Mihaly Mezei
The Mount Sinai School of Medicine, Pharmacological Sciences, Faculty Member
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Abstract We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human... more
Abstract We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH-β variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919-26, 2013 and Endocrinology 157:3658-67, 2016). Furthermore, we have also shown by molecular modelling that both TSH-β and TSH-βv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH-β variant we have inserted the gene sequence together with a FLAG tag into the P-secTag2 vector which was then cloned into mammalian HEK-293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH-β and the TSH-βv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH-β polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH-β and TSH-βv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8-fold increase in luciferase activity with TSH-β and a 4-fold increase with TSH-βv. These data demonstrate the inherent bioactivity of single TSH-β chains which was mirrored by TSH-βv. Local paracrine action by macrophage derived TSH-βv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.
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The aim of this chapter is, instead of reviewing the considerable progress made so far, to discuss the obstacles that prevent the wider use of the Monte Carlo method for macromolecular simulations. Successful adoption of the Monte Carlo... more
The aim of this chapter is, instead of reviewing the considerable progress made so far, to discuss the obstacles that prevent the wider use of the Monte Carlo method for macromolecular simulations. Successful adoption of the Monte Carlo method for conformational sampling of macromolecular assemblies requires solution(s) to the following problems: (1) convince investigators that it is worth it; (2) devise move sets that generate large enough correlated changes that can be accepted with reasonable probability; (3) develop efficient treatment of non-pairwise additive potentials; (4) develop efficient treatment of long-range contributions to the system's energy; and (5) the efficient parallelization of the algorithm. In the remainder of this chapter these issues will be treated one by one.
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Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is... more
Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-D-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23–DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.
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Abstract In patients with Graves’ disease (GD), previous studies have associated SNPs in linkage disequilibrium with the TSHR gene locus with changes in expression of two TSH receptor (TSHR) variants suggesting that receptor variants may... more
Abstract In patients with Graves’ disease (GD), previous studies have associated SNPs in linkage disequilibrium with the TSHR gene locus with changes in expression of two TSH receptor (TSHR) variants suggesting that receptor variants may have an important role in disease etiology. Bioinformatic analyses predicts the existence of several human TSHR isoforms from alternative splicing and the most abundant is the previously described splice variant, TSHR-v1.3. In-silico modeling of TSHR v1.3 confirmed the potential binding of TSH and TSHR autoantibody to the v1.3 protein indicating its potential as an autoantigen and pseudoreceptor. To further characterize the bioactivity of v1.3 we engineered the protein by cloning the entire ORF into a mammalian expression vector. The purified protein was obtained from cell lysate by Ni-affinity chromatography. Immunoprecipitation demonstrated that both TSHR stimulating monoclonal antibody (MS1) and human recombinant TSH were able to bind TSHR v1.3 recombinant protein as predicted by the in-silico studies. TSHR-v1.3 was also able to inhibit the effect of TSH and MS-1 on cAMP generation in a dose-dependent manner. These observations confirmed the ability of purified TSHR-v1.3 protein to interact with TSH and autoantibody and modulate their activity. Using purified IgG fractions from sera of 13 patients with GD with known TSHR antibodies we also looked for TSHR-v1.3 autoantibodies. We used a peptide based ELISA against two different epitopes of the splice variant. 11 out 13 (84.6%) of the GD samples were positive for a carboxy terminal peptide (COOH) and 10 out of 13 (76.9%) were positive to a junction region peptide (JNX). A dose-dependent bocking of signal was observed in the GD samples using the specific peptide. To detemine if the truncated isoform of the receptor could serve as a potential source of antigen and modulate GD we used double transfected cells expressing both GFP tagged TSHR-v1.3 and full-length TSHR. We then induced cell stress and apoptosis using a TSHR-mAb directed to the cleavage region (Tab16) and observed a dose dependent increase in mitochondrial cell stress and increased apoptosis (viability <46%) compared to control isotype specific antibody treated or untreated cells (74/71% viability).The culture supernatant contained released V1.3 tagged GFP protein suggesting that release of a new TSHR receptor antigen by this mechanism could play an important role in modulation of the disease process. These data show that a TSHR isoform that is expressed by alternative splicing can have a duel role by acting as a potential competitor for TSHR autoantibody and TSH itself and also as a likely source of autoantigen. Therefore, expression of such altered receptor protein within tissue depots has the potential to modulate thyroid autoimmunity by serving as an alternative intracellular antigen after apoptosis of thyroid cells.
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ABSTRACT
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Research Interests: Engineering, Chemistry, Monte Carlo Simulation, Monte Carlo, Statistical Physics, and 15 moreChemical Physics, Molecular Dynamics, High Frequency, Physical sciences, CHEMICAL SCIENCES, Langevin Dynamics, Classical Mechanics, Potential Energy Surface, Large Scale, Experimental Data, Molecular Dynamic Simulation, Thermodynamic Properties, Monte Carlo Method, Potential Energy, and Molecular dynamic
Supplementary Table S2. List of genes with expression regulated by selamectin treatment
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Supplementary Table S1 and Fig S1 and S2. Supplementary Table 1: qPCR primers used in this study; Supplementary Figure S1: Structures of compounds screened in Figure 1A and 1B; Supplementary Figure S2: Proximity ligation assay (PLA)... more
Supplementary Table S1 and Fig S1 and S2. Supplementary Table 1: qPCR primers used in this study; Supplementary Figure S1: Structures of compounds screened in Figure 1A and 1B; Supplementary Figure S2: Proximity ligation assay (PLA) analyzing SIN3A-MAD interactions
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We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of... more
We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 ago...
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Background A vast amount of physicochemical experimentation and theoretical work has been carried out on the liquid water system. The multivolume series of review articles edited by Franks (I) is recommended as a point of departure for... more
Background A vast amount of physicochemical experimentation and theoretical work has been carried out on the liquid water system. The multivolume series of review articles edited by Franks (I) is recommended as a point of departure for the original literature. A ...
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Supplementary Table S3. Ingenuity Canonical Pathways modulated by selamectin treatment
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Supplementary Table S3. Ingenuity Canonical Pathways modulated by selamectin treatment
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Biophysical studies have established that the thyrotropin (TSH) receptor (TSHR) undergoes posttranslational modifications including dimerization. Following our earlier simulation of a TSHR–transmembrane domain (TMD) monomer (called... more
Biophysical studies have established that the thyrotropin (TSH) receptor (TSHR) undergoes posttranslational modifications including dimerization. Following our earlier simulation of a TSHR–transmembrane domain (TMD) monomer (called TSHR-TMD-TRIO) we have now proceeded with a molecular dynamics simulation (MD) of TSHR-TMD dimerization using this improved membrane-embedded model. The starting structure was the TMD protein with all extracellular and intracellular loops and internal waters, which was placed in the relative orientation of the model originally generated with Brownian dynamics. Furthermore, this model was embedded in a DPPC lipid bilayer further solvated with water and added salt. Data from the MD simulation studies showed that the dimeric subunits stayed in the same relative orientation and distance during the 1000 ns of study. Comparison of representative conformations of the individual monomers when dimerized with the conformations from the monomer simulation showed sub...
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The receptor for thyroid stimulating hormone (TSHR), a GPCR, is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves’ disease) caused by stimulating TSHR antibodies. To date, only one domain of the... more
The receptor for thyroid stimulating hormone (TSHR), a GPCR, is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves’ disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have now generated a model of the entire TSHR by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane solvated it with water and counterions, and performed 1000ns Molecular Dynamic simulations on it.The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linking region (LR), known more commonly as the “hinge region”, showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also s...
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The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The... more
The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model, which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD-TRIO since 3 representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The 3 models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules...
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Triple negative breast cancers (TNBC) lacking estrogen, progesterone and HER2 receptors account for 10-20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a... more
Triple negative breast cancers (TNBC) lacking estrogen, progesterone and HER2 receptors account for 10-20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly-targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects ...