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Analysis using LRC BMP4

Analysis of BMP4 interactions using LRC

  • The ligand-receptor capture technique allows identification of cell surface receptors which interact with a ligand of interest
  • Importantly, LRC allows detection of these interactions on living cells or tissues
  • The ligand is modified with a trifunctionalcrosslinker (TRICEPS) and incubated with target cells
  • Interaction of the ligand with its target receptor(s) leads to specific crosslinking between ligand and receptor
  • Ligand-receptor complexes are purified and subjected to tandem mass spectrometry to identify the receptor(s) bound by the ligand
  • LRC was used in this study to identify potential novel receptors of BMP4 in the context of FOP (Fibrodysplasia ossificans progressiva)

Labeling of BMP4 with TRICEPS

  • Purified BMP4 was labeled with TRICEPS
  • To determine whether labeling was successful, the BMP4-TRICEPS conjugate was tested via Dot blotting using HRP-conjugated streptavidin
  • As control, insulin-TRICEPS was used
  • As shown, BMP4 was successfully labeled with TRICEPS

fop-65Cluster analysisof LRC datasets

  • Triplicate LRC datasets are shown, either from BMP4 purifications or insulin control purifications

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Volcanoplotofreceptorsidentifiedwith BMP4

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Results: Analysis of BMP4 interactions using LRC

  • LRC analysis using BMP4 yielded a total of 7 receptors
  • ALK2 was not identified
  • 4 receptors are involved in cell surface protein sorting and degradation:

Lysosome membrane protein 2 (SCRB2)
Lysosome-associated membrane glycoprotein 1 (LAMP1)
Endoplasmin (ENPL)
HYOU1

Discussion

The ALK2 interaction network in U2OS cells

Summary of ALK2 interactors identified in U2OS cells

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Discussion: theintracellular ALK2 interaction network in U2OS cells

  • Apart from typical signaling components, the ALK2 interaction network shows a range of regulatory proteins involved in receptor sorting and turnover
  • These include classical chaperones, components of the ubiquitination pathway and the ERAD pathway
  • The presence of these interactors, together with the increase in regulatory interactions after BMP4 stimulations, suggest a tight regulation of ALK2 in U2OS cells
  • The increase in regulatory proteins which bind to ALK2 after BMP4 stimulation also suggests that ALK2 is rapidly internalized and degraded after activation
  • Contrary to wild type ALK2, the FOP (Fibrodysplasia ossificans progressiva) mutant variant R206H apparently does not interact with components of the ubiquitination and ERAD pathways
  • This suggests that ALK2 R206H may be mis-regulated and may not be removed from the cell surface after activation with BMP4

A modelfor ALK2 R206H hyperactivation in FOP

  • The commonly accepted model for FOP postulates a hyperactivation of ALK2 R206H, which leads to differentiation of inflamed tissues into bone
  • Our observation that the R206H mutant fails to interact with many components of the degradation machinery points to a possible explanation for receptor hyperactivity
  • Failure to remove the mutated ALK2 receptor from the surface after BMP4 stimulation may lead to accumulation of active receptor on the cell surface and therefore continued signaling into the downstream network
  • As the ALK2 R206H mutant is capable of correctly inducing downstream signaling in Hek293 cells via both the Smad and p38 pathways, accumulated active ALK2 R206H may „hyperactivate“ these pathways, pushing the cells towards differentiation into bone cells

 

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Analysis U2OS cells

Analysis of ALK2 interactions in U2O Scells

  • To characterize interactions involving ALK2 in U2OS cells, transient transfection was used to express wild type or R206H variants of ALK2
  • Transiently expressing U2OS were either used unstimulated, or were stimulated with BMP4
  • Datasets from unstimulated and BMP4 stimulated cells were compared to identify changes in the protein interaction network after activation of the BMP pathway

Pilot purification experiments using U2OS cells

  • Cells were transiently transfected with ALK2 wild type expression constructs
  • Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
  • Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells


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  • Cells were transiently transfected with ALK2 R206H expression constructs
  • Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
  • Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells

 

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Large scale purification of ALK2 from unstimulated U2OS cells

  • Volcano-Plot of final merged datasets for ALK2 R206H and control
  • Significantly enriched complex partners for ALK2 R206H are boxed

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  • Primary dataset of large scale purifications for ALK2 wild type
  • Triplicate purifications are shown for ALK2 wild type and control
  • Color coding shows variations in relative abundance of complex partners identified

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  • Volcano-Plot of final merged datasets for ALK2 wild type and control
  • Significantly enriched complex partners for ALK2 wild type are boxed

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  • Scatter plots of triplicate purifications are shown
  • Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

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  • Primary dataset of large scale purifications for ALK2 R206H
  • Triplicate purifications are shown for ALK2 R206H and control
  • Color coding shows variations in relative abundance of complex partners identified

  • Volcano-Plot of final merged datasets for ALK2 R206H and control
  • Significantly enriched complex partners for ALK2 R206H are boxed

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  • Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
  • Control purifications and ALK2 wild type purifications are shown to demonstrate reproducibility

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  • Primary dataset of large scale purifications for ALK2 wild type from BMP4 stimulated cells
  • Triplicate purifications are shown for ALK2 wild type and control
  • Color coding shows variations in relative abundance of complex partners identified

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  • Volcano-Plot of final merged datasets for ALK2 wild type and control BMP4 stimulated cells
  • Significantly enriched complex partners for ALK2 wild type are boxed

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  • Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
  • Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

 

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  • Primary dataset of large scale purifications for ALK2 R206H from BMP4 stimulated cells
  • Triplicate purifications are shown for ALK2 R206H and control
  • Color coding shows variations in relative abundance of complex partners identified

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  • Volcano-Plot of final merged datasets for ALK2 R206H and control BMP4 stimulated cells
  • Significantly enriched complex partners for ALK2 R206H are boxed

 

fop-54 Results: Purification of ALK2 complexes from U2OS cells

  • For ALK2 wild type purified from unstimulated U2OS cells, 8 interactors were identified
  • Interactors were mainly involved in receptor sorting, recycling and degradation
  • Stimulation of U2OS cells with BMP4 led to the identification of 15 interactors
  • 9 additional interactors were identified upon BMP4 stimulation, one interaction was lost
  • The ALK2 R206H mutant showed a strongly reduced interaction network: only one interactor was identified
  • Stimulation with BMP4 did not change the interaction network of ALK2 R206H

 Comparison of ALK2 wild type and R206H interactors identified

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Graphical representation of ALK2 interaction networks

  • Merged interaction network of all ALK2 complexes identified (wild type vs R206H; unstimulated vs stimulated)
  • Graphical representation was done using Cytoscape

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Summary: ALK2interaction network in U2OScells

  • The interaction network of ALK2 in U2OS cells shows a large number of proteins involved in receptor sorting and turnover
  • Interactors include chaperones, components of the ubiquitination pathway and components of the ERAD pathway
  • Stimulation with BMP4 leads to additional interaction of ALK2 with several lysosomal proteins and E3 ubiquitin ligases
  • In contrast, the mutant ALK2 R206H does not show interactions with components of either the ubiquitination or ERAD pathways
  • The only identified interaction involving ALK2 R206H is with the calcium binding protein calnexin (CANX)

 

Results cellular models

Characterization of additional cellular models for FOP (Fibrodysplasia ossificans progressiva)

Apart from Hek293 cells, three alternative cell lines were assessed for their use as cellular models of FOP:

  • HUVEC (Human umbilical vein endothelial cells)
  • U2OS (human osteosarcoma cells)
  • Jurkat (immortalized T lymphocytes)

The cell lines were tested for the following parameters:

  • Ease of cultivation
  • Active BMP signaling pathway
  • Efficiency of transfection

Characterization of BMP4 response in U2OS cells

  • A comparison between ALK2-expressing Hek293 cells and U2OS cells is shown
  • Cells were stimulated with BMP4 and samples were taken at indicated time points
  • BMP4 response was assessed using Western blotting with anti phospho-Smad and anti phospho-p38 antibodies
  • U2OS show a proper activation of Smad and p38 in response to BMP4 treatment

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Summary: characterization of additional cellular models for FOP

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Based on the results of the characterization studies, U2OS cells were selected as additional cellular model to study ALK2 interactions

Results

Investigation of ALK2 interactions in Hek293 cells

The following BMP4 signal cascade components were chosen for investigation:

  • ALK2 wild type receptor
  • ALK2 mutated receptors:
    R206H (causes FOP (Fibrodysplasia ossificans progressiva))
    Q207D (constitutive active)
    K235R (dominant negative)
  • ALK6 wild type receptor
  • ALK6 mutated receptor (I200K): causes brachydactyl (shortness of the fingers and toes)
  • BMPRII: type II receptor
  • FKBP12

Construction of novel expression vectors

Novel expression/purification vectors were generated to stably express the selected baits:

  • Strep II and HA epitope tags for detection and purification
  • Inducible tet promoter for regulated expression
  • Selection marker for stable cell line generation

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An additional expressionvector was constructedfortheexpressionof type I integral membraneproteinscarrying a cleavable N-terminal signalsequence:

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Expression constructs

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Transfection and establishment of stable Hek293 cells

  • Hek293 cells were transfected with the following constructs using the CaPo method:
    ALK2wt (wild type)
    ALK2 Q207D
    ALK2 K236R
    ALK2 R206H
  • Stable lines were selected using hygromycine
  • Expressed bait proteins were detected by Western blotting using an antibody against the HA tag
  • Extraction and purification were optimized using different detergent mixes to ensure optimal recovery of transmembrane receptors
  • Establishment of 8 stable Hek293 lines expressing key components of the BMP signaling cascade
  • Optimization of purification procedure to ensure reproducible capture of interactors
  • Western blots of representative purifications are shown below

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  • Optimization of purification procedure to ensure reproducible capture of interactors
  • Western blots comparing purification of ALK6 wild type (wt) and I200K mutant under different conditions from stable Hek293 lines

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Assessing the BMP signaling pathway in stable Hek293 lines

  • Stable Hek293 lines were stimulated with the ligand BMP4 for 30 mins
  • Phosphorylation of Smad proteins was assessed using a phospho-specific antibody at the indicated time points
  • Stimulation was compared across the ALK2 wild type and mutant receptors
  • „Tet“ indicates whether expression of the receptor was induced by tetracycline (+) or whether uninduced cells were used (-)

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Purification of receptor complexes from stable Hek293 lines

  • Protein complexes around the bait proteins were purified from stable Hek293 lines using the optimized large scale affinity purification procedure
  • All purifications were carried out in triplicate using the Strep tag fused to the baits
  • Purified protein complexes were digested with trypsin and peptides were subjected to Nano-LC separation, followed by tandem mass spectrometry analysis on a LTQ Orbitrap
  • Triplicate datasets were analyzed using a custom bioinformatics pipeline and interaction networks were generated using the software Cytoscape

Example of an interactor list generated from a purification of ALK2 wt

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Summary of purifications carried out on 8 stable Hek293 lines

Identifiedinteractorsarelisted in thetablebelow

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  • ALK2 wild type purifications yielded several known and novel interactors
  • ALK2 Q207D purifications yielded several interactors which overlap substantially with ALK2 wild type interactors
  • No interactions were identified for ALK2 R206H and K236R, or the BMPRII and ALK6 receptors
  • FKBP12 purifications yielded one novel interactor, TMX1
  • Interactors identified for ALK2 are mostly components involved in receptor sorting, turnover and receptor internalization

Graphical representation of ALK2 interactions in Hek293 cells

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The mechanisms of disturbances

Introduction to Fibrodysplasia ossificans progressiva (FOP)

  • In 2006, a landmark study showed that a point mutation in the ALK2 receptor is linked to FOP (Shore et al., 2006)
  • ACVR1/ALK2 is part of the family of type I BMP receptors (BMPRs)
  • BMPRs play a crucial role in bone formation during development
  • The study suggests that the identified ALK2 R206H mutation leads to receptor hyperactivation

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The GS- domain of ALK2

  • Structurally defined element within the intracellular part of ALK2
  • Located between the transmembrane segment and the kinase domain of ALK2
  • The GS domain interacts with various intracellular proteins, which form part of the signaling cascade
  • GS domain interactors include Smad proteins and FKBP12

ALK2 hyperactivation hypothesis

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Unanswered questions around the activation of the ALK2 R206H mutant

  • Does the R206H mutation change the interaction pattern of ALK2?
  • Which components of the signal cascade are affected?
  • Can these interactions be identified using a proteomics approach?
  • Do these changed interaction patterns shed any light on the disease mechanism of FOP?

Aim of the study

  • Identification of ALK2 interacting proteins using two proteomics approaches
  • Use of two different cellular model systems, Hek293 cells and U2OS cells
  • Investigation of differences in the interaction patterns of ALK2 wild type and ALK2 R206H
  • Visualization using the network software Cytoscape
  • Identification of possible novel targets for treatment of FOP

 

Introduction FOP

Introduction to Fibrodysplasia ossificans progressiva (FOP, Münchmeyer syndrome)

Fibrodysplasia ossificans progressiva (FOP, Münchmeyer syndrome)

  • First mentioned in England around 1740
  • Orphan disease, approx. one patient per 2 million individuals
  • Approx. 600 cases are documented
  • Autosomal dominant
  • Average life span: 45 years
  • Early indicator: deformed big toes

Feet

  • Progressive incidents of inflammation, followed by differentiation of inflamed tissue into bone
  • Patients evolve a “second skeleton”, leading to progressive immobilization and eventually, death
  • In 2006, a landmark study showed that a point mutation in the ALK2 receptor is linked to FOP (Shore et al., 2006)
  • ACVR1/ALK2 is part of the family of type I BMP receptors (BMPRs)
  • BMPRs play a crucial role in bone formation during development
  • The study suggests that the identified ALK2 R206H mutation leads to receptor hyperactivation

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