Pages

Analysis U2OS cells

/by

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


fop-41

  • 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

 

fop-42

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

fop-43

  • 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

fop-44

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

fop-45

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

fop-49

  • 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

fop-48-1

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

fop-49

  • 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

fop-50

  • 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

fop-51

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

 

fop-52

  • 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

fop-53

  • 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

fop-56

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

fop57

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

/by

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

fop-23

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

fop-24

Expression constructs

Klonierung 34

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

Etablierung 37

  • 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

Etablierung 37

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 (-)

fop-29

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

Aufreinigung42

Summary of purifications carried out on 8 stable Hek293 lines

Identifiedinteractorsarelisted in thetablebelow

Aufreinigung43

  • 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

fop-34

Introduction FOP

/by

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

rücken