白之间的相互作用,为进一步细胞内及体内的糖、脂类代谢等功能研究奠定基础。     

免疫共沉淀结合双向电泳分离相互作用蛋白质的方法优化

目的：建立和优化有针对性的免疫共沉淀（CO-IP）与双向电泳（2DE）-质谱联合的技术来鉴定相互作用蛋白质。方法：利用特异性抗体通过CO-IP对细胞全蛋白中的目标蛋白进行富集,用不同洗脱液洗脱目标及其相互作用蛋白,并行2DE分离。结果：对CO-IP过程、洗脱液成分、上样量等关键步骤进行优化,得到满意的2DE图谱。结论：成功优化CO-IP与2DE分离技术,为通过蛋白质组学研究信号通路中重要蛋白质的相互作用提供有效方法。     

Cochlin Isoforms and Their Interaction with CTL2 (SLC44A2) in the Inner Ear

Choline transporter-like protein 2 (CTL2) is a multi-transmembrane protein expressed on inner ear supporting cells that was discovered as a target of antibody-induced hearing loss. Its function is unknown. A 64 kDa band that consistently co-precipitates with CTL2 from inner ear extracts was identified by mass spectroscopy as cochlin. Cochlin is an abundant inner ear protein expressed as multiple isoforms. Its function is also unknown, but it is suspected to be an extracellular matrix component. Cochlin is mutated in individuals with DFNA9 hearing loss. To investigate the CTL2–cochlin interaction, antibodies were raised to a cochlin-specific peptide. The antibodies identify several cochlin polypeptides on western blots and are specific for cochlin. We show that the heterogeneity of the cochlin isoforms is caused, in part, by in vivo post-translational modification by N-glycosylation and, in part, caused by alternative splicing. We verified that antibody to CTL2 co-immunoprecipitates cochlin from the inner ear and antibody to cochlin co-immunoprecipitates CTL2. Using cochlear cross-sections, we show that CTL2 is more widely distributed than previously described, and its prominent expression on cells facing the scala media suggests a possible role in homeostasis. A prominent but previously unreported ribbon-like pattern of cochlin in the basilar membrane was demonstrated, suggesting an important role for cochlin in the structure of the basilar membrane. CTL2 and cochlin are expressed in close proximity in the inner sulcus, the spiral prominence, vessels, limbus, and spiral ligament. The possible functional significance of CTL2–cochlin interactions remains unknown.     

Status epilepticus induces a TrkB to p75 neurotrophin receptor switch and increases brain-derived neurotrophic factor interaction with p75 neurotrophin receptor: an initial event in neuronal injury induction

In neuronal cultures it has been demonstrated that neurotrophins can elicit neuronal death through the p75 neurotrophic receptor (p75ntr) in the absence of concomitant Trk signaling. However, it was suggested that p75ntr induces neuronal death after status epilepticus (SE) in neuronal populations that express relatively high quantities of tropomyosin receptor kinase B (TrkB). Here, using Western blot and immunohistochemistry analyses in the hippocampus, we found that 3-h SE caused a remarkable decrease in TrkB expression and phosphorylation, and a significant increase in p75ntr. TrkB modification occurs before the overexpression of the tumor suppressor protein p53, accompanies the cell damage taking place in the dentate gyrus, and precedes the CA1 neuronal injury as assessed by Fluoro-Jade B and Nissl staining. Co-immunoprecipitation of brain-derived neurotrophic factor (BDNF) or its immature form proBDNF showed increased interaction with p75ntr after its binding to TrkB was reduced. Interestingly, proBDNF also increases its binding with p75ntr after seizures that do not cause neuronal death (animals injected with pilocarpine that fail to enter SE). However, in those animals, TrkB protein levels remained unchanged and its phosphorylation increased. Our results indicate an intrinsic capacity of neurons in vivo to modify final neurotrophin output by changing the proportion of their receptors' expression and the receptors' interaction with their ligands. These early events support the idea that neurotrophins may be involved in the induction of neuronal death in vivo under pathological conditions.     

RACK1, a novel hPER1-interacting protein

PER1, an important component of circadian clock systems, plays a critical role in regulating the period length and maintaining the precision and stability of the period of circadian rhythms. RACK1 (receptor for activated protein kinase C-1), a member of the WD-40 family of proteins, can interact with numerous signaling proteins and is regarded as a scaffolding, anchor, or adaptor protein in multiple intracellular signal transduction pathways. In the present study, we identified and confirmed RACK1 as a novel protein interacting with human clock protein, hPER1, using the yeast two-hybrid system and co-immunoprecipitation experiment. Further study by RT-PCR showed that RACK1 was expressed widely in tissues and there was no obvious expressional rhythmicity. However, RNA interfering plasmid inhibiting hPER1 (pTER/hPER1-II) could not interfere expression of RACK1. These results together suggested that RACK1 might act as a novel signal molecule to mediate or regulate the functions of PER1 through protein interaction.     

ECHS1 acts as a novel HBsAg-binding protein enhancing apoptosis through the mitochondrial pathway in HepG2 cells

We aimed to confirm the role of ECHS1 as a binding protein of HBsAg (HBs) and investigate its function during the development of hepatocellular carcinoma (HCC). Our results show that both exogenous and endogenous ECHS1 proteins bind to HBs and co-localize in the cytoplasm in vitro. The coexistence of HBs and ECHS1 enhances HepG2 cell apoptosis, affects ECHS1 localization in the mitochondria and induces apoptosis by decreasing the mitochondrial membrane potential (MMP). These findings suggest that ECHS1 may be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.     

不同临产状态的子宫平滑肌组织中与#br# NF-κB相互作用差异蛋白质的分析

目的：分析不同临产状态子宫下段平滑肌组织中与NF-κB相互作用的差异蛋白质,为分娩机制的研究提供新的实验及理论依据。方法：分别采集足月未临产、自然临产和前列腺素E2栓诱导临产(药物临产组)的孕妇子宫下段平滑肌组织。Western印迹验证NF-κB P65表达;联合免疫共沉淀、SDS-PAGE、电喷雾串联质谱(LC-ESI-MS/MS)和蛋白质生物信息分析等技术,分析不同临产状态子宫平滑肌组织中与NF-κB相互作用的差异蛋白质,并利用Western印迹进行验证。结果：人类临产前后子宫下段平滑肌组织中NF-κB表达均为阳性,与NF-κB P65相互作用的差异蛋白,自然临产前后鉴定出9个,药物临产前后鉴定出5个,其中不同临产状态共同差异表达的蛋白3个,分别为热休克蛋白70(heat shock 70 kD protein,HSP70)、膜联蛋白A6和结蛋白,经Western印迹验证与蛋白表达相符。这些差异蛋白分别属于分子伴侣、信号转导、细胞骨架和能量代谢相关的蛋白质。结论：子宫平滑肌细胞中NF-κB通过与分子伴侣、信号传导、细胞骨架和能量代谢等相关的多种蛋白质相互作用,参与分娩的启动或调节。