Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases.

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein-protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

The PDZ protein SCRIB regulates sodium/iodide symporter (NIS) expression at the basolateral plasma membrane.

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.

Gßγ recruits and activates P-Rex1 via two independent binding interfaces.

Gßγ marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, Gßγ recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which Gßγ stimulates P-Rex1 has been debated. We hypothesized that Gßγ activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that Gßγ binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with Gßγ, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to Gßγ, swapped into P-Rex1. Gßγ recruited Q-Rhox to the plasma membrane, indicating that Gßγ/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, Gßγ recruits P-Rex1 via the Gßγ/PDZ-PDZ interface followed by a second contact involving the Gßγ/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane.

Conformational flexibility of GRASPs and their constituent PDZ subdomains reveals structural basis of their promiscuous interactome.

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD-95, DLG, and ZO-1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual ß3 α1 ß4 ß5 α2 ß6 ß1 ß2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable ß2 -strand and a more flexible and unstable α2 -helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.

Viral targeting of PDZ polarity proteins in the immune system as a potential evasion mechanism.

PDZ proteins are highly conserved through evolution; the principal function of this large family of proteins is to assemble protein complexes that are involved in many cellular processes, such as cell-cell junctions, cell polarity, recycling, or trafficking. Many PDZ proteins that have been identified as targets of viral pathogens by promoting viral replication and spread are also involved in epithelial cell polarity. Here, we briefly review the PDZ polarity proteins in cells of the immune system to subsequently focus on our hypothesis that the viral PDZ-dependent targeting of PDZ polarity proteins in these cells may alter the cellular fitness of the host to favor that of the virus; we further hypothesize that this modification of the cellular fitness landscape occurs as a common and widespread mechanism for immune evasion by viruses and possibly other pathogens.-Gutiérrez-González, L. H., Santos-Mendoza, T. Viral targeting of PDZ polarity proteins in the immune system as a potential evasion mechanism.

PDZ proteins are expressed and regulated in antigen-presenting cells and are targets of influenza A virus.

In this work, we identified the expression, regulation, and viral targeting of Scribble and Dlg1 in antigen-presenting cells. Scribble and Dlg1 belong to the family of PDZ (postsynaptic density (PSD95), disc large (Dlg), and zonula occludens (ZO-1)) proteins involved in cell polarity. The relevance of PDZ proteins in cellular functions is reinforced by the fact that many viruses interfere with host PDZ-dependent interactions affecting cellular mechanisms thus favoring viral replication. The functions of Scribble and Dlg have been widely studied in polarized cells such as epithelial and neuron cells. However, within the cells of the immune system, their functions have been described only in T and B lymphocytes. Here we demonstrated that Scribble and Dlg1 are differentially expressed during antigen-presenting cell differentiation and dendritic cell maturation. While both Scribble and Dlg1 seem to participate in distinct dendritic cell functions, both are targeted by the viral protein NS1 of influenza A in a PDZ-dependent manner in dendritic cells. Our findings suggest that these proteins might be involved in the mechanisms of innate immunity and/or antigen processing and presentation that can be hijacked by viral pathogens.

The PDZ-Binding Motif of HPV16-E6 Oncoprotein Modulates the Keratinization and Stemness Transcriptional Profile In Vivo.

OBJECTIVE: The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif. MATERIALS AND METHODS: The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence. RESULTS: The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151. CONCLUSION: The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.

The Gpn3 Q279* cancer-associated mutant inhibits Gpn1 nuclear export and is deficient in RNA polymerase II nuclear targeting.

Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting.

New structural insights into Golgi Reassembly and Stacking Protein (GRASP) in solution.

Among all proteins localized in the Golgi apparatus, a two-PDZ (PSD95/DlgA/Zo-1) domain protein plays an important role in the assembly of the cisternae. This Golgi Reassembly and Stacking Protein (GRASP) has puzzled researchers due to its large array of functions and relevance in Golgi functionality. We report here a biochemical and biophysical study of the GRASP55/65 homologue in Cryptococcus neoformans (CnGRASP). Bioinformatic analysis, static fluorescence and circular dichroism spectroscopies, calorimetry, small angle X-ray scattering, solution nuclear magnetic resonance, size exclusion chromatography and proteolysis assays were used to unravel structural features of the full-length CnGRASP. We detected the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. Our results indicate an unusual behavior of CnGRASP in solution, closely resembling a class of intrinsically disordered proteins called molten globule proteins. To the best of our knowledge, this is the first structural characterization of a full-length GRASP and observation of a molten globule-like behavior in the GRASP family. The possible implications of this and how it could explain the multiple facets of this intriguing class of proteins are discussed.

Allosteric communication pathways and thermal rectification in PDZ-2 protein: a computational study.

Allosteric communication in proteins is a fundamental and yet unresolved problem of structural biochemistry. Previous findings, from computational biology ( Ota, N.; Agard, D. A. J. Mol. Biol. 2005 , 351 , 345 - 354 ), have proposed that heat diffuses in a protein through cognate protein allosteric pathways. This work studied heat diffusion in the well-known PDZ-2 protein, and confirmed that this protein has two cognate allosteric pathways and that heat flows preferentially through these. Also, a new property was also observed for protein structures: heat diffuses asymmetrically through the structures. The underling structure of this asymmetrical heat flow was a normal length hydrogen bond (∼2.85 Å) that acted as a thermal rectifier. In contrast, thermal rectification was compromised in short hydrogen bonds (∼2.60 Å), giving rise to symmetrical thermal diffusion. Asymmetrical heat diffusion was due, on a higher scale, to the local, structural organization of residues that, in turn, was also mediated by hydrogen bonds. This asymmetrical/symmetrical energy flow may be relevant for allosteric signal communication directionality in proteins and for the control of heat flow in materials science.

Protein clustering and RNA phylogenetic reconstruction of the influenza A [corrected] virus NS1 protein allow an update in classification and identification of motif conservation.

The non-structural protein 1 (NS1) of influenza A virus (IAV), coded by its third most diverse gene, interacts with multiple molecules within infected cells. NS1 is involved in host immune response regulation and is a potential contributor to the virus host range. Early phylogenetic analyses using 50 sequences led to the classification of NS1 gene variants into groups (alleles) A and B. We reanalyzed NS1 diversity using 14,716 complete NS IAV sequences, downloaded from public databases, without host bias. Removal of sequence redundancy and further structured clustering at 96.8% amino acid similarity produced 415 clusters that enhanced our capability to detect distinct subgroups and lineages, which were assigned a numerical nomenclature. Maximum likelihood phylogenetic reconstruction using RNA sequences indicated the previously identified deep branching separating group A from group B, with five distinct subgroups within A as well as two and five lineages within the A4 and A5 subgroups, respectively. Our classification model proposes that sequence patterns in thirteen amino acid positions are sufficient to fit >99.9% of all currently available NS1 sequences into the A subgroups/lineages or the B group. This classification reduces host and virus bias through the prioritization of NS1 RNA phylogenetics over host or virus phenetics. We found significant sequence conservation within the subgroups and lineages with characteristic patterns of functional motifs, such as the differential binding of CPSF30 and crk/crkL or the availability of a C-terminal PDZ-binding motif. To understand selection pressures and evolution acting on NS1, it is necessary to organize the available data. This updated classification may help to clarify and organize the study of NS1 interactions and pathogenic differences and allow the drawing of further functional inferences on sequences in each group, subgroup and lineage rather than on a strain-by-strain basis.

Loop-tail phenotype in heterozygous mice and neural tube defects in homozygous mice result from a nonsense mutation in the Vangl2 gene.

N-ethyl-N-nitrosourea (ENU) is a powerful point mutagen that can generate random mutations. It has been used to generate mouse mutations to produce phenotypic models of human disease. Neural tube defects (NTD) are common birth defects in which the brain and/or spinal cord can be exposed; however, the mechanisms of these defects are poorly understood. Craniorachischisis is one type of NTD that bears a close resemblance to the phenotype of the loop-tail (Lp) mouse. Here we describe a C57BL/6J Lp mouse generated by ENU-induced mutagenesis. The mutation was mapped to the Vangl2 gene on chromosome 1, near markers D1Mit113 and D1Mit149. Sequence analysis of Vangl2 heterozygotes (Vangl2(m1Yzcm)/+) revealed a C/T transition mutation that resulted in substitution of a glutamine codon for a stop (nonsense) codon at position 449. The Vangl2 protein is involved in epithelium planar cell polarity. The predicted truncated protein would lack the PDZ-domain binding motif involved in protein-protein interaction; therefore, Vangl2(m1Yzcm) may be a loss-of-function mutant. Morphological and histological examination of homozygous mouse embryos revealed a neural tube closure defect that leads to craniorachischisis. This Vangl2(m1Yzcm) mouse represents a valuable model for the study of NTDs in humans.

PDZ-containing proteins as targets in human pathologies.

PDZ domains are very abundant protein interaction domains widespread in nature. A large amount of evidence has underscored the importance of the PDZ interactions in the control of intracellular pathways whose abnormal regulation may lead to the development of several pathologies. This series of minireviews covers different aspects of human PDZ-containing proteins, underlining and discussing new concepts and findings.

Differential expression of PDZ domain-containing proteins in human diseases - challenging topics and novel issues.

The general features of the PDZ domain structure and functions have been extensively studied during the last decade. PDZ domains are generally present in proteins that are involved in multiple interactions to assemble functional protein complexes that control key cellular processes. One of the best characterized functions of PDZ domain-containing proteins is control of epithelial cell polarity and cell-cell contacts. In the present review, we summarize the current knowledge on regulation of expression of certain PDZ polarity proteins localized at the intercellular junctions. In addition, we provide a critical overview of recent findings regarding the role of these proteins during development of human diseases. Complete understanding of these issues is valuable for the design of novel therapeutic intervention for common pathologies, such as cancer.

Equilibrium unfolding of the PDZ domain of ß2-syntrophin.

ß2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic ß-cells. It contains a PDZ domain (ß2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of ß2-syntrophin allosterically regulates the affinity of ß2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of ß2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, ß2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of ß2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.