Human papillomaviruses and the specificity of PDZ domain targeting

The human papillomavirus (HPV) E6 oncoprotein is fundamental to the ability of these viruses to induce human malignancy. A defining characteristic of the HPV E6 oncoproteins found in cancer-causing HPV types is the presence of a PDZ binding motif at their extreme C-terminus. Through this motif, E6 is able to interact with a large number of cellular proteins that contain PDZ domains. Many of these cellular proteins are involved in regulation of processes associated with the control of cell attachment, cell proliferation, cell polarity and cell signaling. How E6 targets multiple proteins containing the same recognition domain is still an open question. In this review, we highlight aspects of E6 function and biology that help to answer this question, and thereby provide insight into the role of these substrates during development of HPV-induced malignancy.     

Synectin, syndecan-4 cytoplasmic domain binding PDZ protein, inhibits cell migration

Syndecan-4, a member of the syndecan gene family of proteoglycans, is an important regulator of bFGF signaling. In particular, bFGF-dependent regulation of cell growth and migration has been linked to syndecan-4 cytoplasmic domain-mediated interactions. Screening of a yeast two-hybrid library with a cytoplasmic domain of rat syndecan-4 identified a novel binding partner, here termed synectin. Synectin is highly homologous to semaphorin F binding protein semcap1, glucose 1 transporter binding protein glut1cbp, and RGS-GAIP/neuropilin-1 binding protein GIPC. Overexpression of synectin in ECV304 cells in culture led to a dose-dependent inhibition of migration while not affecting cell adhesion or growth rate. We conclude that synectin is involved in syndecan-4-dependent interactions and may play a role in the assembly of syndecan-4 signaling complex.     

Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1. Insights into determinants of PDZ domain specificity

We report a structural comparison of the first PDZ domain of ZO-1 (ZO1-PDZ1) and the PDZ domain of Erbin (Erbin-PDZ). Although the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WV(COOH)), that of ZO1-PDZ1 is similar ([R/K/S/T][T/S][W/Y][V/I/L](COOH)) but broadened by increased promiscuity for three of the last four ligand residues. Consequently, the biological function of ZO-1 is also broadened, as it interacts with both tight and adherens junction proteins, whereas Erbin is restricted to adherens junctions. Structural analyses reveal that the differences in specificity can be accounted for by two key differences in primary sequence. A reduction in the size of the hydrophobic residue at the base of the site(0) pocket enables ZO1-PDZ1 to accommodate larger C-terminal residues. A single additional difference alters the specificity of both site(-1) and site(-3). In ZO1-PDZ1, an Asp residue makes favorable interactions with both Tyr(-1) and Lys/Arg(-3). In contrast, Erbin-PDZ contains an Arg at the equivalent position, and this side chain cannot accommodate either Tyr(-1) or Lys/Arg(-3) but, instead, interacts favorably with Glu/Asp(-3). We propose a model for ligand recognition that accounts for interactions extending across the entire binding site but that highlights several key specificity switches within the PDZ domain fold.     

Identification of small-molecule compounds targeting the dishevelled PDZ domain by virtual screening and binding studies

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/β-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2 μM affinity and had a 0.186 μM K_D value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural–kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.     

Chemically modified peptides targeting the PDZ domain of GIPC as a therapeutic approach for cancer

GIPC (GAIP-interacting protein, C terminus) represents a new target class for the discovery of chemotherapeutics. While many of the current generation of anticancer agents function by directly binding to intracellular kinases or cell surface receptors, the disruption of cytosolic protein-protein interactions mediated by non-enzymatic domains is an underdeveloped avenue for inhibiting cancer growth. One such example is the PDZ domain of GIPC. Previously we developed a molecular probe, the cell-permeable octapeptide CR1023 (N-myristoyl-PSQSSSEA), which diminished proliferation of pancreatic cancer cells. We have expanded upon that discovery using a chemical modification approach and here report a series of cell-permeable, side chain-modified lipopeptides that target the GIPC PDZ domain in vitro and in vivo. These peptides exhibit significant activity against pancreatic and breast cancers, both in cellular and animal models. CR1166 (N-myristoyl-PSQSK(εN-4-bromobenzoyl)SK(εN-4-bromobenzoyl)A), bearing two halogenated aromatic units on alternate side chains, was found to be the most active compound, with pronounced down-regulation of EGFR/1GF-1R expression. We hypothesize that these organic acid-modified residues extend the productive reach of the peptide beyond the canonical binding pocket, which defines the limit of accessibility for the native proteinogenic sequences that the PDZ domain has evolved to recognize. Cell permeability is achieved with N-terminal lipidation using myristate, rather than a larger CPP (cell-penetrating peptide) sequence. This, in conjunction with optimization of targeting through side chain modification, has yielded an approach that will allow the discovery and development of next-generation cellular probes for GIPC PDZ as well as for other PDZ domains.     

Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain

hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ- protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2- mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.     

Interaction partners of the PDZ domain of erbin

In order to identify proteins that bind to the PDZ domain of Erbin, we tested the C-termini of several proteins in a yeast two-hybrid assay. ErbB2, APC, beta-catenin, c-Rel and HTLV-1 Tax were identified as ligands of the PDZ domain of Erbin. The interactions were verified by co-immunoprecipitation experiments. These findings demonstrate the promiscuity of the PDZ domain of Erbin.     

The second PDZ domain of zonula occludens-1 is dispensable for targeting to connexin 43 gap junctions

Zonula occludens (ZO)-1 is emerging as a central player in the control of gap junction (GJ) dynamics. Previously the authors reported that ZO-1 localizes preferentially to the periphery of Cx43 GJs. How ZO-1 arrives at GJ edges is unknown, but this targeting might involve we established interaction between the Cx43 C-terminus and the PDZ2 domain of ZO-1. Here the show that despite blocking the canonical PDZ2-mediated interaction by fusion of GFP to the C-terminus of Cx43, ZO-1 continued to target to domains juxtaposed with the edges of GJs comprised solely of tagged Cx43. This edge-association was not abolished by deletion of PDZ2 from ZO-1, as mutant ZO-1 also targeted to the periphery of GJs composed of either tagged or untagged Cx43. Additionally, ZO-2 was found colocalized with ZO-1 at GJ edges. These data demonstrate that ZO-1 targets to GJ edges independently of several known PDZ2-mediated interactions, including ZO-1 homodimerization, heterodimerization with ZO-2, and direct ZO-1 binding to the C-terminal residues of Cx43.     

Both the N-terminal fragment and the protein-protein interaction domain (PDZ domain) are required for the pro-apoptotic activity of presenilin-associated protein PSAP

Presenilin-associated protein (PSAP) was originally identified as a PS1-associated, PDZ domain protein. In a subsequent study, PSAP was found to be a mitochondrial apoptotic molecule. In this study, we cloned the PSAP gene and found that it is composed of 12 exons and localizes on chromosome 6. To better understand the structure and function of PSAP, we have generated a series of antibodies that recognize different regions of PSAP. Using these antibodies, we found that PSAP is expressed in four isoforms as a result of differential splicing of exon 8 in addition to the use of either the first or the second ATG codon as the start codon. We also found that all these isoforms are localized in the mitochondria and are pro-apoptotic. Furthermore, our data revealed that the PDZ domain and N-terminal fragment are required for the pro-apoptotic activity of PSAP.     

Genome-wide analysis of PDZ domain binding reveals inherent functional overlap within the PDZ interaction network

Binding selectivity and cross-reactivity within one of the largest and most abundant interaction domain families, the PDZ family, has long been enigmatic. The complete human PDZ domain complement (the PDZome) consists of 267 domains and we applied here a Bayesian selectivity model to predict hundreds of human PDZ domain interactions, using target sequences of 22,997 non-redundant proteins. Subsequent analysis of these binding scores shows that PDZs can be divided into two genome-wide clusters that coincide well with the division between canonical class 1 and 2 PDZs. Within the class 1 PDZs we observed binding overlap at unprecedented levels, mediated by two residues at positions 1 and 5 of the second α-helix of the binding pocket. Eight PDZ domains were subsequently selected for experimental binding studies and to verify the basics of our predictions. Overall, the PDZ domain class 1 cross-reactivity identified here implies that auxiliary mechanisms must be in place to overcome this inherent functional overlap and to minimize cross-selectivity within the living cell. Indeed, when we superimpose PDZ domain binding affinities with gene ontologies, network topology data and the domain position within a PDZ superfamily protein, functional overlap is minimized and PDZ domains position optimally in the binding space. We therefore propose that PDZ domain selectivity is achieved through cellular context rather than inherent binding specificity.     

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.