On Classification of PDZ Domains: A Computational Study

Our goal in this present study is to introduce new wavelet based methods for differentiating and classifying Class I and Class II PDZ domains and compare the resulting signals. PDZ domains represent one of the most common protein homology regions playing key roles in several diseases. To perform the classification, we developed two methods. The first of our methods was comparable to the standard wavelet approaches while the second one surpasses it in recognition accuracy. Our models exhibited interesting results, and we anticipate that it can be used as a computational technique to screen out the misfit candidates and to reduce the search space, while achieving high classification and accuracy

Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum

BACKGROUND: Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored. METHODS: We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays. RESULTS: SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system. CONCLUSIONS: In this study, we first characterized a PDZ domain-containing protein GIPC3 in S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections

Putting into Practice Domain-Linear Motif Interaction Predictions for Exploration of Protein Networks

PDZ domains recognise short sequence motifs at the extreme C-termini of proteins. A model based on microarray data has been recently published for predicting the binding preferences of PDZ domains to five residue long C-terminal sequences. Here we investigated the potential of this predictor for discovering novel protein interactions that involve PDZ domains. When tested on real negative data assembled from published literature, the predictor displayed a high false positive rate (FPR). We predicted and experimentally validated interactions between four PDZ domains derived from the human proteins MAGI1 and SCRIB and 19 peptides derived from human and viral C-termini of proteins. Measured binding intensities did not correlate with prediction scores, and the high FPR of the predictor was confirmed. Results indicate that limitations of the predictor may arise from an incomplete model definition and improper training of the model. Taking into account these limitations, we identified several novel putative interactions between PDZ domains of MAGI1 and SCRIB and the C-termini of the proteins FZD4, ARHGAP6, NET1, TANC1, GLUT7, MARCH3, MAS, ABC1, DLL1, TMEM215 and CYSLTR2. These proteins are localised to the membrane or suggested to act close to it and are often involved in G protein signalling. Furthermore, we showed that, while extension of minimal interacting domains or peptides toward tandem constructs or longer peptides never suppressed their ability to interact, the measured affinities and inferred specificity patterns often changed significantly. This suggests that if protein fragments interact, the full length proteins are also likely to interact, albeit possibly with altered affinities and specificities. Therefore, predictors dealing with protein fragments are promising tools for discovering protein interaction networks but their application to predict binding preferences within networks may be limited

PDZ Protein Regulation of β-arrestin Recruitment and GPCR Trafficking

β-arrestins are versatile adaptor proteins that play a vital role in regulation of G protein coupled receptor (GPCR) trafficking and signalling properties. PDZ proteins have previously been shown to modulate β-arrestin2 recruitment and receptor internalization for many GPCRs including Corticotropin-Releasing Factor Receptor 1 (CRFR1), a receptor whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects. Further characterization of the interplay between β-arrestins and PDZ proteins may aid in determining a potential mechanism for PDZ protein regulation of GPCR trafficking. Our findings suggest that PDZ proteins PSD-95, MAGI1, and PDZK1 complex with β-arrestin2 by interacting via the PDZ domain. Using a proteomic approach, mutational analyses were used to reveal that the β-arrestin2 A175F mutant impairs interaction with PSD-95. Additionally, this mutant form of β-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1. Thus, investigating how β-arrestins and PDZ proteins interact could provide further insight into GPCR trafficking properties and the development of novel therapeutics

Molecular basis of class III ligand recognition by PDZ3 in murine protein tyrosine phosphatase PTPN13

Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, represents a large multi-domain non-transmembrane scaffolding protein that contains five consecutive PDZ domains. Here, we report the solution structures of the extended murine PTPN13 PDZ3 domain in its apo form and in complex with its physiological ligand, the carboxy-terminus of protein kinase C-related kinase-2 (PRK2), determined by multidimensional NMR spectroscopy. Both in its ligand-free state and when complexed to PRK2, PDZ3 of PTPN13 adopts the classical compact, globular D/E fold. PDZ3 of PTPN13 binds five carboxy-terminal amino acids of PRK2 via a groove located between the EB-strand and the DB-helix. The PRK2 peptide resides in the canonical PDZ3 binding cleft in an elongated manner and the amino acid side chains in position P0 and P-2, cysteine and aspartate, of the ligand face the groove between EB-strand and DB-helix, whereas the PRK2 side chains of tryptophan and alanine located in position P-1 and P-3 point away from the binding cleft. These structures are rare examples of selective class III ligand recognition by a PDZ domain and now provide a basis for the detailed structural investigation of the promiscuous interaction between the PDZ domains of PTPN13 and their ligands. They will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTPN13 and could ultimately contribute to low molecular weight antagonists that might even act on the PRK2 signaling pathway to modulate rearrangements of the actin cytoskeleton

A MINIMAL CONSERVED REGION OF SHROOM2 AND SHROOM3 MEDIATES ACTIN BINDING AND BUNDLING

Changes in cell morphology are essential for generating the complex tissues that make up all organisms. Regulation of the cell’s morphology is dynamic and is achieved through the coordinated activity of numerous signaling pathways and proteins that converge on the cytoskeleton at many levels. In epithelial cells, the Shroom family of proteins drives anisotropic changes to morphology such as apical constriction and convergent extension in the formation of such structures as the neural tube, vasculature, gut, and eye. Shroom proteins are a class of actin-binding proteins that recruit Rho-associated coiled-coil Kinase (or Rock), to the cytoskeleton to induce contraction of the actin network and effect change. Recent work in this field has focused on understanding the direct interaction of Shroom and Rock, but little work has been done to understand how Shroom proteins directly interact with actin. The actin-binding ability of these proteins has been localized to Shroom Domain 1, a domain unique to Shroom proteins. My work has centered on better understanding the interaction between the SD1 and actin. I have identified small, conserved regions of both Shroom2 and Shroom3 that are capable of binding and bundling actin with similar affinity. With the use of Transmission Electron Microscopy, I observed that both proteins organize actin filaments into similar tightly packed parallel or anti-parallel bundles. While Shroom proteins localize to different populations of actin within the cell, it appears that the ability to bind and bundle actin has been conserved, and that other region of the proteins must drive their unique localization patterns

Interactions and S-acylation of Sprouty and SPRED proteins

Sprouty (Spry)/SPRED proteins are important regulators of the MAPK/ERK signalling pathway, and dysregulation of this pathway can contribute to development of cancer. The defining feature of Spry/SPRED proteins is a highly conserved cysteine-rich Sprouty domain, which for Spry2 contains 26 cysteine residues. Recent work has shown that the SPR domain is S-acylated by zDHHC17. The aim of this thesis is to explore the mechanisms of interaction and S-acylation of Spry/SPRED proteins by zDHHC17 and identify downstream effects of these interactions. The approaches used included expression of zDHHC17 and Spry/SPRED mutants in HEK-293T and PC12 cells, followed by analysis of protein interactions by immunoprecipitation and analysis of S-acylation using click chemistry methodologies. Protein localisation was examined using confocal microscopy and protein stability measured in cycloheximide-chase experiments. The use of AlphaFold structural predictions and other bioinformatic tools was used to inform these analyses. The results indicate that the interaction of Spry2 and zDHHC17 has a unique dual-stabilisation effect on both the enzyme and substrate. Furthermore, a novel mechanism of recognition/S-acylation of Spry/SPRED proteins, which is distinct from the mechanism of SNAP25 recognition/S-acylation by zDHHC17 was identified. Specifically, the zDABM sequence of Spry2 which is the major interaction site for zDHHC17 was shown to be dispensable for S-acylation. Analysis of SPRED3, which lacks a zDABM sequence, showed that the highly conserved SPR domain contains a novel zDHHC17 binding site that facilitates S-acylation. This thesis also identifies a potential effect of Spry2 on the S-acylation of the SARS-CoV-2 accessory protein, Orf3d, which should be investigated further in future work. Collectively, the findings in this thesis provide important new information on substrate recognition by zDHHC enzymes. This new knowledge can assist in identifying new zDHHC17 substrates and could also be used to develop peptide-based inhibitors that disrupt specific substrate interactions of zDHHC17.Sprouty (Spry)/SPRED proteins are important regulators of the MAPK/ERK signalling pathway, and dysregulation of this pathway can contribute to development of cancer. The defining feature of Spry/SPRED proteins is a highly conserved cysteine-rich Sprouty domain, which for Spry2 contains 26 cysteine residues. Recent work has shown that the SPR domain is S-acylated by zDHHC17. The aim of this thesis is to explore the mechanisms of interaction and S-acylation of Spry/SPRED proteins by zDHHC17 and identify downstream effects of these interactions. The approaches used included expression of zDHHC17 and Spry/SPRED mutants in HEK-293T and PC12 cells, followed by analysis of protein interactions by immunoprecipitation and analysis of S-acylation using click chemistry methodologies. Protein localisation was examined using confocal microscopy and protein stability measured in cycloheximide-chase experiments. The use of AlphaFold structural predictions and other bioinformatic tools was used to inform these analyses. The results indicate that the interaction of Spry2 and zDHHC17 has a unique dual-stabilisation effect on both the enzyme and substrate. Furthermore, a novel mechanism of recognition/S-acylation of Spry/SPRED proteins, which is distinct from the mechanism of SNAP25 recognition/S-acylation by zDHHC17 was identified. Specifically, the zDABM sequence of Spry2 which is the major interaction site for zDHHC17 was shown to be dispensable for S-acylation. Analysis of SPRED3, which lacks a zDABM sequence, showed that the highly conserved SPR domain contains a novel zDHHC17 binding site that facilitates S-acylation. This thesis also identifies a potential effect of Spry2 on the S-acylation of the SARS-CoV-2 accessory protein, Orf3d, which should be investigated further in future work. Collectively, the findings in this thesis provide important new information on substrate recognition by zDHHC enzymes. This new knowledge can assist in identifying new zDHHC17 substrates and could also be used to develop peptide-based inhibitors that disrupt specific substrate interactions of zDHHC17

Determinantes moleculares de la interacción entre los canales cardiacos humanos Nav1.5 y Kir2.x

La corriente de sodio cardíaca (INa), generada por los canales Nav1.5, es la responsable de la entrada de Na+ que despolariza la membrana durante el potencial de acción (PA) regulando la excitabilidad y la propagación del impulso cardíaco. Por otro lado, la corriente de salida de K+ con rectificación interna (IK1), generada por los canales Kir2.x, es la responsable de establecer el potencial de reposo así como de modular la fase final de la repolarización y la duración del PA (DPA) en los miocitos cardíacos. Por tanto, la IK1 participa en el control de la excitabilidad y la refractariedad cardíacas. La magnitud de la INa depende del potencial de reposo y de la DPA, por ello se afirma que existe una relación funcional entre la INa y la IK1. Además, se ha demostrado que la relación de las magnitudes de la INa y la IK1 determinan de forma crítica la estabilidad y velocidad de giro de los frentes de reentrada espirales (rotores) responsables de la génesis de las arritmias fibrilatorias tanto a nivel auricular como ventricular. Recientemente se ha demostrado que los canales Nav1.5 y Kir2.1 modulan recíprocamente su expresión en la membrana, de esta forma el aumento o la disminución en la expresión de canales Kir2.1 produce un aumento o disminución en la expresión de canales Nav1.5 y, viceversa. Estos resultados sugerían que la relación de las magnitudes de la INa y la IK1 es tan importante para el control de la excitabilidad y refractariedad cardíacas que su modulación no sólo es funcional. En este momento se desconocen los determinantes moleculares y los mecanismos celulares responsables de la modulación recíproca positiva entre canales Nav1.5 y Kir2.1. Se desconoce también si se produce modulación recíproca positiva entre los canales Nav1.5 y los Kir2.2 y Kir2.3. Este hecho tiene una gran relevancia funcional, considerando que la IK1 ventricular es generada por canales Kir2.1/Kir2.2, mientras que los canales Kir2.3 parecen jugar un papel predominante en la génesis de la IK1 auricular..