A General Framework for Development and Data Analysis of Competitive High-Throughput Screens for Small-Molecule Inhibitors of Protein−Protein Interactions by Fluorescence Polarization

Roehrl, Michael H. A.; Wang, Julia Y.; Wagner, Gerhard

doi:10.1021/bi048233g

Cited by 239 publications

(358 citation statements)

References 31 publications

Supporting

Mentioning

331

Contrasting

Order By: Relevance

“…All SPOP variants bound substrate with an affinity similar to that of SPOP 28–359 (4–8 μM, see Table 2). Experimental data are shown as circles; the solid lines are fits to equation (2) (Roehrl et al , 2004). …”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

View full text Add to dashboard Cite

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

show abstract

Section: Resultsmentioning

confidence: 99%

“…where Puc is the total concentration of peptide, SPOP is the total concentration of SPOP, I max is the maximum anisotropy, and I min is the minimum anisotropy (Roehrl et al , 2004). Three independent experiments were performed for each SPOP, and the average and standard deviation are reported.…”

Section: Methodsmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Fluorescence intensity was measured with a 485 nm excitation filter and a 535 nm emission filter. Normalized data were used to calculate the K d (Roehrl et al, 2004).…”

Section: Proteolysis Assaysmentioning

confidence: 99%

The structure of the TBCE/TBCB chaperones and α-tubulin complex shows a tubulin dimer dissociation mechanism

Serna

Carranza

Martín‐Benito

et al. 2015

Journal of Cell Science

View full text Add to dashboard Cite

BSTRACTTubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in a-tubulin-b-tubulin dissociation. We used electron microscopy and image processing to determine the threedimensional structure of the human TBCE, TBCB and a-tubulin (aEB) complex, which is formed upon a-tubulin-b-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the aEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the a-tubulin-b-tubulin interface that is caused by a steric interaction between b-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucinerich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the aEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating a-tubulin degradation.

show abstract

“…Titration of this peptide with Dcp1-Dcp2 resulted in a fourfold increase in fluorescence anisotropy. Fitting of the data was performed using previously described methods (Roehrl et al 2004). The anisotropy data were converted to fraction bound, and the K d of the labeled peptide with wild-type Dcp1-Dcp2 was determined to be z12 mM (Fig.…”

Section: Lesions In Dcp1 and Edc1 Impair Bindingmentioning

confidence: 99%

“…All assays were performed in triplicate and the average of the readings was plotted using Sigma Plot. Curve-fitting for estimation of K d was performed using equations derived as previously described (Roehrl et al 2004). …”

Section: Fluorescence Anisotropymentioning

confidence: 99%

Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition

Borja

Piotukh²,

Freund³

et al. 2010

RNA

View full text Add to dashboard Cite

Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the K M for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 prolinerich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.

show abstract

A General Framework for Development and Data Analysis of Competitive High-Throughput Screens for Small-Molecule Inhibitors of Protein−Protein Interactions by Fluorescence Polarization

Cited by 239 publications

References 31 publications

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

The structure of the TBCE/TBCB chaperones and α-tubulin complex shows a tubulin dimer dissociation mechanism

Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition

Contact Info

Product

Resources

About