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Sep 27, 2013

Literature Review: Probing Protein Oligomerization

Researchers describe an assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein.

Literature Review: Probing Protein Oligomerization

The authors present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. [© molekuul.be - Fotolia.com]

  • Taking up about a third of cellular proteins, oligomeric proteins may confer several advantages, such as functional control, allosteric regulation, and establishment of higher order complexity (Ali et al., Bioorg Med Chem 2005;13:5013–5020). A number of techniques, including biophysical and fluorescent methods, can be used to detect in vitro/in vivo protein association, and/or provide affinity and stoichiometry determination of the interaction (for example, see Gell et al., Adv Exp Med Biol 2012;747:19–41). Among these different approaches, fluorescence polarization, for instance, not only can measure association constants, but is also high-throughput amenable for potential screening of small molecular modulators of protein stability.

    In their article, Gray and co-workers* report an alternative plate based assay to study regulation of protein self-assembly and its effect on protein interactions. An emerging cancer drug target, pro-oncogenic protein anterior gradient-2 (AGR2), was applied as a model protein for this study due to the existence of a previously reported dimeric species. Using a fluorescently conjugated monoclonal antibody (MAB) and a far infrared emission as the readout, the assay design was based on the MAB recognition of separate epitopes that occurred only when AGR2 was in an oligomeric format (see Figure 1).

  • Click Image To Enlarge +
    Figure 1

    Figure 1. Developing a quantitative microtiter assay to measure AGR2 oligomerization. (A) Evaluation of the bioactivity of fluorescently labeled MAB3.4 in a luminescent-based ELISA. (B) MAB3.4 was left unconjugated or conjugated to DyL800 and after purification of the latter; the monoclonal antibodies were titrated into reactions containing AGR2 protein on the solid phase. The binding of MAB3.4 to AGR2 was measured using an anti-mouse IgG secondary antibody conjugated to peroxidase. The bioactivity of the monoclonal antibody (in relative luminescent units) is measured as a function of increasing MAB3.4 concentration. (C) Evaluation of the bioactivity of fluorescently labeled MAB3.4 in a fluorescence detection assay. (D) MAB3.4 was left unconjugated or conjugated to DyL800 and the monoclonal antibodies were titrated into reactions containing AGR2 protein on the solid phase. The bioactivity of the monoclonal antibody was measured as levels of emission at 800 nm a function of increasing MAB3.4 concentration. (E) Evaluation of the bioactivity of fluorescently labeled MAB3.4 to detect a potential AGR2 oligomer. (F) MAB3.4 was coated onto the solid phase and increasing amounts of oligomeric AGR2 were added to allow capture onto the solid phase. Fixed amounts of DyL800 MAB were added into reactions and the binding of DyL800-MAB3.4. The extent of oligomerization was quantified as levels of emission at 800 nm a function of increasing AGR2 protein concentration and is presented as an average from triplicate titrations. MAB, monoclonal antibody; DyL800, DyLight800; ECL, electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay.

  • Click Image To Enlarge +
    Figure 2. The effects of AGR2 oligomerization on its protein-interaction activity. The indicated AGR2 proteins (wt-AGR2, AGR2E60A, or Δ45-AGR2) were incubated in reactions containing (A) biotinylated TxIYY containing peptide and (B) Reptin. AGR2 bound to either peptide or Reptin was detected using MAB3.4 and quantified using anti-secondary IgG coupled to peroxidase and chemiluminescence. The data are depicted as AGR2 binding activity (in RLU) as a function of increasing AGR2 protein concentration (in triplicate and presented as an average). WT, wild-type (wt-) AGR2; E60A, AGR2E60A; Δ45, Δ45-AGR2; IgG, immunoglobulin G.

    The assay, termed 2SMTA, was further demonstrated as being capable of quantitative measurement of protein oligomerization. More interestingly, the AGR2 protein contains an intrinsically disordered N-terminal region, and testing (de)stabilization effects induced by small molecule modulators or self-peptides generated based on dimer interface or this intrinsically disordered region would be a relevant next step. The authors subsequently demonstrated the assay's ability to differentiate such self-peptides into categories that either acted as stabilizers or disruptors of protein oligomer equilibrium.

    A third relevant utility of the assay was to examine oligomerization effect on protein biochemical activity, such as interaction with other protein substrates. Between two well-characterized AGR2 substrates, a pentapeptide motif and Reptin, no substantial binding activity was observed among wild-type and mutant AGR2 for the former client, while both stabilization and attenuation effects were recognized for the latter (Figure 2). Overall, this work presents a high-throughput friendly assay that can be used to quantitatively measure protein oligomerization, screen for protein stability modulators, and further correlate protein subunit structure with its biochemical activity.

  • *Abstract from Protein Science 2013 Jun 18 [Epub ahead of print]; DOI: 10.1002/pro.2299

    Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2.

    A two site-sandwich microtiter assay (2SMTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the 2SMTA assay. A DSS crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (a more stable dimer), and monomeric AGR2E60A revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2E60A mutant.

    Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its oncogenic potential.



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