Periodic Reporting for period 1 - DIAL-IN (Dynamic Integrated structural and proteomic Analysis of the LUBAC complex and its involvement in NF-κB and inflammation)
Période du rapport: 2016-11-01 au 2018-10-31
OBJECTIVES: As part of my Marie Curie project, I proposed to characterize the TNF-RSC by a variety of complementary methods, based mostly on mass spectrometry, to obtain a comprehensive understanding of the composition, dynamics and regulation of this most relevant complex. A particular emphasis has been placed on the ubiquitin ligase LUBAC, a recently discovered protein complex that is part of the TNF-RSC and that has not yet been, in many respect, thoroughly characterised.
1) DETERMINATION OF TNF-RSC COMPOSITION: We performed a comprehensive, quantitative mapping of the TNF-RSC composition and identification of the new complex members by mass spectrometry using data dependent acquisition (DDA), data-independent acquisition (DIA) and parallel reaction monitoring (PRM) approaches. To achieve this goal, an extensive biochemical optimisation to natively isolate the TNF-RSC from cells has been carried out.
2) DYNAMICS OF TNF-RSC FORMATION: The composition (point #1) has been determined over the course of 4 different time points, providing an unprecedented view of the assembly and disassembly of this signaling platform over time.
3) TNF-RSC NEW MEMBERS FUNCTIONAL CHARACTERIZATION: As part of point #2, we identified two new putative members of the TNF-RSC. We carried out functional validation of these two new factors by several different means, including reciprocal pulldowns, knockdown (siRNA), knockout (CRISPR/Cas9), apoptosis assays and bioinformatics analyses.
4) TNF-RSC STOICHIOMETRY: We performed a first-ever time-resolved stoichiometry determination of the TNF-RSC holo-complex and subcomplexes by mass spectrometry using absolute quantification (AQUA). This is, to our knowledge, one of the the largest mass spectrometry absolute quantification study ever carried out on specific protein complex or signaling platform.
5) TNF-RSC IN SPACE: We developed a protocol to combine native affinity purification of the TNF-RSC wi the separation of protein subcomplexes with blue native page (BNPAGE), in conjunction with targeted mass spectrometry. This has allowed us to define different subcomplexes of the TNF-RSC, define their space-resolved composition and approximate molecular weight.
6) TNF-RSC PROXIMITY LABELING: Systematic interaction mapping by affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling (BioID) of one of the key subcomplexes of the TNF-RSC, LUBAC. Functional validation of targets is ongoing.
7) TNFRI MEMBRANE PROTEOME: We applied an unpublished protocol to label membrane proteins proximal to the TNF-R1 and identification of new candidates that may be involved in the regulation of the receptor’s activity (in collaboration with the Wollscheid Lab, ETH).
Many of the results will be condensed, over the course of the next few months, in two distinct publications. They have been presented internally, but also in the context of collaborations with external groups, and in the context of at least one conference and one workshop."
From a technical point of view, this is to my knowledge one of the most comprehensive integrative attempt to understand a single protein assembly, and taking advantage of several orthogonal techniques has indeed proven an extremely powerful strategies. Of these techniques, several are far from being routine and have required extensive preliminary work and optimisation - including the combination of BNPAGE separation with targeted proteomics, absolute quantification of dozens of different proteins covering several orders of magnitude of abundance, and the combination of DIA, DDA, and PRM approaches to target the same assembly.
From a biological point of view, we have learned already that new previously unreported members of the TNF-RSC exist, and we have characterised some of their properties (e.g. interactors) and started elucidating what effect they may have on TNF signaling and by what means. Furthermore, we have now a complete overview of the relative stoichiometries of all subcomplexes the TNF-RSC is comprised of, and we are in the course of integrating information obtained from the described approaches to define an absolute composition and size of the TNF-RSC.
IMPACT: In line with the results, the impact of the work will branch in two directions: methodologically, it provides an important example of how integrative approaches can be utilised to obtain unique insights into the architecture of large assemblies whose features are otherwise difficult to define by single methods. Biologically, it will provide altogether new details about the composition, dynamics and architecture of this most prominent signaling platform; will help shedding light on some of the most fundamental mechanisms underlying inflammatory signaling; and, in the long term, can aid identifying new important players in inflammation, understanding similar signaling platforms, and define potential targets for therapeutic intervention.