Search Results (112)

Wnt/β-catenin signaling is hyper-activated in several cancer cells and cancer stem cells. Dishevelled/Dvl is a key adapter protein that acts as a bridge between the Wnt receptor Frizzled (Fzd) and other cytosolic factors. In detail, the C-terminal cytosolic region is the ligand of the PSD-95, disks large, and zonula occludens-1 (PDZ) domain of Dvl. Therefore, the PDZ domain (Dvl-PDZ) is thought to be a potential drug target. In this paper, we determined the first crystal structure of the PDZ domain of human Dvl1 (hDvl1-PDZ) at a 2.4 Å resolution. The domain was adapted into a unique trimeric form in which all the canonical ligand-binding clefts were occupied by the β2-β3 loop of the neighbor molecule, like an auto-inhibiting trimer. We used solution nuclear magnetic resonance (NMR) experiments to assess the presence of the self-associated oligomer of hDvl1-PDZ in the solution. Introducing the Ala substitution at Asp 272, the key residue of the β2-β3 loop, partly abolished the concentration-dependent chemical shift change, which suggests that this residue is one of the key residues for formation. Based on these observations, we propose an auto-inhibiting trimer formation of Dvl-PDZ in a Dvl-Axin hetero-oligomerization model of Wnt/β-catenin signal transduction. Full article

In this perspective, we highlight the relevance of the FA-Hippo signaling pathway and its regulation of the Yes-associated protein (YAP) and the transcriptional coactivator with a PDZ-binding domain (TAZ) as main players in the process of implants integration. The modulation and responses of YAP/TAZ triggered by substrate and ECM stiffness are of particular interest in the construction of materials used for medical implants. YAP/TAZ nuclear localization and activity respond to the substrate stiffness by several mechanisms that involve the canonical and non-canonical Hippo signaling and independently of the Hippo cascade. YAP/TAZ regulate the expression of genes involved in several mechanisms of relevance for implant integration such as the proliferation and differentiation of cell precursors and the immune response to the implant. The influence of substrate stiffness on the regulation of the immune response is not completely understood and the progress in this field can contribute to the designing of an adequate implant design. Though the use of nano-biomaterials has been proved to contribute to implant success, the relationship between grain size and stiffness of the material has not been explored in the biomedical field; filling these gaps in the knowledge of biomaterials will highly contribute to the design of biomaterials that could take advantage of the cells sensing and response to the stiffness at the implant interface. Full article

Background/Objectives: The MAST kinases are ancient AGC kinases associated with many human diseases, such as cancer, diabetes, and neurodevelopmental disorders. We set out to describe the origins and diversification of MAST kinases from a structural and bioinformatic perspective to inform future research directions. Methods: We investigated MAST-lineage kinases using database and sequence analysis. We also estimate the functional consequences of disease point mutations on protein stability by integrating predictive algorithms and AlphaFold. Results: Higher-order organisms often have multiple MASTs and a single MASTL kinase. MAST proteins conserve an AGC kinase domain, a domain of unknown function 1908 (DUF), and a PDZ binding domain. D. discoideum contains MAST kinase-like proteins that exhibit a characteristic insertion within the T-loop but do not conserve DUF or PDZ domains. While the DUF domain is conserved in plants, the PDZ domain is not. The four mammalian MASTs demonstrate tissue expression heterogeneity by mRNA and protein. MAST1-4 are likely regulated by 14-3-3 proteins based on interactome data and in silico predictions. Comparative ΔΔG estimation identified that MAST1-L232P and G522E mutations are likely destabilizing. Conclusions: We conclude that MAST and MASTL kinases diverged from the primordial MAST, which likely operated in both biological niches. The number of MAST paralogs then expanded to the heterogeneous subfamily seen in mammals that are all likely regulated by 14-3-3 protein interaction. The reported pathogenic mutations in MASTs primarily represent alterations to post-translational modification topology in the DUF and kinase domains. Our report outlines a computational basis for future work in MAST kinase regulation and drug discovery. Full article

Despite significant progress in characterizing the omics landscape of head and neck squamous cell carcinoma (HNSCC), the development of precision therapies remains limited. One key factor contributing to this challenge is the marked molecular heterogeneity of HNSCC. Further investigation of molecular profiles within HNSCC may facilitate the improvement in more effective precision treatments. Here, we focus on the dysregulation of PDZ and LIM domain protein 3 (PDLIM3) in HNSCC. The expression levels of PDLIM3 were analyzed using public datasets to assess its potential role in tumor progression. We found that PDLIM3 was downregulated in pan–cancer and HNSCC. The prognostic significance of PDLIM3 was evaluated through tissue microarray, and the downregulation of PDLIM3 was correlated with poor HNSCC prognosis. Investigating the implications of PDLIM3 for tumor metastatic ability in vitro, we found that PDLIM3 suppressed the migration and invasion of HNSCC, accompanied by partially impeding the process of epithelial–mesenchymal transition (EMT). Furthermore, PDLIM3 inhibited the transcriptional activity of Yes–associated protein (YAP), suggesting that YAP may be involved in the PDLIM3–mediated suppression of HNSCC metastatic ability. Our findings identify a potential signaling axis wherein PDLIM3 regulates YAP–EMT, thereby influencing tumor metastatic ability, and suggest the potential role of PDLIM3 as a tumor suppressor and prognostic biomarker for HNSCC. Full article

Frmpd3 (FERM and PDZ Domain Containing 3), a scaffold protein potentially involved in excitatory synaptic function, has not been thoroughly characterized in terms of its expression and functional role in vivo. Here, we investigated the distribution of Frmpd3 in the central nervous system and its potential regulatory role in epilepsy, a neurological disorder characterized by disrupted excitatory–inhibitory balance. The distribution of Frmpd3 throughout the mouse brain was investigated by immunofluorescence. Western blotting was conducted to examine potential alterations in Frmpd3 protein expression in the hippocampus of a pentylenetetrazol (PTZ)-induced chronic epilepsy model. Using stereotaxic techniques, we delivered Frmpd3 siRNA-AAV9 into the hippocampal CA1 region to achieve targeted protein knockdown. Then, the functional consequences of Frmpd3 depletion were assessed through behavioral observations and electrophysiological recordings in PTZ-treated mice. Finally, protein–protein interactions were investigated using immunoprecipitation and Western blot analysis. Immunofluorescence analysis revealed Frmpd3 expression in cortical, hypothalamic, cerebellar, and hippocampal neurons of adult mice. Subcellular localization studies demonstrated predominant distribution of Frmpd3 in the excitatory postsynaptic density (PSD) of hippocampal CA1 neurons, with additional expression in inhibitory neurons. Quantitative analysis showed significantly elevated Frmpd3 protein levels in the hippocampus of PTZ-induced epileptic mice compared to controls. Frmpd3 knockdown in the CA1 region resulted in the following: (1) reduced seizure frequency, (2) prolonged seizure latency, and (3) decreased incidence of PTZ-induced generalized seizures. Local field potential (LFP) recordings demonstrated that seizure amplitude tended to be reduced, and epileptic discharge durations tended to be shorter in Frmpd3-depleted mice compared to controls. Furthermore, we observed decreased membrane expression of the AMPA receptor GluA2 subunit in the hippocampus of Frmpd3 knockdown mice. Molecular interaction studies revealed that Frmpd3 forms complexes with glutamate receptor-interacting protein (GRIP) and GluA2. Our findings identify Frmpd3 as a novel regulatory scaffold protein that modulates epileptic susceptibility through molecular interactions with GRIP and GluA2. The underlying mechanism appears to involve Frmpd3-mediated regulation of GluA2 trafficking from the cytoplasm to the membrane, ultimately enhancing neuronal excitability through increased membrane expression of GluA2-containing AMPA receptors. Full article

There is a growing concern worldwide about the potential harmful effects of anesthesia on brain development, based on studies in both humans and animals. In infants, repeated anesthesia exposure is linked to learning disabilities and attention disorders. Similarly, laboratory studies in mice show that neonates exposed to general anesthesia experience long-term cognitive and behavioral impairments. Inhaled anesthetics affect the postsynaptic density (PSD)-95, discs large homolog, and zona occludens-1 (PDZ) domains. The disruption of the synaptic PSD95-PDZ2 domain-mediated protein interactions leads to a loss of spine plasticity and cognitive deficits in juvenile mice. The nitric oxide-mediated protein kinase-G signaling pathway enhances synaptic plasticity also by activating extracellular signal-regulated kinase, which subsequently phosphorylates cAMP-response element binding protein, a crucial transcription factor for memory formation. Exposure to isoflurane or postsynaptic density-95-PDZ2-wildtype peptides results in decreased levels of phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated cAMP-response element binding protein (p-CREB), which are critical for synaptic plasticity and memory formation. Pizotifen treatment after isoflurane or postsynaptic density-95-PDZ2-wildtype peptide exposure in mice prevented decline in p-ERK levels, preserved learning and memory functions at 5 weeks of age, and maintained mushroom spine density at 7 weeks of age. Protein kinase-G activation by components of the nitric oxide signaling pathway leads to the stabilization of dendritic spines and synaptic connections. Concurrently, the ERK/CREB pathway, which is crucial for synaptic plasticity and memory consolidation, is supported and maintained by pizotifen, thereby preventing cognitive deficits caused in response to isoflurane or postsynaptic density-95-PDZ2-wildtype peptide exposure. Activation of ERK signaling cascade by pizotifen helps to prevent cognitive impairment and spine loss in response to postsynaptic density-95-PDZ2 domain disruption. Full article

The oncogenes yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are potent liver oncogenes. Because gene mutations cannot fully explain their nuclear enrichment, we aim to understand which mechanisms cause YAP/TAZ activation in liver cancer cells. The combination of proteomics and functional screening identified numerous apical cell polarity complex proteins interacting with YAP and TAZ. Co-immunoprecipitation (Co-IP) experiments confirmed that membrane protein palmitoylated 5 (MPP5; synonym: PALS1) physically interacts with YAP and TAZ. After removing different MPP5 protein domains, Co-IP analyses revealed that the PDZ domain plays a crucial role in YAP binding. The interaction between YAP and MPP5 in the cytoplasm of cancer cells was demonstrated by proximity ligation assays (PLAs). In human hepatocellular carcinoma (HCC) tissues, a reduction in apical MPP5 expression was observed, correlating with the nuclear accumulation of YAP and TAZ. Expression data analysis illustrated that MPP5 is inversely associated with YAP/TAZ target gene signatures in human HCCs. Low MPP5 levels define an HCC patient group with a poor clinical outcome. In summary, MPP5 facilitates the nuclear exclusion of YAP and TAZ in liver cancer. This qualifies MPP5 as a potential tumor-suppressor gene and explains how changes in cell polarity can foster tumorigenesis. Full article

Proteolytic enzymes play key roles in living organisms. Because of their potentially destructive action of degrading other proteins, their activity must be very tightly controlled. The evolutionarily conserved proteins of the HtrA family are an excellent example illustrating strategies for regulating enzymatic activity, enabling protease activation in response to an appropriate signal, and protecting against uncontrolled proteolysis. Because HtrA homologs play key roles in the virulence of many Gram-negative bacterial pathogens, they are subject to intense investigation as potential therapeutic targets. Model HtrA proteins from bacterium Escherichia coli are allosteric proteins with reasonably well-studied properties. Binding of appropriate ligands induces very large structural changes in these enzymes, including changes in the organization of the oligomer, which leads to the acquisition of the active conformation. Properly coordinated events occurring during the process of HtrA activation ensure proper functioning of HtrA and, consequently, ensure fitness of bacteria. The aim of this review is to present the current state of knowledge on the structure and function of the exemplary HtrA family proteins from Gram-negative bacteria, including human pathogens. Special emphasis is paid to strategies for regulating the activity of these enzymes. Full article

Bipolar disorder’s etiology involves genetics, environmental factors, and gene–environment interactions, underlying its heterogeneous nature and treatment complexity. In 2020, Forstner and colleagues catalogued 378 sequence variants co-segregating with familial bipolar disorder. A notable candidate was an R59Q missense mutation in the PDZ (PSD-95/Dlg1/ZO-1) domain of RGS12. We previously demonstrated that RGS12 loss removes negative regulation on the kappa opioid receptor, disrupting basal ganglia dopamine homeostasis and dampening responses to dopamine-eliciting psychostimulants. Here, we investigated the R59Q variation in the context of potential PDZ domain functional alterations. We first validated a new target for the wildtype RGS12 PDZ domain—the SAPAP3 C-terminus—by molecular docking, surface plasmon resonance (SPR), and co-immunoprecipitation. While initial molecular dynamics (MD) studies predicted negligible effects of the R59Q variation on ligand binding, SPR showed a significant reduction in binding affinity for the three peptide targets tested. AlphaFold2-generated models predicted a modest reduction in protein–peptide interactions, which is consistent with the reduced binding affinity observed by SPR, suggesting that the substituted glutamine side chain may weaken the affinity of RGS12 for its in vivo binding targets, likely through allosteric changes. This difference may adversely affect the CNS signaling related to dynorphin and dopamine in individuals with this R59Q variation, potentially impacting bipolar disorder pathophysiology. Full article

MDA-9/Syntenin, a key scaffolding protein and a molecular hub involved in a diverse range of cell signaling responses, has proved to be a challenging target for the design and discovery of small molecule probes. In this paper, we report on the design and synthesis of small molecule ligands of this key protein. Genetic algorithm-based computational design and the five–eight step synthesis of three molecules led to ligands with affinities in the range of 1–3 µM, a 20–60-fold improvement over literature reports. The design and synthesis strategies, coupled with the structure-dependent gain or loss in affinity, afford the deduction of principles that should guide the design of advanced probes of MDA-9/Syntenin. Full article

Pulmonary fibrosis (PF) is a severe, irreversible lung disease characterized by progressive scarring, with idiopathic pulmonary fibrosis (IPF) being the most prevalent form. IPF’s pathogenesis involves repetitive lung epithelial injury leading to fibroblast activation and excessive extracellular matrix (ECM) deposition. The prognosis for IPF is poor, with limited therapeutic options like nintedanib and pirfenidone offering only modest benefits. Emerging research highlights the dysregulation of the yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathway as a critical factor in PF. YAP and TAZ, components of the Hippo pathway, play significant roles in cell proliferation, differentiation, and fibrosis by modulating gene expression through interactions with TEA domain (TEAD) transcription factors. The aberrant activation of YAP/TAZ in lung tissue promotes fibroblast activation and ECM accumulation. Targeting the YAP/TAZ pathway offers a promising therapeutic avenue. Preclinical studies have identified potential treatments, such as trigonelline, dopamine receptor D1 (DRD1) agonists, and statins, which inhibit YAP/TAZ activity and demonstrate antifibrotic effects. These findings underscore the importance of YAP/TAZ in PF pathogenesis and the potential of novel therapies aimed at this pathway, suggesting a new direction for improving IPF treatment outcomes. Further research is needed to validate these approaches and translate them into clinical practice. Full article

The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein–protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5’s multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5’s structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5’s function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects. Full article

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer. Full article

HPV 16 integration is crucial for the onset and progression of premalignant lesions to invasive squamous cell carcinoma (ISCC) because it promotes the amplification of proto-oncogenes and the silencing of tumor suppressor genes; some of these are proteins with PDZ domains involved in homeostasis and cell polarity. Through a bioinformatics approach based on interaction networks, a group of proteins associated with HPV 16 infection, PDZ domains, and direct physical interaction with E6 and related to different hallmarks of cancer were identified. MAGI-1 was selected to evaluate the expression profile and subcellular localization changes in premalignant lesions and ISCC with HPV 16 in an integrated state in cervical cytology; the profile expression of MAGI-1 diminished according to lesion grade. Surprisingly, in cell lines CaSki and SiHa, the protein localization was cytoplasmic and nuclear. In contrast, in histological samples, a change in subcellular localization from the cytoplasm in low-grade squamous intraepithelial lesions (LSIL) to the nucleus in the high-grade squamous intraepithelial lesion (HSIL) was observed; in in situ carcinomas and ISCC, MAGI-1 expression was absent. In conclusion, MAGI-1 expression could be a potential biomarker for distinguishing those cells with normal morphology but with HPV 16 integrated from those showing morphology-related uterine cervical lesions associated with tumor progression. Full article

Head and neck cancers rank as the sixth most prevalent cancers globally. In addition to traditional risk factors such as smoking and alcohol use, human papillomavirus (HPV) infections are becoming a significant causative agent of head and neck cancers, particularly among Western populations. Although HPV offers a significant survival benefit, the search for better biomarkers is still ongoing. In the current study, our objective was to investigate whether the expression levels of three PDZ-domain-containing proteins (SCRIB, NHERF2, and DLG1), known HPV E6 cellular substrates, influence the survival of HNSCC patients treated by primary surgery (n = 48). Samples were derived from oropharyngeal and oral cancers, and HPV presence was confirmed by PCR and p16 staining. Clinical and follow-up information was obtained from the hospital database and the Croatian Cancer registry up to November 2023. Survival was evaluated using the Kaplan–Meier method and Cox proportional hazard regression. The results were corroborated through the reanalysis of a comparable subset of TCGA cancer patients (n = 391). In conclusion, of the three targets studied, only SCRIB levels were found to be an independent predictor of survival in the Cox regression analysis, along with tumor stage. Further studies in a more typical Western population setting are needed since smoking and alcohol consumption are still prominent in the Croatian population, while the strongest association between survival and SCRIB levels was seen in HPV-negative cases. Full article