Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins

O’Connell, David J.; Bauer, Mikael C.; O’Brien, John; Johnson, Winifred M.; Divizio, Catherine A.; O’Kane, Sara L.; Berggård, Tord; Merino, Alejandro; Åkerfeldt, Karin S.; Linse, Sara; Cahill, Dolores J.

doi:10.1074/mcp.m900324-mcp200

Cited by 44 publications

(64 citation statements)

References 59 publications

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“…5B). This may be because the mutated site for uorophore incorporation, Ser17/Cys, was chosen to be exposed both in free and bound calmodulin, 22 thus the uorophore may be rotating freely both in free CaM and in the complex. We have noticed that the uorescence of CaM increases in the presence of detergent, and it may be that this enhances the anisotropy signal from Alexa 488 itself.…”

Section: Full-length Aqp0 and Cam Interaction Studiesmentioning

confidence: 99%

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

et al. 2018

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Protein-protein interactions play important roles in regulating human aquaporins (AQP) by gating as well as trafficking. While structural and functional studies have provided detailed knowledge of AQP transport mechanisms, selectivity as well as gating by conformational changes of loops or termini, the mechanism behind how protein-protein interactions control AQP-mediated water transport through cellular membranes remains poorly characterized. Here we explore the interaction between two human AQPs and regulatory proteins: the interaction between AQP0 and calmodulin, which mediates AQP0 gating, as well as the interaction between AQP2 and LIP5, which is involved in trafficking. Using microscale thermophoresis (MST) and fluorescence anisotropy, two methods that have the advantage of low sample consumption and detergent compatibility, we show that the interactions can be studied using both full-length AQPs and AQP peptides corresponding to the regulatory protein binding sites. However, full-length AQPs gave better reproducibility between methods and for the first time revealed that AQP0 binds CaM in a cooperative manner, which was not seen in experiments using peptides. Our study highlights that, while peptides are great tools for locating binding sites and pinpointing interacting residues, fulllength proteins may give additional insights, such as binding mechanism, allostery and cooperativity, important parameters for understanding protein-protein mediated regulation in the cellular context. Our work provides a platform for further studies of AQP regulation that may be of interest for designing drugs that target AQP complexes as well as the development of artificial bio-mimetic water channels for water-purification purposes.

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Section: Full-length Aqp0 and Cam Interaction Studiesmentioning

confidence: 99%

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

et al. 2018

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“…In humans it binds to more than 300 targets (1)(2)(3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4)(5)(6)(7)(8), as can altered gene expression (9).…”

mentioning

confidence: 99%

Conserved properties of individual Ca ²⁺ -binding sites in calmodulin

Halling

Liebeskind

Hall

et al. 2016

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

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Calmodulin (CaM) is a Ca 2+ -sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca 2+ concentration into cellular signals by binding to a wide range of target proteins in a Ca 2+ -dependent manner. We do not fully understand how CaM performs its role as a highfidelity signal transducer for more than 300 target proteins, but diversity among its four Ca 2+ -binding sites, called EF-hands, may contribute to CaM's functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca 2+ signals using four specialized EFhands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.calcium signaling | EF-hand | structure | evolution | protein

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“…To identify the global Tp53 and TP63 interacting partners, we used the protein binding microarray approach, allowing the high-throughput screening of the protein interacting candidates among the 16,368 individual human GST-His 6 -tagged proteins spotted on the chip. 20,[22][23][24] We used 25 μg of recombinant glutathione-S-transferase (GST)-Tp53 (1-393 amino acid residues) and GST-TP63 (1-680 amino acid residues) fusions purified from the baculovirus-infected Sf9 insect cells and labeled with Alexa-Flour-647. After each screen that was performed at the stringent conditions in the presence of 5-10 mM DTT, the fluorescence intensity of the spots was normalized against background, and the reproducibility of the duplicate spots was assessed.…”

Section: Resultsmentioning

confidence: 99%

Global tumor protein p53/p63 interactome

et al. 2012

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Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins

Cited by 44 publications

References 59 publications

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

Conserved properties of individual Ca ²⁺ -binding sites in calmodulin

Global tumor protein p53/p63 interactome

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Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins

Cited by 44 publications

References 59 publications

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

Conserved properties of individual Ca 2+ -binding sites in calmodulin

Global tumor protein p53/p63 interactome

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Conserved properties of individual Ca ²⁺ -binding sites in calmodulin