Cyclisation of Cell-Penetrating PDZ-Binding Peptides Directed to PSD95

Suraj Dabhi*

Citation: Cyclisation of Cell-Penetrating PDZ-Binding Peptides Directed to PSD95. American Research Journal of Chemistry, 3(1); pp:1-11.

Copyright This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


 Excitatory glutamatergic neurotransmission via N-methyl-D-aspartate receptor (NMDAR) is critical for synaptic plasticity and survival of neurons. Excessive NMDAR activity, however can cause excitotoxicity and promote cell death, underlying a potential mechanism of neurodegeneration occurring in Alzheimer’s disease (AD). The regulation of calcium levels at synapses is important for pre- and post-synaptic activation in the hippocampus, where glutamate is the primary neurotransmitter, acting via metabotropic and ionotropic receptors.



Many biological activities are regulated through the dynamic interactions of modular protein domains (e.g., WW, SH3, SH2, PH, and PDZ) and their corresponding binding partners. Elucidation of the specificity, selectivity, and regulatory mechanisms involved in these protein-protein interactions can provide important insights into biological processes such as cell proliferation, cell polarity and cell signalling

PDZ domains are abundant protein-protein interaction modules found in various species. In the mouse genome, for example, 928 PDZ domains have been recognized in 328 proteins. PDZ domains exist in single or multiple copies or in combination with other interaction modules. From the abundance and diversity of PDZ domains in cells it is apparent that many cellular and biological functions, especially those involving signal transduction, are affected by PDZ-mediated interactions (Lee & Zheng (2010).

Recently, the postsynaptic density-95 (PSD-95/SAP90) family of cytoskeletal proteins (Topinka& Bredt 1998), which includes SAP97, SAP102 and PSD-93 (Chapsyn-110), has been suggested to mediate receptor clustering at excitatory synapses in brain. PSD-95 contains three PDZ domains, an SH3 domain and a guanylate kinase domain, suggesting that it makes a number of interactions with other proteins. Interactions between NMDA receptors and PSD-95, have been identified at several levels. The dynamic regulation of calcium ions at the synapse demands that a finely-controlled system of Ca2+ transporters and Ca2+ binding proteins collaborate to allow transient increases of calcium during activation of receptors, whilst over the long-term, a low resting state of Ca2+ is achieved, by removing Ca2+ by efflux, or by transporting Ca2+ to intracellular calcium stores. The possibility arises that plasma membrane Ca2+ ATPases (PMCAs) are responsible for some of this fine control. One of these, PMCA2b is expressed at high levels in cerebellar Purkinje neurons. Reciprocal immunoprecipitation and GST pull-down has shown that PMCA2b interacts with the post-synaptic protein PSD95 via its COOHterminal PDZ (PSD95/Dlg/ZO-1) binding domain (Garside et al., 2009). PSD-95 interacts with the NR2a subunit via one of its 3 PDZ domains; this leads to the possibility that binding of glutamate to NMDAR may regulate Ca2+ extrusion via PMCA2b, the interactions being mediated by PSD-95 (Niethammer et al 1998). Each of the 5 protein-binding domains of PSD-95 (3 PDZ domains, an SH3 domain and a guanylate kinase domain) can bind to several other proteins, including ion channel receptors, adhesion and cytoskeleton proteins, and proteins involved in intracellular signalling, including nNOS. An additional scaffold protein NHERF2 also interacts with PMCA2b, via its C-terminus (Demarco & Streher., 2002). SHSY-5Y human neuroblastoma cells have been shown previously to expressNMDA receptors, (Naarala et al., 1993; Nair et al., 1996), albeit at low levels (Sun et al), and thus make a useful model system for this study.Fernandes, et al. (2007) investigated the role of silencing the PMCA2b in neuronal cells, using RNAi techniques, and observed its effects on Ca2+ homeostasis and cell viability. Using fluorescence ratio imaging of free intracellular Ca2+, they determined that hippocampal neurons with reduced expression of PMCA2b took longer to return to their baseline levels of intracellular Ca2+. In addition to this, they identified a greater susceptibility to various stresses, in SH-SY5Y neuroblastoma cells. This study highlights the importance of the PMCA in maintaining intracellular Ca2+ concentrations, and its potential role in the pathogenesis of many diseases.

Four non-allelic genes code for mammalian PMCAs 1, 2, 3,and 4 with additional isoforms arise from alternative RNA splicing. The PMCA-2 variants a and b, differ in the protein sequence C-terminal to the transmembrane domain, and create different C-terminal domains by changes in the translational reading frame. The C-terminal sequence of PMCA2b: IHSLETSL matches the consensus sequence of PDZ-binding ligands E-(T/S}-X-(V/L), where X is any amino acid, and V or L are C-terminal residues. To examine the functional importance of any interaction between PMCA2-b and PSD-95 via its PDZ domain, we synthesized the C-terminal sequence of PMCA2b, as a stabilized cyclic peptide extended with a cell penetration sequence andlabelled with an internal biotin marker. PDZ-binding peptide cyclisation followed the strategy of Pischerio et al (2004) who synthesized a cyclic variant of the CRIPT PDZ-binding sequence, and suggested a 10-fold increase in binding affinity as well as increased protease resistance over the linear form. To investigate the role of a PMCA2b-PSD-95 interaction in calcium regulation and cell death, we synthesized the C-terminal sequence of PMCA2b. Piserchio et al (2004) suggested that cyclisation of a PDZ-binding peptide from CRIPT increased binding to the PDZ3 domain of PSD95 and stabilized structures against proteolytic degradation. We investigated whether a C-terminal peptide from PMCA2b would have these properties, investigating the sequence in the cyclized and linear forms.


Peptide chemicals were obtained from Novabiochem (Nottingham, UK) and solvents from Rathburn (Glasgow, UK). Thus,the C-terminal biotinylated peptide sequence of PMCA2B, was synthesised on solid phase in the linear form with the addition of a DArg6 cell penetration sequence (Balhorn et al., 2009) and Lys-ebiotin as a marker to aid visualisation (AcRRRRRRGG (K-Biotin) SLETSL (R3) (MH+ =2137.5; Calc 2137.52). The purified peptide was characterised by Maldi mass spectrometry. In order to investigate the effects of cyclisation on PDZbinding peptides, which have previously been described by Pischerio et al (2004), an analogue of the PMCA2b sequence, in which the Ser residue at position -2 was substituted by Lys , was synthesised as a cyclised analogue (R2), incorporating β-alanine as a bridge between positions -2 (Lys) and -4 (Glu) in the sequence. Acetyl-DArgDArg-DArg-DArg-DArg-DArg-Gly-Gly-(Lys-Biotin)-Ser-Leu-c[Glu(βAla) Thr-Lys]-Leu-COOH(R2) (MH+ =2190.7; Calc 2190.5). R2 was synthesized on Leu-Peg-PS resin. Fmoc residues were coupled in four fold equivalence with with four-fold equivalents of HATU, and removal of temporary a-amino Fmoc protection, was achieved by 20% piperidine in DMF.a-Fmoc-e-biotinyl lysine was incorporated at step 6of the synthesis(position -6) to allow biotin detection of R2 by microscopy. Orthogonally protected FmocLys(ivDde) and FmocGlu (2-PhiPr) were incorporated at steps 2 and 4 of the synthesis.As a control for the cell-penetrating effect of DArg6 to a cyclic PDZ-binding peptide, a cyclized peptide, without a DArg6 penetration sequence, viz: Acetyl-(Lys-Biotin) Ser-Leu-c[Glu-(βAla)Thr-Lys]-Leu-COOH (R1)was synthesized. (MH+ =1162.0; Calc1157.4).

The N-termini of R2 and R1 were N-acetylated to inhibit proteolytic degradation. To cyclize the peptides, prior to final deprotection and release from resin, the ivDde group of Lys at residue 2 was removed from the peptidylresin by treatment with 2%(v/v) hydrazine in DMF for 3x3min. To create a bridge, Fmocβ-Ala was then coupled to the e NH2 of Lys2 with PyBOP. The 2-PhilPr group on the side chain of Glu residue 4 was removed by mild treatment with 1% TFA and 0.5% ethanedithiol in dichloromethane for 4x3min, and the Fmoc group on β-Ala then removed by treatment with 20% piperidine in DMF for two periods of 5 min and 20 min. Final ring closure, monitored by the loss of ninhydrin colouration from a small peptidyl-resin samples, was catalysed by treatment with 4 equivalents of PyBOP in 0.9M diisopropylethylamine in DMF over a 24hr period. The peptidyl resin was washed with methanol, followed by ethylene dichloride, dried in vacuo, and then cleaved/deprotected with 98% triflouroacetic acid containing 1% (v/v) water and 1% triisopropyl silane for 2hrs, filtered off, evaporated and triturated with diethyl ether. The residue was taken up in 0.1% trifluoroacetic acid and purified by HPLC on Vydac C8,with an acetonitrile gradient, characterised by Maldi mass spectrometry (MH=2190.5; calc 2190.42), which showed that that ring closure had been achieved. The choice of PyBOPas a cyclisation reagent was crucial to avoid guanidination (Alberichio et al, 1998).

As a comparison, the C-terminal sequence of NMDA-R2; Leu-Ser-Ser-Ile-Lys-Ser-Asp-Val-COOH, which binds a third PDZ-domain in Psd95, was synthesised as a cyclic, biotinylated analogue with a DArg6 penetration sequence, by a strategy similar to that above, giving: Ac-DArg-DArg-DArg-DArg-DArg-DArgGly-Gly-(K-Biotin)- Leu-Ser-Ser-Ile-c[Lys-(βAla)Ser-Asp]-Val-COOH (H2) Measured MH was 2350.1. Calc 2349.8

Flourescence Microscopic Studies of PDZ-Binding Peptides

The interaction of biotinylated PDZ-binding peptides with components of SHSY-5Y human neuroblastoma cells were studied by fluorescence microscopy. The presence of ionotropic, and metabotropic glutamate receptors in SHSY-5Y human neuronal cell lines has been shown by Narrala et al (1993)and Nsair et al, 1996. When confluent in 75cm2 culture flasks, the SHSY-5Y cells were transferred onto glass slides in Petri dishes and incubated at 37o C in 5% CO2 /95% O2 for 24 hours. The media was then removed from the slides which were then washed three times with PBS containing 2% horse serum (PBS-HS) (GIBCO, Invitrogen Paisley, Scotland, UK, 16050).

A 27µM solution of the PDZ-binding peptides in PBS-HS was added to the cell-coated slides. The slides were then incubated at 37o C for two hours. Each slide was then washed three times with PBS-HS, and 10ml of methanol, (BDH Chemicals ltd, Poole, England 101586B), cooled to 4o C, was added to each slide. The cells in Petri dishes were incubated at 4o C for ten minutes. The methanol was subsequently removed and the cells were washed again with PBS-HS.

A 1:500 dilution of anti-PSD-95 rabbit antibody (Abcam, Cambridge, UK, AB18258) diluted to 1ml in PBS, was added to each slide and left for one hour at room temperature. The slides were then washed twice with PBS-HS. Solutions diluted with PBS-HS of 1:160 of FITC anti-rabbit IgG (Sigma, Dorset, UK, F9887) (to localize PSD95) and 1:500 ExtrAvidin TRITC conjugate (to localize biotinylated PDZ-binding peptides), was added to the slides, which were left at room temperature for twenty minutes. The slides were then washed 3x with PBSHS, mounted and examined using a Carl Zeiss 240M microscope with fluorescent filters. Internalization of the biotin linked to the PDZ-binding peptides was assessed using a rhodamine filter (550nm), and a GFP filter (495nm) was used to indicate the location of PSD-95.

Intracellular Calcium

SHSY-5Y human neuroblastoma-glioma cells were grown overnight in a 96-well plate, at a starting density of 40,000 cells per well. After 24 hrs, the medium from each well was removed (to eliminate any sources of baseline fluorescence) and PDZ-binding peptides with or without buffered glutamate were added, for incubation for another 16 hrs. Intracellular calcium was measured using the Fluo-4 NW Calcium Assay Kit (Invitrogen, F36206). 100μl of the probenecid stock solution (250mM) to inhibit extrusion of the reagents out of cells by organic anion transporters in 10ml of HBSS was added to one vial of Fluo-4 NW dye mix, and then 100μl added to each well containing cells, and this was then incubated at 37oC for 30 minutes, then left at room temperature for an addition 30 minutes Fluorescence was measured at room temperature using a bottom-read filter, for excitation at 494nm and emission at 516nm.


Molecular Modelling

The PDZ domain is a common folded protein interaction domain that recognises and binds specific C-terminal peptides, and some internal sequences, of target proteins. PDZ domains are (1) found in a number of proteins from different species, (2) share high sequence homology, and (3) are typically composed of 80-90 amino acid residues The domain consists of a central bent five-membered beta-sheet (coloured purple in Figure 1), surrounded by two helices (coloured green). The C-terminal target peptide binds between the C-terminal end of the second beta-strand and the top alpha-helix.

The C-terminal pentapeptide sequence of PMCa2b in a cyclic form with the sequence extended by a biotinylated Lys residue, to act a marker, and six contiguous residues of DArg to act as a cell penetration sequencewas successfully synthesized by solid-phase Fmoc chemistry, demonstrated by close correlation of measured mass with that calculated from the structure, and its homogeneity shown by the single peak obtained on analytical HPLC. An intracellular bridge between Glu2 and Lys4 was incorporated bycoupling Fmoc-b-Ala to Glu2. After removal of the Fmoc from the peptidyl resin, by treatment with piperidine, the ring was closed by formation of a beta-lactam by treatment with excess PyBOP. Each step of the bridge formation was checked by reaction, or loss of reaction of small samples with ninhydrin. The products were >95% purity by analytical HPLC and Maldi mass spectrometry, which confirmed the cyclic structures.

Cell Penetration and Localization of PDZ-Binding Peptides

Both R2 andH2 (27mM),which containDArg6 cell penetration peptide sequences, were efficiently internalised into SHSY-5Y cells after 2 hours incubation (Figure 2), and readily visualised by binding of rhodaminelabelled avidin to the biotin incorporated as side-chain modifications in R2 and H2 (Fig B and E). In contrast, no intracellular staining was observed after incubation with R1, which lacked the 6xDArg sequence (Fig 2F). 

Intracellular co-localisation occurred between R2 or H2 and PSD-95 (Figs C, D and E), as stained by an anti PSD95 rabbit antibody. Significant overlap between R2 (Fig B) and PSD-95 (Fig A) was calculated from the Pearson coefficient of 0.98, and between H2 (Fig E) and PSD-95 (Fig F) (Pearson coefficient 0.95). There was increased staining within certain regions of intracellular membranes, indicating that PSD-95, and bound peptides, may be located in the Golgi apparatus. There was sparse binding to cellular processes in the cellular extremity.To induce partial differentiation, and neurite outgrowth, SHSY-5Y cells were incubated with all-trans retinoic acid (100mM) for 7 days. (Pahlmann et al., 1984)) Retinoic acid induced extension of cells with neurites (Figures D and E), but little change in the distribution of R2 binding, location or levels of expression of PSD-95 and extent of co-localisation was produced (Pearson coefficient 0.97).

SHSY-5Y cells were incubated with R2 (A,B,C,D) or H2 (E) for 2 hours at 37o C in full media, or after preincubation of the cells for 7 days with retinoic acid (0.1mM) to induce differentiation (D and E) , then washed with PBS plus 2% serum followed by methanol at 4o C. PSD-95 was visualised with a 1:500 dilution of PSD-95 rabbit antibody (A) (Abcam, Cambridge, UK, AB18258) and 1:160 of FITC anti-rabbit IgG and the peptides (B) with 1:500 ExtrAvidin TRITC conjugate (Sigma). Fig2 F shows lack of intracellular localization of R1, a version of R2 without the cell penetration sequence, with ExtrAvidin TRITC. Colocalisation analysis (Pearson’s correlation) with retinoic-acid-treated cells revealed verysimilar readings (R2 A=0.971) to cells not treated with retinoic acid (0.98) suggesting no changes to the peptide binding to PSD-95.

Characterization of PSD95 in SHSY-5Y Cells

To analyse any changes to the cellular levels and molecular structure of PSD-95 caused by retinoic acid(RA) treatment, a western blot analysis of RA treated cells and control cells was performed (Figure 3). PSD-95 has a calculated molecular weight of 95kDa, equivalent to the bands seen on the western blot which illustrates the presence of PSD-95 in the SHSY-5Y cells in all conditions tested (Figure 3), with an up-regulation of PSD95 cells treated with retinoic acid for 24 hrs (Figure 3, lane 1).This conflicts with results obtained from the immunohistochemistry, as an increase in PSD-95 staining intensity was not seen. However it is possible that because PSD-95 is partially recruited to neurites the intensity may be similar but the overall concentration of PSD-95 within the cell is greater. It is also clear that PSD-95 is further up-regulated after the longer treatment period of 120 hours (Figure 3 [lane 3]). The broadening of the band seen in lanes 1 and 3 may indicate cellular processing (eg changes in glycolipid post-translational modification)) which is suggestive of changes in cell trafficking or modification during postsynaptic clustering (Topinca and Bredt, 1998) or expression of spliced variants (Niethammer et al.,1998).


Stability of PDZ-Binding Peptides

We compared the stability of the cyclic biotinylated PMCA2b peptide, R3 and its linear counterpart R2, in the presence of SHSY-5Y cells. Separation of the peptides remaining in the cell supernatants or internalized into the cells was obtained from cell supernatants of cell lysates respectively, by binding to avidin-coated plates. The bound peptides were released from the plates after washing by incubation with acetonitrile and 0.1% TFA, and amounts remaining of cyclic R2 or the linear form R3 quantitated from the mass peaks at 2189(R2) and 2137.5(R3), using Lucid (Biorad) software, are compared in Fig.5.