New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains

Ceccarelli, Arianna; Venere, Almerinda Di; Nicolai, Eleonora; Luca, Anastasia De; Rosato, Nicola; Gratton, Enrico; Mei, Giampiero; Caccuri, Anna Maria

doi:10.1016/j.bbalip.2017.05.003

Cited by 12 publications

(12 citation statements)

References 54 publications

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“…demonstrate that the protein modifies the order of the phospholipids in the lipid bilayer in opposite ways, and to a different extent, depending on the temperature. In a recent study , we have reported the effects that TRAF2 exerts on giant unilamellar vesicles, as observed by confocal fluorescence microscopy. In particular, it was shown that the protein induces the formation of intraluminal vesicles, especially in the presence of the ganglioside GM1.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular dynamics simulations have suggested that the most flexible region of the truncated TRAF2 monomers is the N‐terminal domain, which is probably characterized by a loosening of the α‐helix that, in the full‐length TRAF2 oligomer, participates in the coiled coil motif . It was therefore proposed that the mechanical strength exerted by TRAF2 on the membrane occurs through the interaction of this region with ganglioside GM1.…”

Section: Discussionmentioning

confidence: 99%

“…TRAF2 expression and further purification were carried as previously described . The TRAF2 purity was analyzed by SDS–PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we have demonstrated that a truncated form of TRAF2, a TNF receptor‐associated factor, is able to bind giant unilamellar synthetic vesicles and that this capability is strictly dependent on the composition of the bilayer, in the protein nanomolar concentration range . Furthermore, it was also shown that the protein is able to induce vesiculation in the liposomes, a process that resulted to be mediated by GM1 . This ganglioside is a component of membranes of eukaryotic cells and is widely used as a marker of lipid rafts .…”

Section: Introductionmentioning

confidence: 99%

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Studying the TRAF2 binding to model membranes: The role of subunits dissociation

Venere

Nicolai

Sinibaldi

et al. 2018

Biotech and App Biochem

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The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state. These findings have been discussed in terms of their possible role in the activity of TRAF2 in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…TRAF2 expression and further purification were carried as previously described . The TRAF2 purity was analyzed by SDS–PAGE.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Studying the TRAF2 binding to model membranes: The role of subunits dissociation

Venere

Nicolai

Sinibaldi

et al. 2018

Biotech and App Biochem

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“…On the other hand, TRAF2 dimers form hetero-trimeric oligomers with TRAF1 (i.e., TRAF1: [TRAF2] 2 ), which displays an enhanced propensity in cIAPs binding [ 22 ]. Finally, the protein C-terminal domain, TRAF2-C, strongly interacts with membranes when dissociated into monomeric/dimeric forms [ 27 , 28 ]. All these findings suggest that the oligomerization process might have a regulatory function on the TNF signaling in vivo.…”

Section: Introductionmentioning

confidence: 99%

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

Venere

Nicolai

Minicozzi

et al. 2021

IJMS

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TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

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Fluorescence in Protein Science

Mei

2017

Encyclopedia of Life Sciences

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Fluorescence spectroscopy is an optical technique used to study biological systems such as membranes, nucleic acids and proteins, thanks to its high sensitivity and noninvasive nature. In the case of proteins, fluorescence is currently used for analytical detection, to study ligand binding and complex formation and to observe the presence of intermediate species in folding pathways. Resonance energy transfer, lifetime and anisotropy measurements are quantitative applications of fluorescence spectroscopy that allow the characterisation of biological processes in which proteins play fundamental roles, including membrane binding, enzyme catalysis, signalling and the regulation of the function of nucleic acids. The development of new fluorophores, light sources and detectors has opened the possibility to perform such kinds of measurements in vivo , extending the fluorescence technology to cell microscopy. Key Concepts Protein intrinsic fluorescence arises mainly from aromatic amino acids and some cofactors. The timescale of tryptophan lifetime is the same as protein conformational changes. Fluorescence anisotropy decays provide information on protein dynamics. Fluorescence energy transfer is used to measure distances of the order of protein size. Fluorescence correlation spectroscopy is a valid tool for investigating protein dynamics in vivo .

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New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains

Cited by 12 publications

References 54 publications

Studying the TRAF2 binding to model membranes: The role of subunits dissociation

Studying the TRAF2 binding to model membranes: The role of subunits dissociation

The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

Fluorescence in Protein Science

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