PDZ Domain Research Articles

Nonmuscle myosin II proteins, Myh9 and Myh10, interacts with unconventional myosin Myo18A in immortalized mouse thick ascending limb epithelial cells

Conventional nonmuscle myosin II (NM2) and unconventional myosin 18A (Myo18A) belong to an evolutionarily conserved family of molecular motor proteins that slide or walk on the actin cytoskeleton to facilitate a myriad of cellular functions including cargo transport pathways. NM2 and Myo18A are known to copolymerize into filaments in vitro in cell-free systems and in cells. Our previous work demonstrated the crucial role for Myh9&10 proteins in regulating transport of uromodulin (UMOD) and the sodium, potassium, chloride cotransporter (NKCC2) in the thick ascending limb (TAL) of mouse kidney using a Myh9&10 conditional knockout mouse model (Otterpohl, et al; 2020). Myo18A is expressed in the proximal tubule and TAL cells in the murine kidney and localizes to the apical membrane. Our results show reduced expression of Myo18A in the TAL cells of the Myh9&10 cKO mouse kidney sections. Furthermore, we identified Myo18A as a major interaction partner to the Myh10 heavy chain in a BioID screen performed in renal epithelial cells. Given that Myo18A alpha is the only myosin with a PDZ domain, we hypothesize that Myo18A coordinates with Myh9&10 in the TAL cells to regulate membrane remodeling during vesicular transport, including the targeting and stabilization of TAL proteins like UMOD and NKCC2. To test this hypothesis, we stably expressed GFP-tagged Myo18A alpha construct in immortalized, mouse thick ascending limb (imTAL) cells generated in the lab. We performed coimmunoprecipitation (Co-IP) experiments that showed interaction between Myo18A alpha, Myh9 and Myh10 proteins. Immunostaining experiments show colocalization of GFP-Myo18A alpha with Myh9 and Myh10 in imTAL cells. These results strongly support the coordinated functions of Myo18A and Myh9&10 proteins in TAL epithelia and warrants further experiments to determine their role in regulating UMOD and NKCC2 transport in the TAL cells. 1R01DK131020-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A calorimetric and structural analysis of cooperativity in the thermal unfolding of the PDZ tandem of human Syntenin-1

Syntenin-1 is a multidomain protein containing a central tandem of two PDZ domains flanked by two unnamed domains. Previous structural and biophysical studies show that the two PDZ domains are functional both isolated and in tandem, occurring a gain in their respective binding affinities when joined through its natural short linker. To get insight into the molecular and energetic reasons of such a gain, here, the first thermodynamic characterization of the conformational equilibrium of Syntenin-1 is presented, with special focus on its PDZ domains. These studies include the thermal unfolding of the whole protein, the PDZ-tandem construct and the two isolated PDZ domains using circular dichroism, differential scanning fluorimetry and differential scanning calorimetry. The isolated PDZ domains show low stability (ΔG < 10 kJ·mol−1) and poor cooperativity compared to the PDZ-tandem, which shows higher stability (20–30 kJ·mol−1) and a fully cooperative behaviour, with energetics similar to that previously described for archetypical PDZ domains. The high-resolution structures suggest that this remarkable increase in cooperativity is associated to strong, water-mediated, interactions at the interface between the PDZ domains, associated to nine conserved hydration regions. The low Tm value (45 °C), the anomalously high unfolding enthalpy (>400 kJ·mol−1), and native heat capacity values (above 40 kJ·K−1·mol−1), indicate that these interfacial buried waters play a relevant role in Syntenin-1 folding energetics.

Nitric oxide synthases in GtoPdb v.2023.1

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [12] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.

NNOS and Neurological, Neuropsychiatric Disorders: A 20-Year Story.

In the central nervous system, nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS). In the past 20 years, the studies in our group and other laboratories have suggested a significant involvement of nNOS in a variety of neurological and neuropsychiatric disorders. In particular, the interactions between the PDZ domain of nNOS and its adaptor proteins, including post-synaptic density 95, the carboxy-terminal PDZ ligand of nNOS, and the serotonin transporter, significantly influence the subcellular localization and functions of nNOS in the brain. The nNOS-mediated protein-protein interactions provide new attractive targets and guide the discovery of therapeutic drugs for neurological and neuropsychiatric disorders. Here, we summarize the work on the roles of nNOS and its association with multiple adaptor proteins on neurological and neuropsychiatric disorders.

Pharmacologic Comparison of High-Dose Hesperetin and Quercetin on MDCK II Cell Viability, Tight Junction Integrity, and Cell Shape.

The modulation of tight junction (TJ) integrity with small molecules is important for drug delivery. High-dose baicalin (BLI), baicalein (BLE), quercetin (QUE), and hesperetin (HST) have been shown to open TJs in Madin-Darby canine kidney (MDCK) II cells, but the mechanisms for HST and QUE remain unclear. In this study, we compared the effects of HST and QUE on cell proliferation, morphological changes, and TJ integrity. HST and QUE were found to have opposing effects on the MDCK II cell viability, promotion, and suppression, respectively. Only QUE, but not HST, induced a morphological change in MDCK II into a slenderer cell shape. Both HST and QUE downregulated the subcellular localization of claudin (CLD)-2. However, only QUE, but not HST, downregulated CLD-2 expression. Conversely, only HST was shown to directly bind to the first PDZ domain of ZO-1, a key molecule to promote TJ biogenesis. The TGFβ pathway partially contributed to the HST-induced cell proliferation, since SB431541 ameliorated the effect. In contrast, the MEK pathway was not involved by both the flavonoids, since U0126 did not revert their TJ-opening effect. The results offer insight for using HST or QUE as naturally occurring absorption enhancers through the paracellular route.

KIF-Key Interactions Finder: A program to identify the key molecular interactions that regulate protein conformational changes.

Simulation datasets of proteins (e.g., those generated by molecular dynamics simulations) are filled with information about how a non-covalent interaction network within a protein regulates the conformation and, thus, function of the said protein. Most proteins contain thousands of non-covalent interactions, with most of these being largely irrelevant to any single conformational change. The ability to automatically process any protein simulation dataset to identify non-covalent interactions that are strongly associated with a single, defined conformational change would be a highly valuable tool for the community. Furthermore, the insights generated from this tool could be applied to basic research, in order to improve understanding of a mechanism of action, or for protein engineering, to identify candidate mutations to improve/alter the functionality of any given protein. The open-source Python package Key Interactions Finder (KIF) enables users to identify those non-covalent interactions that are strongly associated with any conformational change of interest for any protein simulated. KIF gives the user full control to define the conformational change of interest as either a continuous variable or categorical variable, and methods from statistics or machine learning can be applied to identify and rank the interactions and residues distributed throughout the protein, which are relevant to the conformational change. Finally, KIF has been applied to three diverse model systems (protein tyrosine phosphatase 1B, the PDZ3 domain, and the KE07 series of Kemp eliminases) in order to illustrate its power to identify key features that regulate functionally important conformational dynamics.

PDLIM1 inhibits cell migration and invasion in diabetic retinopathy via negatively regulating Wnt3a

The injury of vascular endothelial cells is a crucial factor in the development of diabetic retinopathy (DR). PDLIM1 (a member of the PDZ and LIM protein family) has been reported to exert an essential function in vascular diseases. This study aimed to elucidate the role of PDLIM1 on retinal vascular endothelial cells in DR. Immunofluorescence staining was used to localize the expression of PDLIM1 in the mouse retina. In some tumor diseases, PDLIM1 has been reported to play a key role in regulating the Wnt pathway. However, no in-depth reports have been found in DR. Retinal capillary endothelial cells (RCECs) were treated with high-glucose and high-lipid (HG/HL) culture medium, and siRNA transfection to investigate the role of PDLIM1 in DR. PDLIM1 and Wnt3a expression was confirmed by qRT-PCR and western blotting. Flow cytometry, Transwell assay, and scratch assay were used to test the ability of cell apoptosis, migration, and invasion. PDLIM1 was mainly expressed in the retinal pigment epithelium (RPE), ganglion cell layer (GCL), inner plexus layer (IPL), and outer plexus layer (OPL). HG/HL increased Wnt3a levels and promoted cell’s ability of apoptosis, migration, and invasion, which were reversed by the knockdown of PDLIM1. PDLIM1 was found to play a protective role in diabetic retinopathy by counter-regulating Wnt3a. PDLIM1 ameliorates cell apoptosis, migration, and invasion by negatively regulating Wnt3a in RCECs of DR, which suggests that PDLIM1 might be a promising therapeutic target for DR treatment.

Abstract 3394: Simultaneous targeting of the PDZ1 and PDZ2 domains of MDA-9 inhibits melanoma metastasis

Abstract Metastasis poses significant obstacles to effective therapy and drug development. Melanoma differentiation-associated gene-9/Syntenin-1/Syndecan Binding Protein (MDA-9) is a pro-metastatic gene robustly and differentially expressed in cancer cells in comparison with corresponding adjacent normal/primary cells and represents an attractive therapeutic target to inhibit cancer cell dissemination and tumor growth. MDA-9 contains two tandem PDZ domains that provide a nexus for interactions with multiple proteins that can initiate the transcription of metastasis-associated genes. The goal of this project was to evaluate the efficiency of a unique first-in-class, second-generation MDA-9 dual-PDZ targeted pharmacological inhibitor generated using a combination of a PDZ1 small molecule and a PDZ2 selective peptide. Fragment-based drug discovery and NMR identified PDZ1i, an inhibitor of the PDZ1 domain, that effectively blocks cancer invasion in vitro and in vivo in multiple experimental animal models. To maximize the disruption of MDA-9 signaling an inhibitor has now been developed that simultaneously binds and blocks the activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in IVMT-Rx-3 that simultaneously engages both PDZ domains of MDA-9. Mechanistic studies involved a combination of molecular and cell biology approaches and included both established and early passaged patient-derived melanoma cell lines. To test the efficacy of potential combinatorial effects in vivo we used a murine syngeneic model and B16F10, an immunotherapy-resistant murine cell line. After confirming synthesis using multiple analytical methods, e.g., HPLC, LC/MS, and NMR, the dissociation constant (determined by microscale thermophoresis (MST) analysis, of the IVMT-Rx-3 recombinant MDA-9 complex was 63 +/- 11 μM. This compound is not toxic to normal melanocytes or melanoma cells, but significantly inhibits invasion in vitro in a wide range of melanomas. IVMT-Rx-3 interrupts c-Src binding to MDA-9 and downregulates NF-kB activation. Inactivation of this transcription factor causes suppression of MMP2/MMP9 and inhibits the secretion of cytokines/growth factors. These molecular changes block cellular invasiveness and facilitate immune cytotoxicity by converting an unfavorable into a favorable tumor microenvironment culminating in the repression of melanoma metastasis. In addition, the in vivo anti-metastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. IVMT-Rx-3 is a unique MDA-9 antagonist in terms of composition that effectively inhibits melanoma metastasis as does our small molecule PDZ1i and holds potential for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9. Citation Format: Anjan K. Pradhan, Jinkal Modi, Santanu Maji, Amit Kumar, Praveen Bhoopathi, Padmanabhan Mannangatti, Chunqing Guo, Daniel K. Afosah, Mark Mochel, Nitai D. Mukhopadhyay, John M. Kirkwood, Xiang-Yang Wang, Umesh R. Desai, Devanand Sarkar, Luni Emdad, Swadesh K. Das, Paul B. Fisher. Simultaneous targeting of the PDZ1 and PDZ2 domains of MDA-9 inhibits melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3394.

Abstract 2566: Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. Tax interacting protein 1 (TIP1) is a molecular target that is overexpressed on the surface of various cancers and correlates with poor overall patient survival. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate cell signaling, cancer development, and progression. We found that antibodies against the PDZ domain of TIP1 induced cytotoxicity of lung cancer cells but not normal cells. A quantitative mass spectrometry-based approach identified a ten-fold increase in Midkine protein expression on blockade of TIP1. Midkine is a heparin-binding growth factor that is involved in cancer progression, therapy resistance, and immune suppression. This study tested the hypothesis that Midkine is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. In co-immunoprecipitation studies we found a direct interaction of TIP1 with β-catenin on the cell surface. Anti-TIP1 antibody treatment led to nuclear translocation of β-catenin in NSCLC cells. We used a luciferase based TOPFlash/FOPFlash reporter assay to study the transcriptional activity of the canonical β-catenin/Wnt pathway following TIP1 blockade. Reporter assays showed a three to four-fold increase (p&amp;lt;0.0001) in the transcriptional activity of β-catenin following TIP1 blockade in NSCLC cells. Genetic silencing of β-catenin inhibited Midkine mRNA transcription (p&amp;lt;0.005), protein induction and its extracellular release (p&amp;lt;0.0001) following anti-TIP1 antibody treatment. In conclusion, the mechanism of Midkine release following TIP1 blockade involves the β-catenin/Wnt signaling pathway. Ongoing studies are evaluating the dual-targeting of TIP1 and Midkine for enhanced efficacy of NSCLC treatment. Citation Format: Minakshi Saikia, Abhay K. Singh, Nathan Cheung, Dennis E. Hallahan, Vaishali Kapoor. Blocking the functional domain of TIP1 upregulates midkine via the β-catenin/Wnt signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2566.

Alignment of human aquaporin 4 and ß-amyloid proteins may indicate involvement of ß-amyloid in brain water homeostasis and prevention of brain edema.

Alignment of human aquaporin 4 and ß-amyloid proteins may indicate involvement of ß-amyloid in brain water homeostasis and prevention of brain edema.

Biological Role and Aberrant Overexpression of Syntenin-1 in Cancer: Potential Role as a Biomarker and Therapeutic Target.

Syntenin-1 is a 298 amino acid protein codified by the melanoma differentiation-associated gene-9 (MDA-9). Structurally, it is composed of four domains: N-terminal, PDZ1, PDZ2, and C-terminal. The PDZ domains of syntenin-1 are involved in the stability and interaction with other molecules such as proteins, glycoproteins, and lipids. Domains are also associated with several biological functions such as the activation of signaling pathways related to cell-to-cell adhesion, signaling translation, and the traffic of intracellular lipids, among others. The overexpression of syntenin-1 has been reported in glioblastoma, colorectal, melanoma, lung, prostate, and breast cancer, which promotes tumorigenesis by regulating cell migration, invasion, proliferation, angiogenesis, apoptosis, and immune response evasion, and metastasis. The overexpression of syntenin-1 in samples has been associated with worst prognostic and recurrence, whereas the use of inhibitors such as shRNA, siRNA, and PDZli showed a diminution of the tumor size and reduction in metastasis and invasion. Syntenin-1 has been suggested as a potential biomarker and therapeutic target in cancer for developing more effective diagnostic/prognostic tests or passive/active immunotherapies.

PDZ interaction of the GABA transporter GAT1 with the syntenin-1 in Neuro-2a cells

The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.

Multiplex measurement of protein-peptide dissociation constants using dialysis and mass spectrometry.

We propose a high-throughput method for quantitatively measuring hundreds of protein-peptide binding affinities in parallel. In this assay, a solution of protein is dialyzed into a buffer containing a pool of potential binding peptides, such that upon equilibration the relative abundance of a peptide species is mathematically related to that peptide's dissociation constant, Kd . We use isobaric multiplexed quantitative proteomics to simultaneously determine the relative abundance, and hence the Kd and its associated error, for an entire peptide library. We apply this technique, which we call PEDAL (parallel equilibrium dialysis for affinity learning), to determine accurate Kd 's between a PDZ domain and hundreds of peptides, spanning an affinity range of multiple orders of magnitude in a single experiment. PEDAL is a convenient, fast, and low-cost method for measuring large numbers of protein-peptide affinities in parallel, providing a rare combination of true in-solution binding equilibria with the ability to multiplex.

Discovery of a PDZ Domain Inhibitor Targeting the Syndecan/Syntenin Protein-Protein Interaction: A Semi-Automated "Hit Identification-to-Optimization" Approach.

The rapid identification of early hits by fragment-based approaches and subsequent hit-to-lead optimization represents a challenge for drug discovery. To address this challenge, we created a strategy called "DOTS" that combines molecular dynamic simulations, computer-based library design (chemoDOTS) with encoded medicinal chemistry reactions, constrained docking, and automated compound evaluation. To validate its utility, we applied our DOTS strategy to the challenging target syntenin, a PDZ domain containing protein and oncology target. Herein, we describe the creation of a "best-in-class" sub-micromolar small molecule inhibitor for the second PDZ domain of syntenin validated in cancer cell assays. Key to the success of our DOTS approach was the integration of protein conformational sampling during hit identification stage and the synthetic feasibility ranking of the designed compounds throughout the optimization process. This approach can be broadly applied to other protein targets with known 3D structures to rapidly identify and optimize compounds as chemical probes and therapeutic candidates.

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets.

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.

A Significant Difference in Core PDZ Interactivity of SARS-CoV, SARS-CoV2 and MERS-CoV Protein E Peptide PDZ Motifs In Vitro.

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.

Computational design of cyclic peptides to inhibit protein-peptide interactions

Many protein-protein interactions result from the binding of one folded protein with one short peptide segment, such as complexes formed by SH3 or PDZ domains. These transient protein-peptide interactions are notably involved in cellular signaling pathways and generally have low affinities, which opens the possibility to design competitive inhibitors of these complexes. We present and assess here our computational approach, called Des3PI, to design de novo cyclic peptides with potential high affinity for protein surfaces involved in interactions with peptide segments. The results were not conclusive for two receptors, the αVβ3 integrin and the CXCR4 chemokine receptor, but were promising in the case of SH3 and PDZ domains: For the former, Des3PI was able to find at least one cyclic sequence with six hotspots that binds a SH3 domain with a better theoretical affinity to the known proline-rich RLP2 peptide. For the latter, Des3PI could identify at least four cyclic sequences with four or five hotspots that have lower binding free energies computed by the MM-PBSA method than the reference peptide GKAP.

Abstract P2-11-35: A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer

Abstract A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer Han Gao1, Fiona Ruge1, Lin Ye1, Jane Lane1, Eleri Davies2, Wen G. Jiang1, Tracey A. Martin1 1Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK 2Wales Breast Centre, University Llandough Hospital, Cardiff CF64 2XX, UK Introduction. MUPP1 (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) or MPDZ is a scaffold protein with multiple PDZ domains, critical for multiple protein docking and protein-protein interactions in cells. MUPP1 is particularly interesting as it offers docking sites for a number of tight junction (TJ) proteins and proteins known to regulate cell junction and motility, including angiomotin (AMOT) and angiomotin-like proteins, the latter being important in regulating cell migration of endothelial cells and an important member in TJ protein complexes. The MUPP1 protein complex at cell junctions also includes CAR/CXADR (coxsackie adenovirus receptor) and members of the ERM (Moesin-Ezrin-Radixin) protein family. It was proposed that the junctional-related MUPP1 docking protein complex may collectively have a role in the development and progression of cancer including breast cancer. The present study examined the profile and integrated expression of these MUPP1 docking molecules in relationship with the outcome, clinical and pathological factors of breast cancer. Methods. Using the datasets of an existing breast cancer cohort at Cardiff, we visited the expression profile of the MUPP1/MPDZ docking molecules including MUPP1, four members of the ERM family, including ezrin, moesin, radixin and EHM2 (also known as NF2) and CAR/CXADR (coxsackie adenovirus receptor). The profile and integrated expression of the profile were analysed against clinical, pathological and outcome factors and also the hormone receptor status of breast cancers. Results. MUPP1 is a weakly linked to the disease progression, so as angiomotin, angiomotin-like1 and angiomotin-like2. Alone, only EHM2 showed significance in linking with the survival of the patient’s OS (p=0.38), neither of the remaining MUPP1 complex molecules, including MUPP1, AMOT, AMOTL1, AMOTL2, ezrin, moesin and radixin was able to offer a significant predictive value for the survival of the patients. However, it was found that MUPP1 has a significant correlation with AMOTs, EHM2 and ezrin. When these significantly correlated molecules were integrated in the analysis, the integrated expression pattern showed a highly significant value in evaluating the overall survival (p=0.003) and disease-free survival (p=0.007). Both of these predictive powers are independent in a multivariate analyses including the clinical and hormone receptors for OS (p=0.022, Hazard Ratio (HR)=1.352 95%CI (1.044-1.752)) and RFS (p=0.023, HR=1.241 (95% CI. 1.031-1.498)). It was interesting to note that the link between the expression pattern and OS was more prominent in ER negative (p=0.018) than in ER positive tumours (p=0.177). The same was observed with disease free survival for ER negative (p=0.011) compared with ER positive (p=0.462) tumours. Non-triple negative breast cancer had shown a similar sensitivity compared with triple negative breast cancer. Discussion. MUPP1, a PDZ domain containing docking protein closed linked with TJ structure, forms an interesting expression profile with its docking molecules including angiomotins and EHM2 that allows evaluation of disease progress and clinical outcome of the patients with breast cancer. Citation Format: Han Gao, Fiona Ruge, Lin Ye, Jane Lane, Eleri Davies, Wen G. Jiang, Tracey A. Martin. A tight junctional scaffold protein MUPP1/MPDZ and its docking proteins, angiomotins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-35.

An X-Domain Phosphoinositide Phospholipase C (PI-PLC-like) of Trypanosoma brucei Has a Surface Localization and Is Essential for Proliferation

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

SDCBP promotes pancreatic cancer progression by preventing YAP1 from β-TrCP-mediated proteasomal degradation

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as syntenin1, as a novel targetable factor in...