PDZscape: a comprehensive PDZ-protein database.

PDZ-containing proteins comprise one of the most widely distributed protein families playing major role in localization and membrane receptor clustering. They are hence important regulators of signal transduction in cellular pathways. Although knowledge on these proteins has increased exponentially, the existing database 'PDZBase' is limited by presence of only 339 proteins as it dates back to 2004 when very little data was available. Thus, lack of exclusive information on this protein family led us to develop PDZscape. 'PDZscape' encompasses the complete available information on 58,648 PDZ-containing proteins with their known and putative binding partners on one platform. It has a user-friendly web interface that can be easily queried with external protein identifiers. With unique integration of prominent databases including NCBI, UniProtKB, Swiss-Prot, Pubmed, PDB, STRING, IntAct, KEGG, Pfam and Protein Mutant Database, it provides detailed information on PDZ interactome apart from the customized BLAST option. Most importantly, this database encompasses the mutations and diseases associated with PDZ containing proteins manually curated by our group, thus making it a comprehensive compilation. It also features tools to query the database using sequence (PDZ-Blast) and to find if protein of interest is a PDZ-binding protein. PDZscape is freely available at http://www.actrec.gov.in:8080/pdzscape .

CFTR-associated ligand is a negative regulator of Mrp2 expression.

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.

Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release.

Antidepressants are one of the first line treatments for neuropathic pain but their use is limited by the incidence and severity of side effects of tricyclics and the weak effectiveness of selective serotonin reuptake inhibitors (SSRIs). Serotonin type 2A (5-HT2A) receptors interact with PDZ proteins that regulate their functionality and SSRI efficacy to alleviate pain. We investigated whether an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT2A receptors and associated PDZ proteins would improve the treatment of traumatic neuropathic allodynia. Tactile allodynia was assessed in spinal nerve ligation-induced neuropathic pain in rats using von Frey filaments after acute treatment with TAT-2ASCV and/or 5-HT2A receptor agonist, alone or in combination with repeated treatment with fluoxetine. In vivo microdialysis was performed in order to examine the involvement of GABA in TAT-2ASCV/fluoxetine treatment-associated analgesia. TAT-2ASCV (100ng, single i.t. injection) improved SNL-induced tactile allodynia by increasing 5-HT2A receptor responsiveness to endogenous 5-HT. Fluoxetine alone (10mg/kg, five i.p. injections) slightly increased tactile thresholds and its co-administration with TAT-2ASCV (100ng, single i.t. injection) further enhanced the anti-allodynic effect. This effect depends on the integrity of descending serotonergic bulbospinal pathways and spinal release of GABA. The anti-allodynic effect of fluoxetine can be enhanced by disrupting 5-HT2A receptor-PDZ protein interactions. This enhancement depends on 5-HT2A receptor activation, spinal GABA release and GABAA receptor activation.

The PDZ-domain protein Whirlin facilitates mechanosensory signaling in mammalian proprioceptors.

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems.

Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFß signaling intermediates, accumulated in the nucleus and Snail, a TGFß target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFß signaling.

Modeling of host PDZ-dependent interactions with SARS-CoV-2 Envelope protein and changes in PDZ proteins expression in macrophages and dendritic cells.

PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks, and mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens which express proteins containing PDZ-binding motifs (PDZbm). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of seven host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of three of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and IL-16), in human monocyte-derived macrophages (MΦ) and in dendritic cells (DCs) upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells (APCs), and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, support the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. Inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed.

Molecular Mechanisms of HIV Protease Inhibitors Against HPV-Associated Cervical Cancer: Restoration of TP53 Tumour Suppressor Activities.

Cervical cancer is a Human Papilloma virus-related disease, which is on the rise in a number of countries, globally. Two essential oncogenes, E6 and E7, drive cell transformation and cancer development. These two oncoproteins target two of the most important tumour suppressors, p53 and pRB, for degradation through the ubiquitin ligase pathway, thus, blocking apoptosis activation and deregulation of cell cycle. This pathway can be exploited for anticancer therapeutic interventions, and Human Immunodeficiency Virus Protease Inhibitors (HIV-PIs) have attracted a lot of attention for this anticancer drug development. HIV-PIs have proven effective in treating HPV-positive cervical cancers and shown to restore impaired or deregulated p53 in HPV-associated cervical cancers by inhibiting the 26S proteasome. This review will evaluate the role players, such as HPV oncoproteins involved cervical cancer development and how they are targeted in HIV protease inhibitors-induced p53 restoration in cervical cancer. This review also covers the therapeutic potential of HIV protease inhibitors and molecular mechanisms behind the HIV protease inhibitors-induced p53-dependent anticancer activities against cervical cancer.

Mechanoregulation of PDZ Proteins, An Emerging Function.

Mechanical forces have emerged as essential regulators of cell organization, proliferation, migration, and polarity to regulate cellular and tissue homeostasis. Changes in forces or loss of the cellular response to them can result in abnormal embryonic development and diseases. Over the past two decades, many efforts have been put in deciphering the molecular mechanisms that convert forces into biochemical signals, allowing for the identification of many mechanotransducer proteins. Here we discuss how PDZ proteins are emerging as new mechanotransducer proteins by altering their conformations or localizations upon force loads, leading to the formation of macromolecular modules tethering the cell membrane to the actin cytoskeleton.

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

Roles of NHERF Family of PDZ-Binding Proteins in Regulating GPCR Functions.

Multicellular organisms are equipped with an array of G-protein-coupled receptors (GPCRs) that mediate cell-cell signaling allowing them to adapt to environmental cues and ultimately survive. This is mechanistically possible through complex intracellular GPCR machinery that encompasses a vast network of proteins. Within this network, there is a group called scaffolding proteins that facilitate proper localization of signaling proteins for a quick and robust GPCR response. One protein family within this scaffolding group is the PSD-95/Dlg/ZO-1 (PDZ) family which is important for GPCR localization, internalization, recycling, and downstream signaling. Although the PDZ family of proteins regulate the functions of several receptors, this chapter focuses on a subfamily within the PDZ protein family called the Na+/H+ exchanger regulatory factors (NHERFs). Here we extensively review the predominantly characterized roles of NHERFs in renal phosphate absorption, intestinal ion regulation, cancer progression, and immune cell functions. Finally, we discuss the future perspectives and possible clinical application of targeting NHERFs in several disorders.

Disruption of 5-HT2A-PDZ protein interaction differently affects the analgesic efficacy of SSRI, SNRI and TCA in the treatment of traumatic neuropathic pain in rats.

Antidepressants remain one of the first line treatments prescribed to neuropathic pain patients despite their limited efficacy and/or their numerous side effects. More and more, pharmacotherapy for neuropathic pain has evolved towards the use of therapeutic combinations. The goal of the present study was to assess the efficacy of the combination of antidepressants - selective serotonin reuptake inhibitors and serotonin-noradrenaline reuptake inhibitors-with a peptide (TAT-2ASCV) able to disrupt the interaction between serotonin type 2A (5-HT2A) receptors and associated PDZ proteins. Mechanical hypersensitivity was assessed in sciatic nerve ligation-induced neuropathic pain in rats using paw pressure test after acute treatment with TAT-2ASCV alone or in combination with repeated treatment with fluoxetine or duloxetine or clomipramine. First, we validated the anti-hyperalgesic effect of TAT-2ASCV on mechanical hypersensitivity at the dose of 100 ng/rat (single i.t. injection). Second, using selective receptor antagonists, we found that the effect of TAT-2ASCV on mechanical hypersensitivity involves 5-HT2A as well as GABAA receptors. Finally, we showed that the association of TAT-2ASCV (100 ng, single i.t. injection) with fluoxetine (10 mg/kg, five i.p. injections) reveals its anti-hyperalgesic effect, while the association with duloxetine (1 mg/kg, five i.p. injections) or clomipramine (2.5 mg/kg, five i.p. injections) is only additive. Those results further accentuate the interest to develop small molecules acting like TAT-2ASCV in order to treat neuropathic pain as a monotherapy or in combination with antidepressants.

Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active ß1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

Regulation of hormone-sensitive renal phosphate transport.

Phosphate is essential for growth and maintenance of the skeleton and for generating high-energy phosphate compounds. Evolutionary adaptation to high dietary phosphorous in humans and other terrestrial vertebrates involves regulated mechanisms assuring the efficient renal elimination of excess phosphate. These mechanisms prominently include PTH, FGF23, and Vitamin D, which directly and indirectly regulate phosphate transport. Disordered phosphate homeostasis is associated with pathologies ranging from kidney stones to kidney failure. Chronic kidney disease results in hyperphosphatemia, an elevated calcium×phosphate product with considerable morbidity and mortality, mostly associated with adverse cardiovascular events. This chapter highlights recent findings and insights regarding the hormonal regulation of renal phosphate transport along with imbalances of phosphate balance due to acquired or inherited diseases states.