Overexpression of the PDZ1 domain of PSD‐95 diminishes ischemic brain injury via inhibition of the GluR6·PSD‐95·MLK3 pathway

Recent studies have shown that kainate (KA) receptors are involved in neuronal cell death induced by seizure, which is mediated by the GluR6·PSD‐95·MLK3 signaling module and subsequent JNK activation. In our previous studies, we demonstrated the neuroprotective role of a GluR6 c‐terminus containing peptide against KA or cerebral ischemia‐induced excitotoxicity in vitro and in vivo. Here, we first report that overexpression of the PDZ1 domain of PSD‐95 protein exerts a protective role against neuronal death induced by cerebral ischemia‐reperfusion in vivo and can prevent neuronal cell death induced by oxygen‐glucose deprivation. Further studies show that overexpression of PDZ1 can perturb the interaction of GluR6 with PSD‐95 and suppress the assembly of the GluR6·PSD‐95·MLK3 signaling module and therefore inhibit JNK activation. Thus, it not only inhibits phosphorylation of c‐Jun and down‐regulates Fas ligand expression but also inhibits phosphorylation of 14‐3‐3 and decreases Bax translocation to mitochondria, decreases the release of cytochrome c, and decreases caspase‐3 activation. Overall, the essential role of the PDZ1 domain of PSD‐95 in apoptotic cell death in neurons provides an experimental foundation for gene therapy of neurodegenerative diseases with overexpression of the PDZ1 domain. © 2009 Wiley‐Liss, Inc.     

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919–922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95–PMCA2 and syntaxin-1A–PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.     

The Legionella HtrA homologue DegQ is a self-compartmentizing protease that forms large 12-meric assemblies

Proteases of the HtrA family are key factors dealing with folding stress in the periplasmatic compartment of prokaryotes. In Escherichia coli, the well-characterized HtrA family members DegS and DegP counteract the accumulation of unfolded outer-membrane proteins under stress conditions. Whereas DegS serves as a folding-stress sensor, DegP is a chaperone-protease facilitating refolding or degradation of defective outer-membrane proteins. Here, we report the 2.15-Å-resolution crystal structure of the second major chaperone-protease of the periplasm, DegQ from Legionella fallonii. DegQ assembles into large, cage-like 12-mers that form independently of unfolded substrate proteins. We provide evidence that 12-mer formation is essential for the degradation of substrate proteins but not for the chaperone activity of DegQ. In the current model for the regulation of periplasmatic chaperone-proteases, 6-meric assemblies represent important protease-resting states. However, DegQ is unable to form such 6-mers, suggesting divergent regulatory mechanisms for DegQ and DegP. To understand how the protease activity of DegQ is controlled, we probed its functional properties employing designed protein variants. Combining crystallographic, biochemical, and mutagenic data, we present a mechanistic model that suggests how protease activity of DegQ 12-mers is intrinsically regulated and how deleterious proteolysis by free DegQ 3-mers is prevented. Our study sheds light on a previously uncharacterized component of the prokaryotic stress-response system with implications for other members of the HtrA family.     

Unraveling the biological mechanisms in Alzheimer's disease--lessons from genomics

Alzheimer's disease (AD) is the most common form of dementia and the most common neurodegenerative disease, with a complex genetic background. Genome-wide association studies (GWAS) have yielded important new insights into genetic mechanisms of AD pathology. Current results unequivocally confirm apolipoprotein E (APOE) as a major genetic risk factor for development of late onset AD. Additional associations of more than twenty genes have also been identified and replicated in subsequent genetic studies. Despite the exciting new GWAS data which have emerged in the last few years, it has become clear that common variants within the genome cannot fully explain the underlying genetic risk for AD. Novel approaches such as genome-wide analysis of copy number variations (CNV) or low-frequency rare functional gene variants may provide additional insight into genetic basis of AD. In this review we summarize the findings of eighteen GWAS studies in AD performed to date, with an emphasis on potential future developments in the quest for genetic risk factors of AD.     

Human papillomavirus (HPV)‐18 E6 oncoprotein interferes with the epithelial cell polarity Par3 protein

High‐risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD‐95/DLG/ZO‐1 (PDZ) domain‐containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ‐dependent manner without significant reduction in Par3 protein levels. In addition, the high‐risk HPV‐18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV‐associated tumours.     

Cancer Stem Cells and Circulatory Tumor Cells Promote Breast Cancer Metastasis

Breast cancer (BC) is a highly metastatic, pathological cancer that significantly affects women worldwide. The mortality rate of BC is related to its heterogeneity, aggressive phenotype, and metastasis. Recent studies have highlighted that the tumor microenvironment (TME) is critical for the interplay between metastasis mediators in BC. BC stem cells, tumor-derived exosomes, circulatory tumor cells (CTCs), and signaling pathways dynamically remodel the TME and promote metastasis. This review examines the cellular and molecular mechanisms governing the epithelial to mesenchymal transition (EMT) that facilitate metastasis. This review also discusses the role of cancer stem cells (CSCs), tumor-derived exosomes, and CTs in promoting BC metastasis. Furthermore, the review emphasizes major signaling pathways that mediate metastasis in BC. Finally, the interplay among CSCs, exosomes, and CTCs in mediating metastasis have been highlighted. Therefore, understanding the molecular cues that mediate the association of CSCs, exosomes, and CTCs in TME helps to optimize systemic therapy to target metastatic BC.     

Disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect

PSD-95 binds to and co-localizes with NMDA receptors at postsynaptic sites. Their co-expression in COS7 cells induces the formation of aggregates containing both proteins. These findings have lead to the hypothesis that PSD-95 helps to cluster NMDA receptors at postsynaptic sites. In addition, PSD-95 binds various regulatory proteins including Src, Pyk2, SynGAP, and nNOS and may recruit signaling proteins to NMDA receptors. We tested whether synaptic transmission or plasticity was affected by acute dissociation of the PSD-95-NMDA receptor interaction with various peptides that bound to the first two PDZ domains of PSD-95 and its homologs and with antibodies directed against the very C-terminus of the NR2A and NR2B subunits of the NMDA receptor. Membrane-impermeable peptides injected via whole cell patch electrodes distributed within minutes throughout dendritic branches into spines in acute hippocampal slices and membrane-permeable peptides containing 11 arginine residues effectively accumulated in neurites in slices and primary hippocampal cultures. Neither peptides nor antibodies showed any effect on basal synaptic transmission or induction of long-term potentiation (LTP) in hippocampal slices. Pharmacologically isolated NMDA receptor activity was also not affected. However, the membrane-permeable peptide disrupted the NMDA receptor-PSD-95 interaction in slices as tested by immunoprecipitation and subsequent immunoblotting. These findings suggest that acute dissociation of PSD-95 and its homologs from the NMDA receptor and likely from other protein complexes does not result in any easily detectable physiological effects in hippocampal slices. However, we cannot exclude a role of PSD-95 in early events that lead to clustering of NMDA receptors or of other proteins including stargazin and AMPA receptors at postsynaptic sites nor do these experiments address the possibility of long-term changes in the slices. In fact, incubation of primary hippocampal cultures with the membrane-permeable peptide lead to a moderate decrease in the number of dendritic clusters of PSD-95 and NMDA receptors and their colocalization by 20-30%, suggesting some role of PSD-95 and its homologs in NMDA receptor clustering.     

Differential roles of GRIP1a and GRIP1b in AMPA receptor trafficking

Regulated trafficking controls AMPA receptor (AMPAR) number at the postsynaptic membrane to modify the efficiency of synaptic transmission. The PDZ proteins GRIP1 and the related ABP-L/GRIP2 bind AMPAR subunit GluA2, and have been proposed to play a role in AMPAR trafficking associated with Long Term Depression (LTD) of synaptic transmission. Both GRIP1 and ABP-L/GRIP2 exist in different splice isoforms, including alternative 18 amino acid domains at the extreme N-terminus, which determine whether the protein can be palmitoylated. The implications of this differential splicing for AMPAR trafficking is unknown. Here, we use surface biotinylation and quantitative Western blotting to show that the N-terminal splice variants GRIP1a and GRIP1b have differential effects in NMDA-induced AMPAR internalization in cultured hippocampal neurons. GRIP1a inhibits, but GRIP1b enhances this trafficking event. We further demonstrate that GRIP1a and GRIP1b have dramatically different subcellular distributions in cultured neurons and exhibit NMDA-dependent colocalisation with early endosomes. We propose that GRIP1 palmitoylation modulates NMDA-induced AMPAR internalisation by differential regulation of the early endosomal system.     

中药治疗中风的分子靶点

“中风”是中医学对急性脑血管疾病的统称,具有极高的死亡率和致残率。中医药治疗中风有着悠久的历史,临床实验也证实了其疗效。然而由于中药常常需要多味药物联合使用,其有效成分很难明确,对其有效性评价具有挑战性,因此很难将中药制剂推向世界。综述中药治疗中风的分子靶点作用,列举了一些可能起效的天然化学物质,调查结果表明多成分多靶点的中药理论具有科学性。现代分子医学和传统中医理论的结合能促进医学界的广泛交流,值得深入探讨。     

Characterization of interactions of Na+/H+ exchanger regulatory factor‐1 with the parathyroid hormone receptor and phospholipase C*

Abstract: The Na⁺/H⁺ exchanger regulatory factor‐1 (NHERF1) is a molecular scaffold important for the signaling of the G‐protein coupled receptor for the parathyroid hormone (PTH1R). The two PDZ (PSD‐95, Discs‐large, ZO1) domains of NHERF1 through association with the C‐termini of PTH1R and phospholipase C enhance the signaling pathway associated with PTH. To examine these interactions, we have produced the individual PDZ1 and PDZ2 domains as well as the tandem PDZ1–PDZ2 domains (PDZ12) of NHERF1 and have characterized the binding affinities of the C‐terminal motifs of PTH1R and PLCβ using fluorescence anisotropy. Circular dichroism indicates that the PDZ1 and PDZ2 are properly folded. Based on fluorescence anisotropy we find that the C‐terminus of PTH1R, containing ETVM, has similar affinities (approximately 10 μm ) for both PDZ1 and PDZ2. The PTH1R displayed reduced binding affinity for the tandem PDZ12 (16 μm ) compared with the individual domains or a solution of equal molar concentrations of PDZ1 and PDZ2 (5.8 μm ), suggesting negative cooperativity between the PDZ domains or intervening region. The C‐termini of PLCβ (both β1 and β2 isozymes were examined, containing DTPL and ESRL, respectively) displayed a diminished affinity for PDZ2 (approximately 30 μm ) over that of PDZ1 (approximately 8 μm ). Finally, we demonstrate trans PDZ1–PDZ2 association that is enhanced in the presence of the C‐terminus of PTH1R or PLCβ, suggesting oligomerization of NHERF as a mode for enhancing the signaling associated with PTH.