Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1–PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.     

Developmental expression of CAPON and Dexras1 in spinal cord of rats

To study the expression of the carboxy-terminal PSD-95/DLG/ZO-1 ligand of nNOS (CAPON) and Dexras1 mRNA during development in the spinal cord of rats, real-time polymerase chain reaction (Real-time PCR), as a quantitative method, was used to study the developmental expression of CAPOn and Dexras1 mRNA level in the spinal cord. The spatial expression of CAPON and Dexras1 mRNA was examined by a combination of in situ hybridization (ISH) and immunofluorescence. During the development of the spinal cord, CAPON mRNA was expressed in low levels from embryo day 14 to day 18. At postnatal day 1, it reached the peak and was expressed in the part which will become the dorsal horn when mature. It then decreased gradually until postnatal week 12, when it presented in the ventral horn. At embryo day 14, Dexras1 mRNA was expressed at low levels, increased during embryo day 16 to day 18 and peaked at postnatal day 1. Spatiotemporal expression of Dexras1 mRNA was similar to CAPOn as confirmed by correlation analysis and colocalization. CAPOn and neuronal nitric oxide synthase (nNOS) was expressed within the same cells of the dorsal horn at postnatal day 1 but had different subcellular localizations. Co-expression of CAPOn and Dexras1 mRNA in myeloid tissue during development process of rat indicates that the adaptor protein, CAPON may play a probable role in differentiation of neurons, synaptic plasticity and synaptogenesis by regulating nNOS to activate Dexras1. __________ Translated from Chinese Journal of Neuroanatomy, 2007, 23(4): 349–354 [译自: 神经解剖学杂志]     

The role of glutamate receptors in traumatic brain injury: implications for postsynaptic density in pathophysiology

Traumatic brain injury (TBI) is the major cause of death and disability, and the incidence of TBI continues to increase rapidly. In recent years, increasing attention has been paid to an important structure at the postsynaptic membrane: the postsynaptic density (PSD). Glutamate receptors, as major components of the PSD, are highly responsive to alterations in the glutamate concentration at excitatory synapses and activate intracellular signal transduction via calcium and other second messengers following TBI. PSD scaffold proteins (PSD-95, Homer, and Shank), which anchor glutamate receptors and form a network structure, also have potential effects on these downstream signaling pathways. The changes in the function and structure of these major PSD proteins are also induced by TBI, indicating that there is a more complicated mechanism associated with PSD proteins in the pathophysiological process of TBI.     

The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses.     

Family-based association studies of CAPON and schizophrenia in the Chinese Han population

Although there is evidence pointing to CAPON as a susceptible gene for schizophrenia, the results of independent association studies have so far been inconsistent. A recent case-control study by Zheng et al. supported CAPON as a susceptible site for the disease in the Chinese Han population. In their study both the single polymorphism (rs348624) and individual haplotypes showed significant association with schizophrenia. Our study further investigates this relationship this time using a family-based association. We selected 5 SNPs including rs348624 and performed a Transmission Disequilibrium Test (TDT) in 319 Chinese Han trios. Our results identified no single marker nor haplotype associated with schizophrenia, which did not suggest that CAPON was a susceptible site in the Chinese Han population, or it appeared unlikely that the CAPON played a major role in the aetiology of schizophrenia. Since there is consistent evidence pointing to 1q21-22 as a positional candidate region for schizophrenia, we suggest that further research should focus on other genes located in this region.     

Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca/calmodulin-dependent protein kinase II

We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.     

结构域蛋白质组学研究进展

蛋白质的结构域是蛋白质中具有特异结构和独立功能的区域。与蛋白质本身的数量相比，结构域的数量是有限，每个结构域所包含的成员数目也是有限的。在蛋白质组学研究的现阶段，研究方法上的限制尚无法全面实时地研究哺乳动物细胞的整个蛋白质组。由于结构域在功能上的重要性和其数量的有限性决定了以结构域为研究对象在蛋白质组学规模上进行研究不失为现阶段蛋白质组学研究的一种现实的选择。本文综述一些在这些方面所做出的早期的尝试。研究人员分别以SH3、PDZ和WW等结构域为对象进行蛋白质组学规模上的研究，并建立和完善了一些研究方法。总之，在结构域基础上研究蛋白质组是研究蛋白质组学的一个独特的视角。     

Activin induces long-lasting N-methyl-D-aspartate receptor activation via scaffolding PDZ protein activin receptor interacting protein 1

Calcium entry into the postsynaptic neuron through N-methyl-D-aspartate-type glutamate receptors (NMDARs) triggers the induction of long-term potentiation (LTP), which is considered to contribute to synaptic plasticity and plays a critical role in behavioral learning. We report here that activin, a member of the transforming growth factor-beta (TGF-beta) superfamily, promotes phosphorylation of NMDARs and increases the Ca2+ influx through these receptors in primary cultured rat hippocampal neurons. This signal transduction occurs in a functional complex of activin receptors, NMDARs, and Src family tyrosine kinases, including Fyn, formed on a multimer of postsynaptic scaffolding postsynaptic density protein 95/Dlg/ZO-1 (PDZ), activin receptor interacting protein 1 (ARIP1). Activin-induced NMDAR activation persists for more than 24 h, which is complimentary to the activation time of NMDARs by brain-derived neurotrophic factor (BDNF). Our results suggest that activin is a unique and powerful potentiator for NMDAR-dependent signaling, which could be involved in the regulatory mechanisms of synaptic plasticity.     

PDZ1-腺病毒重组体的构建及其对KA诱导的海马神经元凋亡的拮抗作用

目的构建PSD-95结构域PDZ1与腺病毒重组体并观察其对海人藻酸(KA)诱导的海马神经元凋亡的作用。方法采用RT-PCR法获得PDZ1的cDNA;与腺病毒穿梭载体pAdTrack-CMV用T4 DNA连接酶连接;连接产物与骨架载体pAdEasy-1共转染至原核包装细胞BJ5183;将原核重组体用脂质体转入真核包装细胞293H;纯化包装后的病毒颗粒并测定其滴度;腺病毒感染培养的海马神经元,加入KA,用DAPI染色后荧光显微镜观察细胞凋亡;用流式细胞仪定量分析细胞凋亡率。结果①经RT-PCR得到了PDZ1的cDNA;②电泳鉴定得到原核重组体;③得到包装完整的具感染能力的病毒颗粒;④纯化的病毒颗粒滴度为1.08×109ifu/mL⑤病毒表达的PDZ1使KA诱导的海马神经元凋亡数量减少(P<0.05)。结论获得了能表达PDZ1-GFP的病毒颗粒,并且PDZ1过表达能拮抗KA诱导的海马神经元凋亡。     

调控黑色素瘤转移的新信号蛋白: Syntenin

Syntenin蛋白在多种肿瘤中表达增强,最近被认为是一个新的黑色素瘤转移调节因子.作为一类支架信号蛋白,Syntenin通过它的两个PDZ 功能基团可与许多细胞膜受体胞内末端或细胞内的信号分子结合,调控多种重要的细胞生理过程和信号传导途径,包括细胞膜受体的聚集,细胞内蛋白质的转运,细胞骨架的重建,转录因子的激活,以增强肿瘤细胞的生长、黏附以及肿瘤的血管生成、侵袭和转移能力.本文简要综述了syntenin的结构和功能,相关的信号途径,及其在黑色素瘤研究领域的最新进展.