Quantification of Protein–Protein Interactions Using Fluorescence Polarization

Jameson, David M.; Seifried, Steven E.

doi:10.1006/meth.1999.0853

Cited by 99 publications

(94 citation statements)

References 28 publications

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“…To test this hypothesis, we measured RNAP binding to a Cy3-labeled duplex vapB10 promoter template (Fig. S4A) using a fluorescence anisotropy assay (26). We found that addition of RbpA decreased the dissociation constant, K d , for binding to the promoter DNA nearly twofold (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Hubin

Tabib-Salazar

Humphrey

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping σ-factor, σ A . The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ A as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.RbpA | X-ray crystallography | transcription | actinobacteria | mycobacteria

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Section: Resultsmentioning

confidence: 99%

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Hubin

Tabib-Salazar

Humphrey

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…FP is a method allowing the evaluation of the binding between a molecule of small molecular mass and an acceptor molecule due to their molecular movement in solution (16). Besides protein-protein interactions (16), FP can be applied to the analysis of carbohydrate-protein interactions (17).…”

Section: Resultsmentioning

confidence: 99%

“…Besides protein-protein interactions (16), FP can be applied to the analysis of carbohydrate-protein interactions (17). In this study, we applied FP on the analysis of the carbohydrate-protein interaction between C4-S and cathepsin K or collagen and monitored the displacement of C4-S from the cathepsin K/C4-S complex by potential inhibitors.…”

Section: Resultsmentioning

confidence: 99%

Selective Inhibition of the Collagenase Activity of Cathepsin K

Selent

Kaleta

et al. 2007

Journal of Biological Chemistry

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Cathepsin K, the main bone degrading protease, and chondroitin 4-sulfate (C4-S) form a complex with enhanced collagenase activity. In this report, we demonstrate the specific inhibition of the collagenase activity of cathepsin K by negatively charged polymers without affecting the overall proteolytic activity of the protease. Three different mechanisms to interfere with cathepsin-catalyzed collagen degradation are discussed: 1) inhibition of the formation of the cathepsin K/C4-S complex, 2) inhibition of the attachment of C4-S to collagen, and 3) masking of the collagenase cleavage sites in collagen. By targeting these interaction sites, collagen degradation can be modulated while the non-collagenolytic activities of cathepsin K remain intact. The main inhibitory effect on collagen degradation is due to the impeding effect on the active cathepsin K/C4-S complex. Essential structural elements in the inhibitor molecules are negative charges which compete with the sulfate groups of C4-S in the cathepsin K/C4-S complex. The inhibitory effect can be controlled by length and charge of the polymers. Longer negatively charged polymers (e.g. polyglutamates, oligonucleotides) tend to inhibit all three mechanisms, whereas shorter ones preferentially affect the cathepsin K/C4-S complex.An imbalance between bone formation and resorption can cause various bone diseases such as osteoporosis, certain forms of arthritis, and Paget disease. Type I collagen represents an essential part (90%) of the organic bone mass (1). It has been shown that cathepsin K, a cysteine protease predominantly expressed in osteoclasts (2-4), is an efficient collagenase that cleaves type I collagen at multiple sites in its helical domain (5, 6). Based on the physiological role of cathepsin K in bone resorption, cathepsin K inhibitors are being developed for the treatment of osteoporosis (7). Those inhibitors may also serve as drugs for rheumatoid arthritis (8). Presently, conventional cathepsin K inhibitors target the active site, thus causing a complete inhibition of enzymatic activity. An active site inhibition is associated with the loss of other physiological functions of cathepsin K activity which may result in undesirable side effects. Ablation of the matrix-degrading function in tandem with preservation of its non-collagenolytic protease activity would be greatly advantageous and will be addressed in this report.Collagen consists of three intertwining ␣-chains of ϳ1000 residues each (9). The three-dimensional structures of triplehelical collagen and cathepsin K suggest that the active cleft of cathepsin K does not provide sufficient space to accommodate intact triple-helical collagen. The entrance to the catalytic site of cathepsin K is only 5 Å wide (10), while triple-helical collagen has a diameter of 15 Å. This leads to the assumption that prior to hydrolysis by cathepsin K, the triple-helical collagen has to be unwound to expose its single chains. It was recently demonstrated that bone-and cartilage-resident glycosaminoglycans such as chondr...

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“…(Fig. 2)-To determine binding affinity between Tec kinase and FGF2, steady-state fluorescence polarization was used (54). His-tagged FGF2 was labeled with fluorescein-5-maleimide (F150, Thermo Fisher Scientific) covalently modifying a cysteine residue on the molecular surface of FGF2.…”

Section: Methodsmentioning

confidence: 99%

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Venuta

Wegehingel

Sehr

et al. 2016

Journal of Biological Chemistry

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Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.Although the majority of secretory proteins carry signal peptides for endoplasmic reticulum/Golgi-dependent secretion, a set of important growth factors and cytokines involved in tumor-induced angiogenesis and inflammatory responses makes use of alternative routes. These processes have collectively been termed "unconventional protein secretion" (1-5). FGF2 and IL1␤ are prominent examples for secretory proteins that make use of such pathways (4 -12). The molecular mechanism by which IL1␤ is secreted from cells is debated and may differ in some aspects between various kinds of immune cells (4,5,10,(13)(14)(15)(16). By contrast, the mechanism of the unconventional secretory pathway of FGF2 is emerging with great molecular detail (12). It is based upon direct translocation of folded species of FGF2 across plasma membranes (9,(17)(18)(19)(20). Hallmarks of this process are (i) FGF2 recruitment at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P 2 2 (9, 21, 22), (ii) FGF2 oligomerization and membrane pore formation (11,12,23,24), and (iii) extracellular trapping of FGF2 mediated by membrane-proximal heparan sulfate proteoglycans (1,2,25,26). Recently, these hallmarks of FGF2 secretion have also been implicated in unconventional secretion of HIV-Tat (HIV trans-activator of transcription) (12, 27-30). Based on a genome-wide RNAi screening approach, two additional factors physically associated with the plasma membrane have been identified to play a role in FGF2 secretion, Tec kinase (9, 11, 12, 24, 31) and ATP1A1 (12, 32-34), the ␣ subunit of the Na/K-ATPase (35). Although t...

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Quantification of Protein–Protein Interactions Using Fluorescence Polarization

Cited by 99 publications

References 28 publications

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Selective Inhibition of the Collagenase Activity of Cathepsin K

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

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