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The demand for oil and its derivatives has driven the oil and gas sector to explore deep waters. Nickel-based alloys, like Inconel, are used due to their corrosion resistance properties. Cladding structural steels with these alloys is a cost-effective solution. However, dissimilar metal welding poses challenges, including the formation of Partially Diluted Zones (PDZ). This work highlights the use of Electron Backscatter Diffraction (EBSD) as a very adequate technique for the microstructural characterization of dissimilar anticorrosive materials. The MIG process was utilized to perform cladding of Inconel 625 onto the A36 steel substrate. The samples were prepared for analysis using techniques such as EDS, FEG-SEM, EBSD, and Vickers microhardness testing. The results demonstrate the effectiveness of the EBSD technique in characterizing dissimilar metal cladding. The combination of EBSD with EDS analysis and microhardness testing provided comprehensive insights into the microstructure and mechanical properties of the material. EDS mapping analysis confirmed the formation of PDZ with a distinct beach-like morphology, indicating localized compositional variations. The uniform thickness of the PDZ, approximately 20 µm, highlights the convective motion and solidification behavior of the partially diluted zone. Phase map EBSD analysis revealed different crystal structures in the cladding zone, including a FCC structure for the nickel cladding layer and a BCC structure for the ferritic steel base metal. The PDZ exhibited the same crystal structure as the base metal, indicating chemical element diffusion without altering the ferritic steel structure. Contrast band EBSD analysis provided detailed microstructural information, revealing solidification structures and potential hardness variations. The observed martensite formation in the PDZ highlights its susceptibility to cracking and detachment of the coated layer. The hardness results support the microstructural findings, demonstrating significant variations in hardness across the cladding structure, particularly in the PDZ and at the interface between the Inconel alloy and the base metal. These findings contribute valuable insights into the microstructural and compositional gradients within the joint region, reinforcing their implications for material performance.

The surge in deep and ultra-deep water oil exploration in Brazil, particularly in the pre-salt layer, necessitates the advancement of materials and engineering components to meet sectoral challenges. To enhance the longevity and corrosion resistance of pipelines and structures, welded cladding of stainless alloys onto structural steels has been deployed. However, dissimilar welding often leads to the formation of Partially Diluted Zones (PDZ), marked by discontinuous, high-hardness regions at the interface. This study employs Electron Microscopy via Backscattered Electron Diffraction (EBSD) to characterize these zones, addressing limitations in conventional metallographic techniques. By cladding ASTM A36 steel test plates with AWS E 309L (309L stainless steel) through automated MIG welding, metallurgical-mechanical properties were assessed using Field Emission Scanning Electron Microscopy (FEG-SEM), EBSD, SEM-EDS, and Vickers Microhardness. The Heat-Affected Zone (HAZ) displayed low hardness, while the PDZ exhibited varied morphologies with significant concentrations of iron, chromium, and nickel, accompanied by elevated microhardness and a characteristic martensitic microstructure. In comparison between single and double-layer depositions, the latter substantially reduced hardness in the PDZ and HAZ. This study provides critical insights into the microstructural attributes of welded cladding, offering valuable guidance for optimizing materials in deepwater applications.

Simple Summary: There are three subfamilies of human PDZ-LIM family proteins with a total of 10 protein molecules, and PDZ-LIM family proteins serve as a class of scaffolding proteins that assume the function of signal transduction. In this paper, we describe the signature structural domains and major regulatory signals of PDZ-LIM family proteins and provide an overview and discussion of their functions in various tumors and major diseases, aiming to provide directions for future disease (mainly tumor) prevention and drug development. PDZ-LIM family proteins (PDLIMs) are a kind of scaffolding proteins that contain PDZ and LIM interaction domains. As protein–protein interacting molecules, PDZ and LIM domains function as scaffolds to bind to a variety of proteins. The PDLIMs are composed of evolutionarily conserved proteins found throughout different species. They can participate in cell signal transduction by mediating the interaction of signal molecules. They are involved in many important physiological processes, such as cell differentiation, proliferation, migration, and the maintenance of cellular structural integrity. Studies have shown that dysregulation of the PDLIMs leads to tumor formation and development. In this paper, we review and integrate the current knowledge on PDLIMs. The structure and function of the PDZ and LIM structural domains and the role of the PDLIMs in tumor development are described. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are membrane-limited organelles mediating cell-to-cell communication in health and disease. EVs are of high medical interest, but their rational use for diagnostics or therapies is restricted by our limited understanding of the molecular mechanisms governing EV biology. Here, we tested whether PDZ proteins, molecular scaffolds that support the formation, transport, and function of signal transduction complexes and that coevolved with multicellularity, may represent important EV regulators. We reveal that the PDZ proteome (ca. 150 proteins in human) establishes a discrete number of direct interactions with the tetraspanins CD9, CD63, and CD81, well-known EV constituents. Strikingly, PDZ proteins interact more extensively with syndecans (SDCs), ubiquitous membrane proteins for which we previously demonstrated an important role in EV biogenesis, loading, and turnover. Nine PDZ proteins were tested in loss-of-function studies. We document that these PDZ proteins regulate both tetraspanins and SDCs, differentially affecting their steady-state levels, subcellular localizations, metabolism, endosomal budding, and accumulations in EVs. Importantly, we also show that PDZ proteins control the levels of heparan sulfate at the cell surface that functions in EV capture. In conclusion, our study establishes that the extensive networking of SDCs, tetraspanins, and PDZ proteins contributes to EV heterogeneity and turnover, highlighting an important piece of the molecular framework governing intracellular trafficking and intercellular communication. [ABSTRACT FROM AUTHOR]

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus's pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response. [ABSTRACT FROM AUTHOR]

Small structural E protein of coronaviruses uses its C-terminal PDZ motif to compromise the cellular PDZ interactome. In this work we compared core PDZ interactivity of small (seven amino acids) peptide PDZ motifs, originating from the envelope proteins of recently transmitted coronaviruses SARS-CoV, SARS-CoV2, and MERS-CoV. As the interaction targets we used 23 domains of the largest PDZ proteins MUPP1/MPDZ and PATJ/INAD. Results revealed exceptional affinity and interaction promiscuity of MERS-CoV PDZ motif in vitro, suggesting an increased probability of potential PDZ targets in vivo. We hypothesize that together with its known ability to enter the cells from both apical and basolateral sites, this might further contribute to its elevated disruption of cellular PDZ pathways and higher virulence. [ABSTRACT FROM AUTHOR]

Abstract Background Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV. Methods We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7. Results We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis. Conclusions The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.

Protein–protein interactions that involve recognition of short peptides are critical in cellular processes. Protein–peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide‐binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are a family of peptide‐binding domains located in several intracellular signaling and trafficking pathways. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ‐binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain‐containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting‐sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ–peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had marginal effects on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell. [ABSTRACT FROM AUTHOR]

Scribble (Scrib) is a highly conserved cell polarity regulator that harbours potent tumour suppressor activity and plays an important role in cell migration. Dysregulation of polarity is associated with poor prognosis during viral infections. Human T‐cell lymphotrophic virus‐1 (HTLV‐1) encodes for the oncogenic Tax1 protein, a modulator of the transcription of viral and human proteins that can cause cell cycle dysregulation as well as a loss of genomic integrity. Previous studies established that Scribble interacts with Tax1 via its C‐terminal PDZ‐binding motif (PBM), leading to aggregation of polarity regulators and subsequent perturbation of host cell adhesion, proliferation, and signalling. Using isothermal titration calorimetry, we now show that all four PDZ domains of Scribble bind to Tax1 PBM. We then determined crystal structures of Scribble PDZ1, PDZ2 and PDZ3 domains bound to Tax1 PBM. Our findings establish a structural basis for Tax1‐mediated subversion of Scribble‐mediated cell polarity signalling and provide the platform for mechanistic studies to examine Tax1 induced mislocalization of Scribble and the associated changes in cellular architecture and subsequent tumorigenesis. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the high-risk HPV E6 oncoprotein PDZ binding motifs (PBMs) can interact with PDZ proteins or members of the 14-3-3 family, depending upon the E6 phosphorylation status. However, different HPV E6 oncoproteins are subjected to phosphorylation by different cellular kinases. We have therefore been interested in determining whether we can dissect E6's PDZ and 14-3-3 interactions at the molecular level. Using HPV-18 E6, we have found that its Chk1 phosphorylation requires residues both upstream and downstream of the phospho-acceptor site, in addition to the Chk1 consensus recognition motif. Furthermore, we demonstrate that different high-risk HPV E6 types are differentially phosphorylated by Chk1 kinases, potentially due to the differences in their carboxy-terminal residues, as they are critical for kinase recognition. Moreover, differences in the E6 phosphorylation levels of different HR HPV types directly link to their ability to interact with different 14-3-3 isoforms, based on their phospho-status. Interestingly, 14-3-3 recognition appears to be less dependent upon the precise sequence constraints of the E6 carboxy terminal region, whilst minor amino acid variations have a major impact upon PDZ recognition. These results demonstrate that changes in E6 phospho-status during the life cycle or during malignant progression will modulate E6 interactions and, potentially, inversely regulate the levels of PDZ and 14-3-3 proteins.

Abstract Background Recurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC). Thus, it is of great significance to clarify the underlying mechanisms and identify predictors for tailoring adjuvant chemotherapy to improve the outcome of CRC. Methods By screening differentially expressed genes (DEGs), constructing random forest classification and ranking the importance of DEGs, we identified membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) as an important gene in CRC recurrence. Immunohistochemical and western blot assays were employed to further detect MAGI3 expression in CRC tissues and cell lines. Cell counting kit-8, plate colony formation, flow cytometry, sub-cutaneous injection and azoxymethane plus dextran sulfate sodium induced mice CRC assays were employed to explore the effects of MAGI3 on proliferation, growth, cell cycle, apoptosis, xenograft formation and chemotherapy resistance of CRC. The underlying molecular mechanisms were further investigated through gene set enrichment analysis, quantitative real-time PCR, western blot, co-immunoprecipitation, ubiquitination, GST fusion protein pull-down and immunohistochemical staining assays. Results Our results showed that dysregulated low level of MAGI3 was correlated with recurrence and poor prognosis of CRC. MAGI3 was identified as a novel substrate-binding subunit of SKP1-Cullin E3 ligase to recognize c-Myc, and process c-Myc ubiquitination and degradation. Expression of MAGI3 in CRC cells inhibited cell growth, promoted apoptosis and chemosensitivity to fluoropyrimidine-based chemotherapy by suppressing activation of c-Myc in vitro and in vivo. In clinic, the stage II/III CRC patients with MAGI3-high had a significantly good recurrence-free survival (~ 80%, 5-year), and were not necessary for further adjuvant chemotherapy. The patients with MAGI3-medium had a robustly good response rate or recurrence-free survival with fluoropyrimidine-based chemotherapy, and were recommended to undergo fluoropyrimidine-based adjuvant chemotherapy. Conclusions MAGI3 is a novel E3 ubiquitin ligase by degradation of c-Myc to regulate CRC development and may act as a potential predictor of adjuvant chemotherapy for CRC patients. Graphical Abstract

The PDZ family is comprised of small modular domains that play critical roles in the allosteric modulation of many cellular signaling processes by binding to the C‐terminal tail of different proteins. As dominant modular proteins that interact with a diverse set of peptides, it is of particular interest to explore how different binding partners induce different allosteric effects on the same PDZ domain. Because the PICK1 PDZ domain can bind different types of ligands, it is an ideal test case to answer this question and explore the network of interactions that give rise to dynamic allostery. Here, we use all‐atom molecular dynamics simulations to explore dynamic allostery in the PICK1 PDZ domain by modeling two PICK1 PDZ systems: PICK1 PDZ‐DAT and PICK1 PDZ‐GluR2. Our results suggest that ligand binding to the PICK1 PDZ domain induces dynamic allostery at the αA helix that is similar to what has been observed in other PDZ domains. We found that the PICK1 PDZ‐ligand distance is directly correlated with both dynamic changes of the αA helix and the distance between the αA helix and βB strand. Furthermore, our work identifies a hydrophobic core between DAT/GluR2 and I35 as a key interaction in inducing such dynamic allostery. Finally, the unique interaction patterns between different binding partners and the PICK1 PDZ domain can induce unique dynamic changes to the PICK1 PDZ domain. We suspect that unique allosteric coupling patterns with different ligands may play a critical role in how PICK1 performs its biological functions in various signaling networks. [ABSTRACT FROM AUTHOR]

PDZ-LIM family proteins (PDLIMs) are a kind of scaffolding proteins that contain PDZ and LIM interaction domains. As protein–protein interacting molecules, PDZ and LIM domains function as scaffolds to bind to a variety of proteins. The PDLIMs are composed of evolutionarily conserved proteins found throughout different species. They can participate in cell signal transduction by mediating the interaction of signal molecules. They are involved in many important physiological processes, such as cell differentiation, proliferation, migration, and the maintenance of cellular structural integrity. Studies have shown that dysregulation of the PDLIMs leads to tumor formation and development. In this paper, we review and integrate the current knowledge on PDLIMs. The structure and function of the PDZ and LIM structural domains and the role of the PDLIMs in tumor development are described.

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus’s pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response.

Human papillomavirus type 18 (HPV18) has high carcinogenic power in invasive cervical cancer (ICC) development. However, the underlying mechanism remains elusive. The carcinogenic properties of HPV18 require the PDZ‐binding motif of its E6 oncoprotein (HPV18 E6) to degrade its target PSD95/Dlg/ZO‐1 (PDZ) proteins. In this study, we demonstrated that the PDZ protein membrane‐associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) inhibited the Wnt/β‐catenin pathway, and subsequently cervical cancer (CC) cell migration and invasion, via decreasing β‐catenin levels. By reducing MAGI3 protein levels, HPV18 E6 promoted CC cell migration and invasion through activation of Wnt/β‐catenin signaling. Furthermore, HPV18 rather than HPV16 was preferentially associated with the downregulation of MAGI3 and activation of the Wnt/β‐catenin pathway in CC. These findings shed light on the mechanism that gives HPV18 its high carcinogenic potential in CC progression.

One of the most intriguing features of multicellular animals is their ability to move. On a cellular level, this is accomplished by the rearrangement and reorganization of the cytoskeleton, a dynamic network of filamentous proteins which provides stability and structure in a stationary context, but also facilitates directed movement by contracting. The ALP/Enigma family proteins are a diverse group of docking proteins found in numerous cellular milieus and facilitate these processes among others. In vertebrates, they are characterized by having a PDZ domain in combination with one or three LIM domains. The family is comprised of CLP-36 (PDLIM1), Mystique (PDLIM2), ALP (PDLIM3), RIL (PDLIM4), ENH (PDLIM5), ZASP (PDLIM6), and Enigma (PDLIM7). In this review, we will outline the evolution and function of their protein domains which confers their versatility. Additionally, we highlight their role in different cellular environments, focusing specifically on recent advances in muscle research using Drosophila as a model organism. Finally, we show the relevance of this protein family to human myopathies and the development of muscle-related diseases.

Friction stir welding is a brand-new welding technique that was discovered in 1991 by Wayne Thomas in United Kingdom. It is a form of solid state welding in which a non-consumable electrode is used to unite two work components without melting either of them. The mechanical characteristics and numerous microscopic studies of materials used for friction stir welding, such as duplex stainless steel UNS S31803, SA 213 tube to SA387, Al7075 alloy, and AA6061 metal, are reviewed. Here, welded areas were the primary focus of the microscopic study, and the diverse outcomes are also discussed. [ABSTRACT FROM AUTHOR]

Background: Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV.Methods: We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7.Results: We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis.Conclusions: The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation. [ABSTRACT FROM AUTHOR]

Centrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, ciliogenesis, and signaling. Aberrant centrosome number, structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the conserved Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and possibly cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 regulation and function is poorly understood. Here, we combined computational analysis with cell biology approaches to uncover possible structure–function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains: a GEF domain, an Ig-like domain, and a PDZ-like domain. In addition to these predicted structural features, CeSPD-2 is also predicted to be intrinsically disordered. Surface electrostatic maps identified a large basic region unique to the ASH domain of CeSPD-2. This basic region overlaps with most of the residues predicted to be involved in protein–protein interactions. In vivo, ASH::GFP localized to centrosomes and centrosome-associated microtubules. Our analysis groups ASH domains, PapD, Usher chaperone domains, and Major Sperm Protein (MSP) domains into a single superfold within the larger Immunoglobulin superfamily. This study lays the groundwork for designing rational hypothesis-based experiments to uncover the mechanisms of CeSPD-2 function in vivo. Abbreviations: AIR, Aurora kinase; ASH, Aspm-SPD-2 Hydin; ASP, Abnormal Spindle Protein; ASPM, Abnormal Spindle-like Microcephaly-associated Protein; CC, coiled-coil; CDK, Cyclin-dependent Kinase; Ce, Caenorhabditis elegans; CEP, Centrosomal Protein; CPAP, centrosomal P4.1-associated protein; D, Drosophila; GAP, GTPase activating protein; GEF, GTPase guanine nucleotide exchange factor; Hs, Homo sapiens/Human; Ig, Immunoglobulin; MAP, Microtubule associated Protein; MSP, Major Sperm Protein; MDP, Major Sperm Domain-Containing Protein; OCRL-1, Golgi endocytic trafficking protein Inositol polyphosphate 5-phosphatase; PAR, abnormal embryonic PARtitioning of the cytosol; PCM, Pericentriolar material; PCMD, pericentriolar matrix deficient; PDZ, PSD95/Dlg-1/zo-1; PLK, Polo like kinase; RMSD, Root Mean Square Deviation; SAS, Spindle assembly abnormal proteins; SPD, Spindle-defective protein; TRAPP, TRAnsport Protein Particle; Xe, Xenopus; ZYG, zygote defective protein. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is a direct cell-cell communication system involved in a wide variety of biological processes, and its disruption is observed in several pathologies. The pathway is comprised of a ligand-expressing (sender) cell and a receptor-expressing (receiver) cell. The canonical ligands are members of the Delta/Serrate/Lag-1 (DSL) family of proteins. Their binding to a Notch receptor in a neighboring cell induces a conformational change in the receptor, which will undergo regulated intramembrane proteolysis (RIP), liberating the Notch intracellular domain (NICD). The NICD is translocated to the nucleus and promotes gene transcription. It has been demonstrated that the ligands can also undergo RIP and nuclear translocation, suggesting a function for the ligands in the sender cell and possible bidirectionality of the Notch pathway. Although the complete mechanism of ligand processing is not entirely understood, and its dependence on Notch receptors has not been ruled out. Also, ligands have autonomous functions beyond Notch activation. Here we review the concepts of reverse and bidirectional signalization of DSL proteins and discuss the characteristics that make them more than just ligands of the Notch pathway. [ABSTRACT FROM AUTHOR]

Introduction: The PICK1 PDZ domain has been identified as a potential drug target for neurological disorders. After many years of effort, a few inhibitors, such as TAT-C5 and mPD5, have been discovered experimentally to bind to the PDZ domain with a relatively high binding affinity. With the rapid growth of computational research, there is an urgent need for more efficient computational methods to design viable ligands that target proteins., Method: Recently, a newly developed program called AfDesign (part of ColabDesign) at https:// github.com/sokrypton/ColabDesign), an open-source software built on AlphaFold, has been suggested to be capable of generating ligands that bind to targeted proteins, thus potentially facilitating the ligand development process. To evaluate the performance of this program, we explored its ability to target the PICK1 PDZ domain, given our current understanding of it. We found that the designated length of the ligand and the number of recycles play vital roles in generating ligands with optimal properties., Results: Utilizing AfDesign with a sequence length of 5 for the ligand produced the highest comparable ligands to that of prior identified ligands. Moreover, these designed ligands displayed significantly lower binding energy compared to manually created sequences., Conclusion: This work demonstrated that AfDesign can potentially be a powerful tool to facilitate the exploration of the ligand space for the purpose of targeting PDZ domains., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Recurrence and chemoresistance constitute the leading cause of death in colorectal cancer (CRC). Thus, it is of great significance to clarify the underlying mechanisms and identify predictors for tailoring adjuvant chemotherapy to improve the outcome of CRC. Methods: By screening differentially expressed genes (DEGs), constructing random forest classification and ranking the importance of DEGs, we identified membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) as an important gene in CRC recurrence. Immunohistochemical and western blot assays were employed to further detect MAGI3 expression in CRC tissues and cell lines. Cell counting kit-8, plate colony formation, flow cytometry, sub-cutaneous injection and azoxymethane plus dextran sulfate sodium induced mice CRC assays were employed to explore the effects of MAGI3 on proliferation, growth, cell cycle, apoptosis, xenograft formation and chemotherapy resistance of CRC. The underlying molecular mechanisms were further investigated through gene set enrichment analysis, quantitative real-time PCR, western blot, co-immunoprecipitation, ubiquitination, GST fusion protein pull-down and immunohistochemical staining assays. Results: Our results showed that dysregulated low level of MAGI3 was correlated with recurrence and poor prognosis of CRC. MAGI3 was identified as a novel substrate-binding subunit of SKP1-Cullin E3 ligase to recognize c-Myc, and process c-Myc ubiquitination and degradation. Expression of MAGI3 in CRC cells inhibited cell growth, promoted apoptosis and chemosensitivity to fluoropyrimidine-based chemotherapy by suppressing activation of c-Myc in vitro and in vivo. In clinic, the stage II/III CRC patients with MAGI3-high had a significantly good recurrence-free survival (~ 80%, 5-year), and were not necessary for further adjuvant chemotherapy. The patients with MAGI3-medium had a robustly good response rate or recurrence-free survival with fluoropyrimidine-based chemotherapy, and were recommended to undergo fluoropyrimidine-based adjuvant chemotherapy. Conclusions: MAGI3 is a novel E3 ubiquitin ligase by degradation of c-Myc to regulate CRC development and may act as a potential predictor of adjuvant chemotherapy for CRC patients. [ABSTRACT FROM AUTHOR]

The PDZ (PSD95, Dlg and ZO-1) genes encode proteins that primarily function as scaffolds of diverse signaling pathways. To date, 153 PDZ genes have been identified in the human genome, most of which have multiple protein isoforms widely studied in epithelial and neural cells. However, their expression and function in immune cells have been poorly studied. Herein, we aimed to assess the transcriptional profiles of 83 PDZ genes in human macrophages (Mɸ) and dendritic cells (DCs) and changes in their relative expression during cell PRR stimulation. Significantly distinct PDZ gene transcriptional profiles were identified under different stimulation conditions. Furthermore, a distinct PDZ gene transcriptional signature was found in Mɸ and DCs under the same phagocytic stimuli. Notably, more than 40 PDZ genes had significant changes in expression, with potentially relevant functions in antigen-presenting cells (APCs). Given that several PDZ proteins are targeted by viral products, our results support that many of these proteins might be viral targets in APCs as part of evasion mechanisms. Our results suggest a distinct requirement for PDZ scaffolds in Mɸ and DCs signaling pathways activation. More assessments on the functions of PDZ proteins in APCs and their role in immune evasion mechanisms are needed. [ABSTRACT FROM AUTHOR]

Pathobiont expansion, such as that of adherent-invasive Escherichia coli (AIEC), is an emerging factor associated with inflammatory bowel disease. The intestinal epithelial barrier is the first line of defense against these pathogens. Inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of AIEC. AIEC in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier. One critical element of the epithelial barrier is the tight junction. Increasing evidence suggests that AIEC may selectively target protein components of tight junctions, leading to increased barrier permeability. This may represent one mechanism by which AIEC could contribute to the development of inflammatory bowel disease. This review article discusses potential mechanisms by which AIEC can disrupt epithelial tight junction function and intestinal barrier function.

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2. • Vangl1/2 harbor a remarkably conserved PDZ binding motif at their C-terminus • Extension and quantification of the PDZ interaction network of VANGL2 • Identification of PBM residues with a critical role for PDZ interactions • A structural basis for the VANGL2 PBM/SYNJBP2 PDZ interaction • VANGL2 S517 and S520 phosphorylations differentially impact its PDZ interactome [ABSTRACT FROM AUTHOR]

High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes. [ABSTRACT FROM AUTHOR]

Scribble is a highly conserved regulator of cell polarity, a process that enables the generation of asymmetry at the cellular and tissue level in higher organisms. Scribble acts in concert with Disc-large (Dlg) and Lethal-2-giant larvae (Lgl) to form the Scribble polarity complex, and its functional dysregulation is associated with poor prognosis during viral infections. Viruses have been shown to interfere with Scribble by targeting Scribble PDZ domains to subvert the network of interactions that enable normal control of cell polarity via Scribble, as well as the localisation of the Scribble module within the cell. The influenza A virus NS1 protein was shown to bind to human Scribble (SCRIB) via its C-terminal PDZ binding motif (PBM). It was reported that the PBM sequence ESEV is a virulence determinant for influenza A virus H5N1 whilst other sequences, such as ESKV, KSEV and RSKV, demonstrated no affinity towards Scribble. We now show, using isothermal titration calorimetry (ITC), that ESKV and KSEV bind to SCRIB PDZ domains and that ESEV unexpectedly displayed an affinity towards all four PDZs and not just a selected few. We then define the structural basis for the interactions of SCRIB PDZ1 domain with ESEV and ESKV PBM motifs, as well as SCRIB PDZ3 with the ESKV PBM motif. These findings will serve as a platform for understanding the role of Scribble PDZ domains and their interactions with different NS1 PBMs and the mechanisms that mediate cell polarity within the context of the pathogenesis of influenza A virus. [ABSTRACT FROM AUTHOR]

Diverse lines of evidence suggest that amyloid-β (Aβ) peptides causally contribute to the pathogenesis of Alzheimer disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wild-type or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity but not on its kinase activity. We further present evidence that the protective effect of EphB2 may be mediated by the AMPA-type glutamate receptor subunit GluA2, which can become associated with the PDZ-binding motif of EphB2 through PDZ domain-containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrP(C)), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPA-type glutamate receptors.

Human papillomavirus type 18 (HPV18) has high carcinogenic power in invasive cervical cancer (ICC) development. However, the underlying mechanism remains elusive. The carcinogenic properties of HPV18 require the PDZ‐binding motif of its E6 oncoprotein (HPV18 E6) to degrade its target PSD95/Dlg/ZO‐1 (PDZ) proteins. In this study, we demonstrated that the PDZ protein membrane‐associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) inhibited the Wnt/β‐catenin pathway, and subsequently cervical cancer (CC) cell migration and invasion, via decreasing β‐catenin levels. By reducing MAGI3 protein levels, HPV18 E6 promoted CC cell migration and invasion through activation of Wnt/β‐catenin signaling. Furthermore, HPV18 rather than HPV16 was preferentially associated with the downregulation of MAGI3 and activation of the Wnt/β‐catenin pathway in CC. These findings shed light on the mechanism that gives HPV18 its high carcinogenic potential in CC progression. [ABSTRACT FROM AUTHOR]

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls., Competing Interests: Competing interestsThe authors declare that they have no conflicts of interest., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

PDZ domains are modular domains that conventionally bind to C terminal or internal motifs of target proteins to control cellular functions through the regulation of protein complex assemblies. Almost all reported structures of PDZ-target protein complexes rely on fragments or peptides as target proteins. No intact target protein complexed with PDZ was structurally characterized. In this study, we used NMR spectroscopy and other biochemistry and biophysics tools to uncover insights into structural coupling between the PDZ domain of protein interacting with C-kinase 1 (PICK1) and α7 nicotinic acetylcholine receptors (α7 nAChR). Notably, the intracellular domains of both α7 nAChR and PICK1 PDZ exhibit a high degree of plasticity in their coupling. Specifically, the MA helix of α7 nAChR interacts with residues lining the canonical binding site of the PICK1 PDZ, while flexible loops also engage in protein-protein interactions. Both hydrophobic and electrostatic interactions mediate the coupling. Overall, the resulting structure of the α7 nAChR-PICK1 complex reveals an unconventional PDZ binding mode, significantly expanding the repertoire of functionally important PDZ interactions.

The intracellular signaling molecule Dishevelled (Dvl) mediates canonical and non-canonical Wnt signaling via its PDZ domain. Different pathways diverge at this point by a mechanism that remains unclear. Here we show that the peptide-binding pocket of the Dvl PDZ domain can be occupied by Dvl's own highly conserved C-terminus, inducing a closed conformation. In Xenopus, Wnt-regulated convergent extension (CE) is readily affected by Dvl mutants unable to form the closed conformation than by wild-type Dvl. We also demonstrate that while Dvl cooperates with other Wnt pathway elements to activate canonical Wnt signaling, the open conformation of Dvl more effectively activates Jun N-terminal kinase (JNK). These results suggest that together with other players in the Wnt signaling pathway, the conformational change of Dvl regulates Wnt stimulated JNK activity in the non-canonical Wnt signaling.

CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

The PDZ (PSD95, Dlg and ZO-1) genes encode proteins that primarily function as scaffolds of diverse signaling pathways. To date, 153 PDZ genes have been identified in the human genome, most of which have multiple protein isoforms widely studied in epithelial and neural cells. However, their expression and function in immune cells have been poorly studied. Herein, we aimed to assess the transcriptional profiles of 83 PDZ genes in human macrophages (Mɸ) and dendritic cells (DCs) and changes in their relative expression during cell PRR stimulation. Significantly distinct PDZ gene transcriptional profiles were identified under different stimulation conditions. Furthermore, a distinct PDZ gene transcriptional signature was found in Mɸ and DCs under the same phagocytic stimuli. Notably, more than 40 PDZ genes had significant changes in expression, with potentially relevant functions in antigen-presenting cells (APCs). Given that several PDZ proteins are targeted by viral products, our results support that many of these proteins might be viral targets in APCs as part of evasion mechanisms. Our results suggest a distinct requirement for PDZ scaffolds in Mɸ and DCs signaling pathways activation. More assessments on the functions of PDZ proteins in APCs and their role in immune evasion mechanisms are needed.

Exosomes support cell‐to‐cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF‐7 breast carcinoma cells, this compound is non‐toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin‐exosomal pathway, of potential interest for exosome research and oncology. [ABSTRACT FROM AUTHOR]

Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular‐like "rosette" state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide‐binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high‐resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG‐3 PDZ domains from M. brevicollis. Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG‐3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity. PDB Code(s): 6X1X, 6X20, 6X22, 6X23, 6X1P, 6X1R and 6X1N; [ABSTRACT FROM AUTHOR]

Abstract Background Synaptic cell adhesion molecule 1 (SynCAM1) also known as cell adhesion molecule 1 (CADM1) is a transmembrane cell adhesion protein that operates in a variety of physiological and pathological cellular contexts, and its interaction with the PDZ signalling protein MUPP1 have been previously implicated in autism spectrum disorder (ASD). Methods We used in vitro pull‐down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions. Results So far, the investigated interaction of SynCAM1/CADM1 with MUPP1 has been mostly attributed to an unspecified region of MUPP1 PDZ domains 1–5 or exclusively to domain 2, using a yeast two‐hybrid system. We also confirmed the single interaction of native synaptosomal CADM1 with PDZ domain 2. However, in this work, using recombinant proteins overexpressed in bacteria, we found an in vitro pull‐down conditions in which all first five domains and, to a much lesser extent, MUPP1 domains 7 and 11 significantly interacted with the whole C‐terminal domain of SynCAM1/CADM1. These PDZ interactions were confirmed by a pull‐down assay using the last seven amino acids of the SynCAM1/CADM1 PDZ motif and using two fusion partners. Multiple interactions were additionally replicated using the continuous N‐terminal MUPP1 protein fragment, which included first five PDZ domains, containing either intact or mutated domain 2. Conclusions We hypothesize that multiple interactions might exist in vivo, representing transient low‐affinity interactions or alternative binding sites on MUPP1 when domain 2 is occupied or occluded by the interaction with other ligands. This newly identified interactions extend the potential genetic mutations, possibly affecting SynCAM1/CADM1/MUPP1 function. Possible reasons for the absence of some of the identified CADM1 PDZ interactions in mammalian extracts are discussed.

Globular PDZ domains typically serve as protein–protein interaction modules that regulate a wide variety of cellular functions via recognition of short linear motifs (SLiMs). Often, PDZ mediated-interactions are essential components of macromolecular complexes, and disruption affects the entire scaffold. Due to their roles as linchpins in trafficking and signaling pathways, PDZ domains are attractive targets: both for controlling viral pathogens, which bind PDZ domains and hijack cellular machinery, as well as for developing therapies to combat human disease. However, successful therapeutic interventions that avoid off-target effects are a challenge, because each PDZ domain interacts with a number of cellular targets, and specific binding preferences can be difficult to decipher. Over twenty-five years of research has produced a wealth of data on the stereochemical preferences of individual PDZ proteins and their binding partners. Currently the field lacks a central repository for this information. Here, we provide this important resource and provide a manually curated, comprehensive list of the 271 human PDZ domains. We use individual domain, as well as recent genomic and proteomic, data in order to gain a holistic view of PDZ domains and interaction networks, arguing this knowledge is critical to optimize targeting selectivity and to benefit human health.

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1. [ABSTRACT FROM AUTHOR]

The E3 region of all simian and human types classified within species Human mastadenovirus B (HAdV-B) encodes two unique highly conserved ORFs of unknown function designated E3-CR1β and E3-CR1γ. We generated a HAdV-3 mutant encoding small epitope tags at the N-termini of both E3-CR1β and E3-CR1γ (HAdV-3 N-tag wt) and a double knock out (HAdV-3 N-tag DKO) mutant virus that does not express either protein. Our studies show that HAdV-3 E3-CR1β and E3-CR1γ are type I transmembrane proteins that are produced predominantly at late times post infection, are glycosylated, co-localize at the plasma membrane of non-polarized epithelial cells, and interact with each other. At their extreme C-termini HAdV-B E3-CR1β and E3-CR1γ possess a conserved di-leucine motif followed by a class II PDZ domain binding motif (PBM). HAdV-3 E3-CR1β and E3-CR1γ are dispensable for virus growth, progeny release, spread, and plaque formation in A549 cells. • HAdV-B E3-CR1β and E3-CR1γ are type I transmembrane proteins. • HAdV-B E3-CR1β and E3-CR1γ possess a C-terminal class II PDZ binding motif. • HAdV-3 E3-CR1β and E3-CR1γ interact and co-localize at the plasma membrane. • HAdV-3 E3-CR1β and E3-CR1γ are dispensable for virus progeny release and spread. [ABSTRACT FROM AUTHOR]

Background: Synaptic cell adhesion molecule 1 (SynCAM1) also known as cell adhesion molecule 1 (CADM1) is a transmembrane cell adhesion protein that operates in a variety of physiological and pathological cellular contexts, and its interaction with the PDZ signalling protein MUPP1 have been previously implicated in autism spectrum disorder (ASD). Methods: We used in vitro pull‐down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions. Results: So far, the investigated interaction of SynCAM1/CADM1 with MUPP1 has been mostly attributed to an unspecified region of MUPP1 PDZ domains 1–5 or exclusively to domain 2, using a yeast two‐hybrid system. We also confirmed the single interaction of native synaptosomal CADM1 with PDZ domain 2. However, in this work, using recombinant proteins overexpressed in bacteria, we found an in vitro pull‐down conditions in which all first five domains and, to a much lesser extent, MUPP1 domains 7 and 11 significantly interacted with the whole C‐terminal domain of SynCAM1/CADM1. These PDZ interactions were confirmed by a pull‐down assay using the last seven amino acids of the SynCAM1/CADM1 PDZ motif and using two fusion partners. Multiple interactions were additionally replicated using the continuous N‐terminal MUPP1 protein fragment, which included first five PDZ domains, containing either intact or mutated domain 2. Conclusions: We hypothesize that multiple interactions might exist in vivo, representing transient low‐affinity interactions or alternative binding sites on MUPP1 when domain 2 is occupied or occluded by the interaction with other ligands. This newly identified interactions extend the potential genetic mutations, possibly affecting SynCAM1/CADM1/MUPP1 function. Possible reasons for the absence of some of the identified CADM1 PDZ interactions in mammalian extracts are discussed. [ABSTRACT FROM AUTHOR]

Even though the abundance of GlyT1 in postsynaptic aspects of forebrain neurons is low, its previously reported interaction with postsynaptic density protein PSD95 represents a prototype of interaction, which might uncover the binding mechanism and regulation of the GlyT1 C-terminal PDZ motif. We used a phosphomimetic approach to mimic the potential phosphorylation of GlyT1 C-terminal serines 640, 643, 644, and 649 of the mouse GlyT1b subtype (mGlyT1b) (Uniprot P28571-2) and its effect on GlyT1-PSD95 PDZ interaction. Among them, only phosphomimetic mutation of serine 649 to aspartate, which resides in the minimal PDZ motif -SRI, significantly eliminated the interaction of the GlyT1 C-terminus with PSD95 PDZ domain 2. The effect was observed with recombinant fusion proteins as well as with GlyT1 and PSD95 expressed in tissue culture. Results indicate that phosphorylation of mouse GlyT1b serine 649 and equivalent serines of GlyT1a and GlyT1c subtypes might regulate the PDZ interaction of the GlyT1 C-terminal PDZ binding motif. [ABSTRACT FROM AUTHOR]

In the present study, low alloy steel AISI 4140 pipe in reheated, quenched and tempered (RQT) condition was friction-welded using 6.4 MPa upset pressure followed by post-weld heat treatment as per the manufacturing practice prevailing in the drill pipe industry and compared the same with friction welds made at different upset pressures in the RQT condition and in the as-received condition to evaluate bond quality. The weld region consisted of a mixture of tempered martensite and ferrite in case of RQT condition and mixture of lath martensite/upper bainite and ferrite in the as-received condition. As the upset pressure was increased, the martensite lath size decreased in both as-received and RQT conditions. In general, as the upset pressure was increased, the average microhardness increased. The average microhardness of the material adjacent to partially deformed zone showed lower hardness due to tempering effect because of weld heat. The upset pressure 6.4 MPa showed better notch tensile strength ratio confirming better ductility of the weld zone. The impact strength decreased as the upset pressure increased in both as-received and RQT conditions. But as-received condition showed significantly lower impact strength due to higher carbide content at the weld interface. [ABSTRACT FROM AUTHOR]

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor. [ABSTRACT FROM AUTHOR]