Caracterización del dominio transmembrana del receptor de neurotrofinas p75 y su papel en la dimerización, localización subcelular, procesamiento y señalización

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 15-07-2016Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu

TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition

20 páginas, 8 figurasγ-secretase inhibitors (GSI) were developed to reduce the generation of Aβ peptide to find new Alzheimer's disease treatments. Clinical trials on Alzheimer's disease patients, however, showed several side effects that worsened the cognitive symptoms of the treated patients. The observed side effects were partially attributed to Notch signaling. However, the effect on other γ-secretase substrates, such as the p75 neurotrophin receptor (p75NTR) has not been studied in detail. p75NTR is highly expressed in the basal forebrain cholinergic neurons (BFCNs) during all life. Here, we show that GSI treatment induces the oligomerization of p75CTF leading to the cell death of BFCNs, and that this event is dependent on TrkA activity. The oligomerization of p75CTF requires an intact cholesterol recognition sequence (CRAC) and the constitutive binding of TRAF6, which activates the JNK and p38 pathways. Remarkably, TrkA rescues from cell death by a mechanism involving the endocytosis of p75CTF. These results suggest that the inhibition of γ-secretase activity in aged patients, where the expression of TrkA in the BFCNs is already reduced, could accelerate cholinergic dysfunction and promote neurodegeneration.This study was supported by the Spanish Minister of Economy and Competitiveness grant SAF2017-84096-R and by the Generalitat Valenciana 2018-55 to M Vilar. I García-Carpio was supported by an Formación de Personal Investigador (FPI) pre-doctoral fellowship (BFU2013/42746-P) and a mobility grant (EEBB-I-15-10278) from the Spanish Minister of Economy and Competitiveness. This work was funded by the Stichting Alzheimer Onderzoek (S16013) and the Fonds voor Wetenschappelijk Onderzoek or Flanders Research Foundation (FWO) research project (G0B2519N) to L Chavez-GutiérrezPeer reviewe

Heterodimerization of p45–p75 Modulates p75 Signaling: Structural Basis and Mechanism of Action

<div><p>The p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily, is required as a co-receptor for the Nogo receptor (NgR) to mediate the activity of myelin-associated inhibitors such as Nogo, MAG, and OMgp. p45/NRH2/PLAIDD is a p75 homologue and contains a death domain (DD). Here we report that p45 markedly interferes with the function of p75 as a co-receptor for NgR. P45 forms heterodimers with p75 and thereby blocks RhoA activation and inhibition of neurite outgrowth induced by myelin-associated inhibitors. p45 binds p75 through both its transmembrane (TM) domain and DD. To understand the underlying mechanisms, we have determined the three-dimensional NMR solution structure of the intracellular domain of p45 and characterized its interaction with p75. We have identified the residues involved in such interaction by NMR and co-immunoprecipitation. The DD of p45 binds the DD of p75 by electrostatic interactions. In addition, previous reports suggested that Cys257 in the p75 TM domain is required for signaling. We found that the interaction of the cysteine 58 of p45 with the cysteine 257 of p75 within the TM domain is necessary for p45–p75 heterodimerization. These results suggest a mechanism involving both the TM domain and the DD of p45 to regulate p75-mediated signaling.</p></div

Determination of p75ICD dimer interface by NMR.

<p>(A) Concentration-dependent chemical shift changes of ¹⁵N-labeled p75ICD observed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001918#pbio.1001918-Grzesiek1" target="_blank">[42]</a>-TROSY spectra at p75ICD concentrations of 10 µM, 100 µM, and 500 µM. (B) Some examples of residue peaks of p75ICD at the different concentrations of 10 µM (purple), 100 µM (green), and 500 µM (red). The cross-peaks are labeled to the corresponding residues. (C) Determination of monomer-dimer k_d using the changes in the chemical shift of different residues from the dimer interface plotted versus p75ICD concentration. (D) Concentration-dependent chemical shift changes from data in (B) are mapped onto the 3D structure of p75DD (PDB code 1NGR; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001918#pbio.1001918-Liepinsh1" target="_blank">[45]</a>). The 3D structure is represented by a ribbon diagram and by a surface representation. Residues with chemical shift perturbations ΔCS, larger than 0.2 ppm, are displayed and colored in green. ΔCS  = 25[Δ(δ(¹H))² + Δ(δ(¹⁵N))²]^0.5, where δ(¹H) and δ(¹⁵N) are the chemical shifts in part per million (ppm) along the ω₂(¹H) and ω₁(¹⁵N) dimensions, respectively. (E) Immunoprecipitation experiments of wild-type or some p75 mutants in the dimer interface demonstrating the role of those residues in p75 homodimerization.</p

Three-dimensional NMR structure of p45DD.

<p>(A) Superposition of 20 conformers representing the 3D NMR structure (left) and ribbon diagram of the lowest energy conformer highlighting the α-helices in red and yellow (right). (Bottom) ¹³C^α chemical shift deviation from their corresponding "random coil" values Δδ(¹³C^α) of p45ICD (residues 130–228). Segments of positive deviations are indicative of helical secondary structure. The location of the six α-helices of p45 are represented by cylinders and labeled accordingly. The asterisk indicates the unusual chemical shift of R211 attributed to the salt bridge between R211 and D213 as well as E160. (B) Electrostatic potential of p45DD and p75DD in a surface representation indicates a highly negative patch around helix α3 of p45DD. The same orientation as in (A) is used. (C) Sequence alignment of DEDs of mouse PEA-15 (Q62048), human FADD-DED (Q13158), human Caspase-8 (Q14790), molluscum contagiosum virus MCV-159 (Q98325), and death domain from rat p75DD (NP_036742) and mouse p45DD (NP_080288). The positions of helices are indicated by the diagram below the p45DD sequence. The conserved motif RxDΦ at the beginning of helix 6 and the conserved residues (EL) in helix 2 are indicated in bold.</p

Model illustrating p45 inhibition of p75/NgR signaling.

<p>p75 is a constitutive dimer in the membrane, stabilized by the Cys257 at the TM domain, where it can bind to NgR complex and activate RhoA signaling and axonal growth collapse. When p45 binds to p75 through both the TM domain (Cys257–Cys58) and DD interactions, p75 downstream signaling is inhibited.</p

p75ICD homodimerization.

<p>(<b>A</b>) Size exclusion gel-filtration chromatography of p75ICD. The elution profile reveals the presence of a mixture of monomers and dimers in the case of p75ICD (black lines). In the presence of DTT, only one elution peak in the gel filtration chromatogram is seen (gray line). Molecular weight standards are shown above the chromatogram. (B) Coomassie blue staining of reducing and nonreducing SDS-PAGE of the fractions collected in gel filtration as shown in (A). The presence of a protein band corresponding to a p75ICD dimer in the nonreducing SDS-PAGE is shown with an arrowhead. The migration of p45ICD is shown as a reference. (C) Analytical ultracentrifugation data on p75ICD in PBS (pH 8.0). (Top) Overlay of successive sedimentation velocity profiles recorded at ∼10 min intervals, represented by different colors. The solid lines represent the direct fitting of the data to a two-species model by the Svedberg program. (Bottom) Sedimentation velocity AUC profiles and the <i>c(S</i>) distributions for p75ICD (at 0.1, 0.3, and 1.0 mg ml⁻¹). The residual differences between the experimental data and the fit for each point are shown above. Theoretical p75ICD MW  = 16.5 kDa. Fitting data MW  = 30.7±1.2 kDa.</p

Interactions between p75 and p45.

<p>(A) A schematic diagram showing domains of p45 in comparison with p75. TM, transmembrane domain; DD, putative death domain; PDZ, putative PDZ binding domain. The degrees of identity and homology in amino acid residues between mouse p75 and p45 are shown in percentages. (B) P45 forms a complex with FADD and p75. V5-tagged TNFR1, human Fas, mouse Fas, p75, FADD, or Caspase-8 was transfected into CrmA/Flag-p45/293 stable cells. The lysates were immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-V5 antibodies. (C) P7 cerebellum extracts were immunprecipitated with anti-p75 ECD antibody (9651) or anti-p45 ECD antibody (6750) followed by immnuoblotting with anti-p75 antibody (Buster). T, total lysate. (D) Western blotting analysis of p45 and p75 expression in the brain, spinal cord, and DRG of P0 and adult mice. (E) Increased expression of p45 and p75 in the spinal cord and sciatic nerve following sciatic nerve injury. (Top) Spinal cord sections were immunostained with anti-p45 or -p75 antibodies. p45 and p75 immunoreactivities were markedly increased in the ipsilateral side as compared to the contralateral side. Higher magnification indicated that expression of p45 and p75 is increased in motor neurons. (F) Longitudinal sections of crushed and uncrushed sciatic nerves were immunostained with antibodies against p45, p75, or neurofilament. The levels of p45 and p75 were markedly increased in the distal (D) portion of sciatic nerves as compared to the proximal (P) end and the uncrushed (UC) nerve.</p

p75/p45 interaction is promoted by DD and TM domain.

<p>(A) Co-immunoprecipitation experiments of wild-type or mutant p45 with Myc-p75 in HEK293 cells. p45 mutants show differential binding to p75. The p75DD-dependent chemical shift changes of p45DD are mapped onto the 3D structure of p45DD. (B) Co-immunoprecipitation experiments of wild-type or p75 mutants with Flag-p45 in HEK293 cells showed differential binding to p45. The p45DD-dependent chemical shift changes of p75DD are mapped onto the 3D structure of p45DD. (C) Protein sequences of p75 and p45 TM domains are highly conserved. The conserved cysteine residue is highlighted in a red box. (D) The cysteine residues in the TM domain of both p75 and p45 form a covalent disulfide dimer between p75 and p45. Co-immunoprecipitations of either p75 wild-type or the p75 TM domain mutant (p75-C257A) with p45 wild type were analyzed in HEK293 cells and in reducing and nonreducing SDS-PAGE followed by Western blot. p75 and p45 form a heterodimer sensitive to DTT (arrow). (E) Co-immunoprecipitations of either p75 wild type or the p75 TM domain mutant (p75-C257A) with p45 wild type or p45 C58A mutant were analyzed in HEK293 cells, indicating that both p75-C257A and p45-C58A TM domain mutants diminish the interaction between p75 and p45.</p

p75 stabilized dimers through both DD and TM domain are required for NgR interaction.

<p>(A) Immunoprecipitation experiments of wild-type or mutant p75 with NgR that is Flag-tagged in HEK293 cells. The same mutants described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001918#pbio-1001918-g004" target="_blank">Figure 4</a> that are not co-immunoprecipitated with Flag-ΔECD-p75 are not able to be co-immunoprecipitated with Flag-tagged NgR either, suggesting a role of DD dimer stabilization in NgR binding. WT, 100%; D372R, 61.8%±3.64%, <i>N</i> = 5; *** <i>p</i><0.0001; H376E, 125.4%±9.13%, <i>N</i> = 5; * <i>p</i><0.1; E377R, 27.4%±4.79%, <i>N</i> = 5; *** <i>p</i><0.0001. The data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001918#pbio.1001918.s012" target="_blank">Table S1</a>. (B) Immunoprecipitation experiments of wild-type or p75-C257A with NgR-Flag in HEK293 cells. The p75-C257A interaction with NgR is impaired in comparison to p75 wild type. WT,100%, C257A, 17.67%±4.67%, <i>N</i> = 3; *** <i>p</i><0.0001. The data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001918#pbio.1001918.s012" target="_blank">Table S1</a>. (C) Immunoprecipitation experiments of wild-type or p75-C257A with NgR-Flag in HEK293 cells and nonreducing SDS-PAGE followed by Western blot. The NgR interaction to p75 dimers is preferred to p75 monomers. The presence of dimers (d) and monomers (m) is labeled in the blot.</p