Biophysical studies of properties and interaction of three proteins involved in Alzheimer's disease : estrogen receptor α, calmodulin and FKBP52

Nous nous sommes intéressés à plusieurs protéines impliquées dans la maladie d’Alzheimer, notamment la protéine FKBP52, la calmoduline et le ROα. Nous nous sommes attachés à apporter quelques éléments de réponse quant à la formation d'un éventuel hétérocomplexe ROα/Ca4CaM/FKBP52. Dans une première partie, nous avons voulu étudier quelques bases moléculaires de l'interaction entre FKBP52 et la Ca4CaM, afin de mieux comprendre la pertinence biologique de cette affinité. Après avoir produit différents domaines de la protéine FKBP52 et la Ca4CaM, différentes techniques d’interaction protéine/protéine ont été utilisées. L'approche protéique de ce travail a été confortée par une approche peptidique. Elles ont permis de cibler le troisième domaine comme lieu de l’interaction. Pour la première fois, il a été totalement attribué par RMN et les sites concernés par l’interaction ont pu être discriminés. Par ailleurs, il a été montré que le premier domaine de FKBP52 pouvait interagir intermoléculairement avec ROα, par un motif en coude β de type II. Le ROα est un facteur de transcription dont l'activité dépend d'un certain nombre de coactivateurs parmi lesquels Ca4CaM. Le peptide issu de la séquence de recrutement de la calmoduline au sein du ROα (séquence 298-310) a fait l’objet au sein du groupe de nombreuses publications. Il a été montré que ce peptide possédait un caractère amyloïde. Bien qu’il n’existe aucun lien apparent entre cette caractéristique et une quelconque pathologie associée, la cinétique de formation des fibres issues de ce peptide dans différentes conditions de pH et de concentrations a été étudiée.We are interested in several proteins involved in the Alzheimer disease, in particular the FKBP52, calmodulin and ERα. We have provided some answers concerning the formation of a possible ROα/Ca4CaM/FKBP52 heterocomplex. In a first part, we wanted to study the molecular basis of the interaction between FKBP52 and Ca4CaM, to better understand the biological relevance of this affinity. After producing different domains of the FKBP52 protein and Ca4CaM, various techniques such as ITC, SPR, fluorescence or NMR were used. The protein approach of this work was supported by a peptide based study. These approaches have made it possible to target the third domain as the place of interaction. For the first time, the TPR domain was assigned by NMR spectroscopy and the sequences involved in the interaction could be discriminated. Furthermore, it was shown that the first domain of FKBP52 could interact intermolecularly with the ROα, by a type II β-turn motif. ROα is a transcription factor whose activity depends on a number of coactivators including Ca4CaM. The peptide resulting from the recruitment sequence of calmodulin within ROα (sequence 298-310) has been the subject of numerous publications within the group. It has been shown that this peptide has an amyloid character. Although there is no apparent link between this feature and any associated pathology, the kinetics of fiber formation from this peptide under different pH and concentration conditions has been studied

Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology

International audienceThe peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into β sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 μM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects

A β-Turn Motif in the Steroid Hormone Receptor’s Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52

International audienceThe immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor alpha (hER alpha) and using nuclear magnetic resonance, we show here that the short V(364)PGF(367) sequence, which is located within its ligand-binding domain and adopts a type II beta-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein

Bio-inspired hybrid fluorescent ligands for the FK1 domain of FKBP52

Publisher: American Chemical SocietyABSTRACT: The protein FKBP52 is a steroid hormone receptor co-activator likely involved in neurodegenerative disease. A series of small, water-soluble, bioinspired, pseudopeptidic fluorescent ligands for the FK1 domain of this protein are described. The design is such that engulfing of the ligand in the pocket of this domain is accompanied by hydrogen-bonding of the dansyl chromophore which functions as both an integral part of the ligand and a fluorescent reporter. Binding is concomitant with a significant wavelength shift and an enhancement of the ligand fluorescence signal. Excitation of FK1 domain native tryptophan residues in the presence of bound ligand results in Förster Resonance Energy Transfer. Variation of key ligand residues within the short sequence was undertaken and the interaction of the resulting library with the protein was measured by techniques including isothermal calorimetry analysis, fluorescence and FRET quenching and a range of Kd’s was determined. Co-crystallization of a protein ligand complex at 2.30 Å resolution provided detailed information at the atomic scale while also providing insight into native substrate binding

Identification of a human estrogen receptor α tetrapeptidic fragment with dual antiproliferative and anti-nociceptive action

International audienceThe synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295–311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain

Acute and Chronic Sarcoid Arthropathies: Characteristics and Treatments From a Retrospective Nationwide French Study

International audienceIntroduction: We aimed to analyze patients with acute and chronic joint involvements in sarcoidosis.Methods: This is a retrospective multicenter analysis of patients with proven sarcoidosis, as defined by clinical, radiological, and histological criteria, with at least one clinical and/or ultrasonographic synovitis.Results: Thirty-nine patients with sarcoid arthropathy were included, and among them 19 had acute sarcoidosis (Lofgren's syndrome). Joint involvement and DAS44-CRP were not significantly different in acute and chronic sarcoid arthropathies. Acute forms were more frequent than chronic sarcoid arthropathy in Caucasians, without any difference of sex or age between these 2 forms. Joint involvement was frequently more symmetrical in acute than chronic forms (100 vs. 70%; p < 0.05), with a more frequent involvement in wrists and ankles in acute forms, whereas the tender and swollen joint counts and the DAS44-CRP were similar between the 2 groups. Skin lesions were significantly more frequent in patients with acute forms [17 (89%) vs. 5 (25%); p < 0.05] and were erythema nodosum in all patients with Löfgren's syndrome and sarcoid skin lesions in those with chronic sarcoidosis. Among 20 patients with chronic sarcoidosis, treatment was used in 17 (85%) cases, and consisted in NSAIDs alone (n = 5; 25%), steroids alone (n = 5; 25%), hydroxychloroquine (n = 2; 20%), methotrexate (n = 3; 15%), and TNF inhibitors (n = 2; 10%). A complete/partial joint response was noted in 14 (70%) cases with a DAS44-CRP reduction of 2.07 [1.85–2.44] (from 3.13 [2.76–3.42] to 1.06 [0.9–1.17]; p < 0.05).Conclusion: Sarcoid arthropathies have different clinical phenotypes in acute and chronic forms and various treatment regimens such as hydroxychloroquine and methotrexate could be used in chronic forms

A β‑Turn Motif in the Steroid Hormone Receptor’s Ligand-Binding Domains Interacts with the Peptidyl-prolyl Isomerase (PPIase) Catalytic Site of the Immunophilin FKBP52

The immunophilin FKBP52 interacts with nuclear steroid hormone receptors. Studying the crystal structure of human estrogen receptor α (hERα) and using nuclear magnetic resonance, we show here that the short V³⁶⁴PGF³⁶⁷ sequence, which is located within its ligand-binding domain and adopts a type II β-turn conformation in the protein, binds the peptidyl-prolyl isomerase (PPIase or rotamase) FK1 domain of FKBP52. Interestingly, this turn motif displays strong similarities with the FKBP52 FK1 domain-binding moiety of macrolide immunomodulators such as rapamycin and GPI-1046, an immunophilin ligand with neuroprotective characteristics. An increase in the hydrophobicity of the residue preceding the proline and cyclization of the VPGF peptide strengthen its recognition by the FK1 domain of FKBP52. Replacement of the Pro residue with a dimethylproline also enhances this interaction. Our study not only contributes to a better understanding of how the interaction between the FK1 domain of FKBP52 and steroid hormone receptors most likely works but also opens new avenues for the synthesis of FKBP52 FK1 peptide ligands appropriate for the control of hormone-dependent physiological mechanisms or of the functioning of the Tau protein. Indeed, it has been shown that FKBP52 is involved in the intraneuronal dynamics of the Tau protein