Structural Basis of Tau Interaction With BIN1 and Regulation by Tau Phosphorylation

Bridging integrator-1 (BIN1) gene is associated with an increased risk to develop Alzheimer’s disease, a tauopathy characterized by intra-neuronal accumulation of phosphorylated Tau protein as paired helical filaments. Direct interaction of BIN1 and Tau proteins was demonstrated to be mediated through BIN1 SH3 C-terminal domain and Tau (210–240) peptide within Tau proline-rich domain. We previously showed that BIN1 SH3 interaction with Tau is decreased by phosphorylation within Tau proline-rich domain, of at least T231. In addition, the BIN1/Tau interaction is characterized by a dynamic equilibrium between a closed and open conformations of BIN1 isoform 1, involving an intramolecular interaction with its C-terminal BIN1 SH3 domain. However, the role of the BIN1/Tau interaction, and its potential dysregulation in Alzheimer’s disease, is not yet fully understood. Here we showed that within Tau (210–240) peptide, among the two proline-rich motifs potentially recognized by SH3 domains, only motif P216TPPTR221 is bound by BIN1 SH3. A structural model of the complex between BIN1 SH3 and Tau peptide (213–229), based on nuclear magnetic resonance spectroscopy data, revealed the molecular detail of the interaction. P216 and P219 within the proline-rich motif were in direct contact with the aromatic F588 and W562 of the BIN1 SH3 domain. The contact surface is extended through electrostatic interactions between the positively charged R221 and K224 residues of Tau peptide and those negatively charged of BIN1 SH3, corresponding to E556 and E557. We next investigated the impact of multiple Tau phosphorylations within Tau (210–240) on its interaction with BIN1 isoform 1. Tau (210–240) phosphorylated at four different sites (T212, T217, T231, and S235), contrary to unphosphorylated Tau, was unable to compete with the intramolecular interaction of BIN1 SH3 domain with its CLAP domain. In accordance, the affinity of BIN1 SH3 for phosphorylated Tau (210–240) peptide was reduced, with a five-fold increase in the dissociation constant, from a Kd of 44 to 256 μM. This study highlights the complexity of the regulation of BIN1 isoform 1 with Tau. As abnormal phosphorylation of Tau is linked to the pathology development, this regulation by phosphorylation might have important functional consequences

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

Sam68, also known as KHDRBS1, is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, nuclear magnetic resonance spectroscopy and cell biology techniques to provide a comprehensive post-translational modification mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells

Etude structurale et fonctionnelle de la protéine HasS, un facteur anti-sigma impliqué dans la régulation de l'acquisition de l'hème chez Serratia marcescens

Chez les bactéries Gram-négatif, les systèmes d acquisition des différentes sources de fer sont généralement soumis à une régulation très fine. Ils sont régulés en fonction de la concentration intracellulaire en fer. Pour certains, il existe un niveau de régulation supplémentaire qui s effectue par une signalisation transmembranaire. Cette signalisation nécessite l interactions entre trois protéines spécifiques d un système donné : le récepteur membranaire et le facteur sigma de type ECF (extracytoplasmic fonction) et son anti-sigma qui une protéine de membrane interne. Les mécanismes moléculaires de ce processus de signalisation sont inconnus. Durant cette étude, nous nous sommes intéressés à la voie de signalisation régulant l acquisition de l hème via le système Has (heme acquisistion system) de Serratia marcescens. Nous nous sommes focalisés sur la première étape de cette signalisation transmembranaire à savoir, l interaction entre le domaine périplasmique du récepteur HasR et le facteur anti-sigma HasS. Nous avons étudié les aspects structuraux et fonctionnels de ces deux protéines. Nous avons déterminé par RMN la structure 3D du domaine périplasmique de HasR et identifié les résidus impliqués dans la voie de signalisation. De plus, nous avons produit pour la première fois le domaine périplasmique de HasS. Nous avons ensuite déterminé son état de repliement et étudié son interaction avec le domaine périplasmique de HasRIron uptake systems in gram-negative bacteria are generally tightly regulated by iron intracellular concentration. Some of them are also controlled by a transmembrane signaling. Three specific proteins are involved in the latter process : the outer membrane receptor and the ECF (extracytoplasmic function) sigma and antisigma factors. The data about these proteins and of their molecular interactions are sparse and the mechanisms governing this transmembrane signalisation are not understood. We present here the results of the study of the transmembrane signaling in the Has system (heme acquisition system) of Serratia marcescens. We focused on the interaction between the periplasmic domain of the receptor HasR and the ECF anti-sigma factor HasS, two proteins controlling the first step of this signaling process. We carried out structural and functional studies of these protiens. We solved the structure of the periplasmic domain of HasR by NMR and determined which of its residues were involved in the transmembrane signaling. We produced, for the fisrt time, the periplasmic domain of HasS and carried out its characterized regarding its structural features and its interaction with the periplasmic domain of HasRPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Conformation and Affinity Modulations by Multiple Phosphorylation Occurring in the BIN1 SH3 Domain Binding Site of the Tau Protein Proline-Rich Region

International audienceAn increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau

Interaction of a partially disordered antisigma factor with its partner, the signaling domain of the TonB-dependent transporter HasR.

International audienceBacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS-HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling

Influence of HasS_CTD expression on the regulation of the expression of HasR and HasA in <i>S. marcescens</i>.

<p>Immunodetection with anti-HasR (left panel) and anti-HasA (right panel) antibodies of whole cell extracts of <i>S.marcescens</i> SM365 either transformed with pBAD24 (control) or pBADHas_CTD for periplasmic expression of HasS_CTD. Lane 1, SM365pBAD24 grown in presence of glucose and iron; lane 2, SM365pBAD24 grown in presence of glycerol, dipyridyl and heme; lane 3, SM365pBAD24 grown in presence of arabinose, dipyridyl and heme; lane 4, SM365pBADHasS_CTD grown in presence of glucose and iron; lane 5, SM365pBADHasS_CTD grown in presence of glycerol, dipyridyl and heme; lane 6, SM365pBADHasS_CTD grown in presence of arabinose, dipyridyl and heme.</p

Structure and backbone dynamics of the periplasmic domain of HasR.

<p>(A) Overall structure of the HasR periplasmic domain. Top: cartoon diagram of the family of ten structures with lowest energy values (the flexible N- and C-termini are not presented). Bottom: (A) representative structure of the family (all residues are shown) with the linker containing the HasB/TonB box (magenta). The structure is rotated 180° around the Y axis. (B) Steady-state ¹⁵N-¹H NOE (C) relaxation rates R2 and (D) R1 were measured at 600 MHz at 20°C. (E) Structural comparison of the signaling domain of HasR with its two structural homologous. The loop 35–47 is surrounded.</p