An extensive body of literature suggested a possible role of the microtubule-associated protein Tau in chromatin functions and/or organization in neuronal, non-neuronal, and cancer cells. How Tau functions in these processes remains elusive. Here we report that Tau expression in breast cancer cell lines causes resistance to the anti-cancer effects of histone deacetylase inhibitors, by preventing histone deacetylase inhibitor-inducible gene expression and remodeling of chromatin structure. We identify Tau as a protein recognizing and binding to core histone when H3 and H4 are devoid of any post-translational modifications or acetylated H4 that increases the Tau's affinity. Consistent with chromatin structure alterations in neurons found in frontotemporal lobar degeneration, Tau mutations did not prevent histone deacetylase-inhibitor-induced higher chromatin structure remodeling by suppressing Tau binding to histones. In addition, we demonstrate that the interaction between Tau and histones prevents further histone H3 post-translational modifications induced by histone deacetylase-inhibitor treatment by maintaining a more compact chromatin structure. Altogether, these results highlight a new cellular role for Tau as a chromatin reader, which opens new therapeutic avenues to exploit Tau biology in neuronal and cancer cells.

The MUC4 membrane-bound mucin is a large O-glycoprotein involved in epithelial homeostasis. At the cancer cell surface MUC4 interacts with ErbB2 receptor via EGF domains to promote cell proliferation and migration. MUC4 is highly regarded as a therapeutic target in pancreatic cancer as it is not expressed in healthy pancreas, while it is neoexpressed in early preneoplastic stages (PanINs). However, the association/dissociation constant of MUC4-ErbB2 complex is unknown. Protein-protein interactions (PPIs) have become a major area of research in the past years and the characterization of their interactions, especially by biophysical methods, is intensively used in drug discovery. To characterize the MUC4-ErbB2 interaction, we used MicroScale Thermophoresis (MST), a powerful method for quantitative protein interaction analysis under challenging conditions. We worked with CHO cell lysates containing either the transmembrane β subunit of MUC4 (MUC4β) or a truncated mutant encompassing only the EGF domains (MUC4EGF3+1+2). MST studies have led to the characterization of equilibrium dissociation constants (Kd) for MUC4β-ErbB2 (7-25 nM) and MUC4EGF3+1+2/ErbB2 (65-79 nM) complexes. This work provides new information regarding the MUC4-ErbB2 interaction at the biophysical level and also confirms that the presence of the three EGF domains of MUC4 is sufficient to provide efficient interaction. This technological approach will be very useful in the future to validate small molecule binding affinities targeting MUC4-ErbB2 complex for drug discovery development in cancer. It will also be of high interest for the other known membrane mucins forming oncogenic complexes with ErbBs at the cancer cell surface.

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.

Intrinsically disordered protein YAP (yes-associated protein) interacts with TEADs transcriptional factors family (transcriptional enhancer associated domain) creating three interfaces. Interface 3, between the Ω-loop of YAP and a shallow pocket of TEAD was identified as the most important TEAD zone for YAP-TEAD interaction. Using the first X-ray structure of the hYAP50⁻71-hTEAD1209⁻426 complex (PDB 3KYS) published in 2010, a protein-protein interaction inhibitors-enriched library (175,000 chemical compounds) was screened against this hydrophobic pocket of TEAD. Four different chemical families have been identified and evaluated using biophysical techniques (thermal shift assay, microscale thermophoresis) and in cellulo assays (luciferase activity in transfected HEK293 cells, RTqPCR in MDA-MB231 cells). A first promising hit with micromolar inhibition in the luciferase gene reporter assay was discovered. This hit also decreased mRNA levels of TEAD target genes.

CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer.

The quest for small molecule inhibitors of the PD-1/PD-L1 checkpoint continues in parallel to the extensive development of monoclonal antibodies directed against this immune checkpoint. Drug screening strategies are being set up to identify novel PD-L1 inhibitors.

The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their "growth factor-like" biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and β-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

Orally-active anticancer small molecules targeting the PD-1/PD-L1 immune checkpoint are actively searched. Phenyl-pyrazolone derivatives with a high affinity for PD-L1 have been designed and characterized. In addition, the phenyl-pyrazolone unit acts as a scavenger of oxygen free radicals, providing antioxidant effects. The mechanism is known for the drug edaravone (1) which is also an aldehyde-reactive molecule. The present study reports the synthesis and functional characterization of new molecules (2-5) with an improved anti-PD-L1 activity. The leading fluorinated molecule 5 emerges as a potent checkpoint inhibitor, avidly binding to PD-L1, inducing its dimerization, blocking PD-1/PD-L1 signaling mediated by phosphatase SHP-2 and reactivating the proliferation of CTLL-2 cells in the presence of PD-L1. In parallel, the compound maintains a significant antioxidant activity, characterized using electron paramagnetic resonance (EPR)-based free radical scavenging assays with the probes DPPH and DMPO. The aldehyde reactivity of the molecules was investigated using 4-hydroxynonenal (4-HNE), which is a major lipid peroxidation product. The formation of drug-HNE adducts, monitored by high resolution mass spectrometry (HRMS), was clearly identified and compared for each compound. The study leads to the selection of compound 5 and the dichlorophenyl-pyrazolone unit as a scaffold for the design of small molecule PD-L1 inhibitors endowed with antioxidant properties.

Gloves represent an essential feature for hand protection because it is a requirement in the professional framework to comply with both hand hygiene standards and the principles of good laboratory practice. Despite their wide use, there is a knowledge gap regarding their composition, including phthalates. The purpose of the present study was to develop two orthogonal methods, GC-MS and HPLC-DAD, for the screening of plasticizers in gloves. Performances of these two methods were compared in terms of ease of use, number of analyzed plasticizers, and sample preparation. The two methods were validated and applied for the identification and quantification of plasticizers in ten gloves made with different materials (vinyl, nitrile, latex, and neoprene). Results revealed the presence of three main ones: DEHP, DEHT, and DINP. Additionally, the contents of plasticizers were extremely variable, depending on the glove material. As expected, the results point out a predominant use of plasticizers in vinyl gloves with an amount that should be of concern. While DEHP is classified as a toxic substance for reproduction 1B, it was, however, quantified in the ten different glove samples studied. This study provides new data regarding the plasticizers' content in protective gloves, which could be useful for risk assessment.

The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.