Cyclic PDZ-binding peptides as neuroprotective agents against excitotoxic brain damage

June 23, 2017 | Autor: R. Empson | Categoria: Clinical Sciences, Neurosciences, Brain Damage
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PDZ-Binding Peptides as Neuroprotective agents against Excitoxic Brain damage BrianAusten1, Kate Duberley1,Paul R. Turner2, Molly Garside3, Ruth M. Empson2

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1 Department of Basic Medical Sciences, St George’s University of London, UK. 2 Department of Physiology, University of Otago, Dunedin, New Zealand. 3 School of Biological Sciences, Royal Holloway College, University of London, UK.

Introduction

dichloromethane for 4x3min, and the final Fmoc group by 20% piperidine in DMF. Final ring closure was by treatment with 4 equivalants of PyBOP in 0.9M diisopropylamine in DMF. The peptide was purified by HPLC and characterised by Maldi mass spectrometry (MH=.2189.05.1; calc 2193).

Figure 3. a) Representative Western blot of GST pulldown experiment with mouse forebrain homogenate and R2. The concentration of R2 is shown at the top of the figure and the GST fusion protein bait is shown at the bottom. Note the similarity between the degree of inhibition of binding of NMDA R1 and PSD-95. b) Densitometric analysis of the inhibition of binding of NMDA R1 and PSD-95 by R2. The concentration of R2 reducing binding levels to 50% of controls is about 18µM for both NMDA R1 and PSD-95.

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The plasma membrane calcium pump isoform 2 (PMCA2) is expressed in neuronal tissue and is crucially important in precise spatial and temporal control of intracellular Ca2+ levels (1, 2). Following activation, PMCA2 utilises ATP to pump Ca2+ across the plasma membrane and into the extracellular space. At synapses it forms part of a multi-protein complex (3) that modulates neurotrasmission and intracellular signalling, processes that are implicated in learning and memory as well as disease states such as Alzheimer’s and post stroke neurotoxicity (4). We have recently identified an interaction between PSD95 (a post-synaptic scaffolding protein), PMCA2 and the NMDA receptor at Figure 1. Proposed coupling of the Ca2+ importing synapses in mouse brain (5). Given that synaptic NMDA receptor to the Ca2+ exporting PMCA2 pump by PSD-95 (3, 5). Both NR2A and NR1 subunits have been plasticity relies on exquisite control of localized intra-cellular Ca2+ flux, we hypo-thesize that this shown to bind PSD-95 (6). linkage of a Ca2+ export pump to the Ca2+ importing NMDA receptor is critical to normal synaptic function (Figure 1). To further investigate the importance of the inter-action between PMCA2 and PSD-95 we have designed and synthesised a cyclic peptide inhibitor (R2), targeted to the PDZ1 interaction domain of PSD-95, Figure 2. We report here initial studies describing the design, synthesis, cellular uptake and biochemical activity of R2.

Using a complementary technique, R2 inhibits the co-immunoprecipitation of PSD-95 but not syntaxin (which binds to PMCA2 at a site other than the carboxy-terminal tail) using anti-PMCA2 antibody, suggesting R2 is acting in a specific manner, Figure 4.

Materials and Methods

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R2 protects neuronal cells against toxicity of 2mM glutamate.

Figure 5. Immunohistochemical analysis of 27µM Ruth 2 internalisation in SHSY-5Y cells and PSD-95 at 63x magnification. A- Combined Rhodamine and GFP filtered images B- GFP filter for FITC anti-rabbit, binding to rabbit Ab to PSD-95 C- Rhodamine filter for ExtrAvidin-TRITC, binding to Ruth 2 A.

A schematic of the modelling of R2 binding in the PDZ1 domain is shown in Figure 2 and shows the stabilising efect of the ß-alanine bridge which protrudes from the diffuse surface of PSD-95.

Figure 6. Internalisation of R2 in HEK-293 cells. R2 at 10µM overnight is detected with secondary ant-biotin antibody (left frame), while cells without R2 (right frame) remain unlabelled with secondary antibody.

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Results

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Cellular Uptake HEK-293 cells grown on slides were incubated overnight with 10µM R2, then fixed with methanol for 20 minutes at -20̊C. Cells were permeabilised with 0.01% triton x-100 and R2 detected using an anti-biotin-alexaflur 488 antibody conjugate. Slides were examined using a Bio Rad radiance 2100 confocal microscope.

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Immunoprecipitation Synaptosomal preparations were prepared from forebrain homogenate by centrifugation in sucrose gradients. Synaptosomal proteins were solubilisied using SDS and Triton X-100 before incubation with R2 (100µM) followed by the immunoprecipitating antibody pan-PMCA. After overnight incubation at 4̊C with rotation, protein G Dynabeads (Invitrogen) were added to precipitate antibody / antigen complexes and the resulting proteins separated and visualized by Western blotting.

Figure 4. Immunoprecipitation of PSD-95 and syntaxin using anti-pan PMCA antibody. Addition of 100µM R2 prevents precipitation of PSD-95 but not syntaxin.

R2 effectively enters SHSY-5Y neuronal cells at concentrations of 27mM, and stain colocalises with PSD-95 antibody as shown in Figure 5. While Hek cells are not neuronal R2 entry found to be similar (Fig6). The long exposure period (16 hours) demonstrates the stability of R2 in the intracellular environment.

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Peptide Modelling and Synthesis The structure of R2 binding to the PDZ1 domain of OSD-95 was modelled from the NMR structure of Piserchio et al., 2004 (7). Aside from the PSD-95 targetting sequence (the cyclised PMCA2b, PSD-95 binding domain), R2 also possesses a membrane penetrating sequence (hexa-Arg) to promote cellular entry and a biotin tag to allow visualisation in vitro Fmoc protection, and orthogonally protected residues, which were deprotected and reacted with a bridging residue, prior to final deprotection. Purification by HPLC was followed by verification using mass spectrometry (ms) and ms2 by Maldi TOF-TOF. GST pull-down GST fusion constructs were created with pGex-4T1 and encoded the PSD-95 binding domain of PMCA2b (amino acids 1139-1243), GST alone or the protein bound to GSH-agarose beads was mixed with mouse brain tissue homogenate and R2. Following this GST pull-down, PSD95 was detected in the purified material by Wester blot.

Conclusion

Inhibition of binding of PSD-95 to the PMCA2b binding by R2 using a GST pull down assay is shown in Figure 3. The concentration of R2 required to reduce binding to about 50% is approximately 18µM. In a similar manner R2 also inhibits the pull-down of the NMDA receptor with a comparable dose response curve. The C-terminal sequence of PMCA2b Ser-Leu-Glu-Thr-Ser-Leu-COOH.was synthesised as a cyclic variant; R2, acetyl-arg-arg-arg-arg-arg-arg-Gly-Gly-Lys(Biotin)Ser-Leu-c[Glu(ßAla)Thr-Lys]Leu-COOH. R2 was synthesized on Leu Peg-PS resin. using temporary a-amino Fmoc protected residues. The cyclised C-terminal sequence of PMCA2 was extended at the N-terminus by six D-arginine residues to act as a cell penetration sequence, and finally N-acetylated to block the N-terminus against protease degradation. Orthogonally protected residue FmocLys(ivDde) was coupled at step 2) and FmocGlu((2-PhiPr) at step4. Pior to final deprotection, the ivDde group was removed with 2% hydrazine in DMG for 3x3min. Fmocß-ala was coupled with PyBOP. The 2-PhilPr group was removed by treatment with 1% TFAand 0.5% ethanedithiol in

References 1. Empson RM, Garside ML, Knopfel T. Plasma membrane Ca2+ atpase 2 contributes to short term synapse plasticity at the parallel fiber to purkinje neuron synapse. J Neurosci. (2007) 27:3753-3758. 2. Jensen TP, Filoteo AG, Knopfel T, Empson RM. Presynaptic plasma membrane Ca2+ atpase isoform 2a regulates excitatory synaptic transmission in a rat hippocampal ca3. L Physiol. (2007) 579:85-99. 3. DeMarco SJ, Strehler EE. Plasma membrane Ca2+ atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/DIg/ZO-1) domain-contaning proteins. J Biol Chem. (2001) 276:21594-600. 4. Kalia LV, Kalia SK, Salter MW. Nmda receptors in clinical neurology: Excitatory times ahead. Lancet Neurol. (2008) 7:742-755. 5. Garside ML, Turner PR, Ausetn B, Strehler EE, Beesley PW, Empson RM. Molecular interactions of the plasma membrane calcium ATPase 2 pre-and-post-synaptic sites in rat cerebellum. Neorosience. (2009) 162:383-95. 6. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH. Domian interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science. (1995) 269:1737-40. 7. Piserchio A, Salinas GD. Li T, Marshall J, Spaller MR, Mierke DF. Targeting specific PDZ domains of PSD-95; structual basis for enhanced affinity and enzymatic stability of a cyclic peptide. Chem Biol. (2004) 11:469-73.

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Figure 2. a) Chemical structure of R2. The ß-alanine bridge is designed to stabilise the peptide backbone and confer protease resistance, the hexa-arginine sequence enables cell penetration and the biotin moiety allows tracking and visualisation. The remaining peptide backbone is modelled on the carboxyl-terminus of PMCA2b which binds PSD-95. b) The peptide backbone and ß-alanine bridge of R2 binding to PSD-95 (structure based on Piserschio et al., 2004).

Inhibition of binding of PSD-95 to the PMCA2b binding domain by R2 using a GST pull-down assay suggests the concentration of R2 required to reduce binding to about 50% is approximately 18µM. In a similar manner R2 also inhibits the pull-down of the NMDA receptor with a comparable dose response curve. Using a complimentary technique R2 inhibits the coinmmunoprecipitation of PSD-95 using anti-PMCA2 antibody. Taken together these results indicate that R2 selectively targets the PMCA2b-PSD-95-NMDA receptor complax and combined with its cell penetrating capability will be useful in examining the functional effects of dissociating this complex in vivo.

We acknowledge the BBSRC, Otago University and Swan

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