Tools used to study how protein complexes are assembled in signaling cascades

Dwane, Susan; Kiely, Patrick A.

doi:10.4161/bbug.2.5.17844

Cited by 26 publications

(9 citation statements)

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“…This technique includes antibody/antigen purification complexes at conditions that specifically bind antibodies. Rare proteins can be accumulated up to 10,000-fold by IP (Dwane and Kiely, 2011). Luciferase immunoprecipitation systems (LIPS) have been developed for the rapid detection of antibodies against peste des petits ruminants virus (PPRV) (Berguido et al, 2016), varicella-zoster virus (VZV) (Cohen et al, 2014), zinc transporter (ZnT8) autoantibodies (Ustinova et al, 2014), and pancreatic and duodenal homeobox 1 autoantibodies (PAA) (Donelan et al, 2013).…”

Section: Immunoprecipitationmentioning

confidence: 99%

Antibody Engineering for Pursuing a Healthier Future

et al. 2017

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Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

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Section: Immunoprecipitationmentioning

confidence: 99%

Antibody Engineering for Pursuing a Healthier Future

et al. 2017

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“…The use of biosensor technology for elucidation of function, via detailed kinetic and mechanistic studies of protein-protein is not common. Instead, it is sometimes simply recognized as one of many methods for identifying and confirming novel interactions (Berggard et al, 2007;Dwane and Kiely, 2011), as we and others have also performed for other scaffolding proteins (Fukunaga et al, 2005;Larsson et al, 2003;Wang et al, 2009) and in the initial study of caldendrin interactions with AKAP79 (Gorny et al, 2012). The quantification is often limited to determination of affinities, assuming simple interaction models even when the interaction mechanism is clearly more complex (Fukunaga et al, 2005).…”

Section: Kinetic Characteristics and Functional Consequencesmentioning

confidence: 99%

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function

Seeger

Gorny

Reddy

et al. 2012

J of Molecular Recognition

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The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (K(D) = 20 nM) and calmodulin (K(D) = 30 nM), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca(2+). Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca(2+), but with different kinetic rate constants. The data are consistent with a similar initial Ca(2+)-dependent binding step for the two proteins. For caldendrin, a second Ca(2+)-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein-protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.

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“…Peptide arrays of immobilised overlapping 23-mer peptides, each shifted to the right by 5 amino acids encompassing the entire PP2A-C sequence were generated as previously described [12,31,43]. Arrays were probed with GST alone or GST-RACK1, and bound GST was detected by immunoblotting with anti-GST antibody as described in Materials and Methods (Fig.…”

Section: Rack1 and Pp2a-c Complex Was Confirmed (Fig 1a (I) And (Ii))mentioning

confidence: 99%

RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells

Kiely

Adams

Hayes

et al. 2017

Cellular Signalling

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. (2017) RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells. Cellular Signalling, 35, pp. 290-300. (doi:10.1016Signalling, 35, pp. 290-300. (doi:10. /j.cellsig.2016 This is the author's final accepted version.There may be differences between this version and the published version. You are advised to consult the publisher's version if you wish to cite from it.http://eprints.gla.ac.uk/128660/ RACK1/PP2A complex is required for cell adhesion, proliferation, migration, invasion.Potential role for the RACK1/PP2A complex in the progression of breast cancer. KeywordsBreast Cancer, RACK1, PP2A, Protein-Protein Interactions. ABSTRACTConflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilize PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer.

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Tools used to study how protein complexes are assembled in signaling cascades

Cited by 26 publications

References 148 publications

Antibody Engineering for Pursuing a Healthier Future

Antibody Engineering for Pursuing a Healthier Future

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function

RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells

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