Optical biosensing: Kinetics of protein A‐IGG binding using biolayer interferometry

Wilson, Jo Leanna; Scott, Israel M.; McMurry, Jonathan L.

doi:10.1002/bmb.20442

Cited by 29 publications

(24 citation statements)

References 37 publications

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“…Mitochondrial Thiolase Directly Interacts with p46Shc with 55 nM K D -We determined that p46Shc and thiolase directly interact in vitro using BLI to detect real-time protein-protein interactions (12)(13)(14). We expressed in HEK cells and purified biotinylated ACAA2, p46Shc, p46Shc␦SH2 (p46Shc lacking the SH2 domain), and GFP as a non-binding control (Fig.…”

Section: P46shc Is the Only Mitochondrial Shc Isoform Under Physiologmentioning

confidence: 99%

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p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Tomilov

Tomilova

Shan

et al. 2016

Journal of Biological Chemistry

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Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steadystate flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shcdepleted mice in vivo.Shc proteins have three isoforms: p46, p52, and p66, and have major effects on metabolism (1-4). p52Shc contacts the insulin receptor and regulates the signaling between the IRS1 and Ras pathway (5). In 2004, it was demonstrated that the major mitochondrial Shc isoform is p46Shc (6), but since that time the mitochondrial partner and physiological function of p46Shc has been a mystery. Here we show the mitochondrial partner and function of p46Shc, and suggest how this could contribute to the obesity-resistance observed in ShcKO mice.There are three isoforms at the mammalian Shc locus, the highly expressed isoforms p46Shc and p52Shc, and the minor p66Shc. All three Shc isoforms are derived from a single DNA locus. First, two mRNA are produced: p66Shc and p52/46Shc by means of trans-splicing. The p66Shc mRNA has start codons for all three Shc isoforms. The p52/46Shc-mRNA does not have a start codon for p66Shc and only produces p52Shc and p46Shc (7). For this reason, knockdowns of either p66Shc, or all three Shc isoforms together have been achieved to date. There are two mouse models of Shc depletion produced to date: ShcL, also known as p66ShcKO developed by the Tom Prolla group (ShcProlla, Ref. 4), and Shc mice developed by the Pelicci group (ShcP or ShcPelicci, also known as p66Shc (Ϫ/Ϫ, Refs. 3,4,8,9). Names in literature for these two models can be described by these definitions: ShcL, ShcProlla; ShcKO, ShcP ϭ ShcPelicci ϭ p66Shc(Ϫ/Ϫ).Briefly, ShcL or ShcProlla mice have a deletion of only the minor p66Shc isoform and have no health benefits: ShcL mice are not lean, do not resist weight gain on high fat diets (HFD), 2 and are not longer-lived on HFD, and do not have improved insulin sensitivity (4). Thus p66Shc deletion by itself does not result in health benefits.By contrast, ShcKO have...

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Section: P46shc Is the Only Mitochondrial Shc Isoform Under Physiologmentioning

confidence: 99%

“…We carried out mass spectrometry, two-dimensional gel electrophoresis, and bio-layer interferometry (BLI) (12)(13)(14) assays to identify the major mitochondrial partner of p46Shc. These assays identified mitochondrial 3-ketoacylCoA thiolase (ACAA2) as the p46Shc major partner.…”

mentioning

confidence: 99%

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Tomilov

Tomilova

Shan

et al. 2016

Journal of Biological Chemistry

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“…The biotinylated CobB was tethered on the tip of a streptavidin-coated sensor. The binding partners in SD buffer was then exposed to the tethered one, and the binding was measured by coincident change in the interference pattern [27,28].…”

Section: Kinetic Assaymentioning

confidence: 99%

Global identification of CobB interactors by an <italic>Escherichia coli</italic> proteome microarray

Liu¹,

Wu²,

Jiang³

et al. 2014

ABBS

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Protein acetylation is one of the most abundant posttranslational modifications and plays critical roles in many important biological processes. Based on the recent advances in mass spectrometry technology, in bacteria, such as Escherichia coli, tremendous acetylated proteins and acetylation sites have been identified. However, only one protein deacetylase, i.e. CobB, has been identified in E. coli so far. How CobB is regulated is still elusive. One right strategy to study the regulation of CobB is to globally identify its interacting proteins. In this study, we used a proteome microarray containing ∼4000 affinity-purified E. coli proteins to globally identify CobB interactors, and finally identified 183 binding proteins of high stringency. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in carboxylic acid metabolic process and hexose catabolic process, and also enriched in transferase and hydrolase. We further used bio-layer interferometry to analyze the interaction and quantify the kinetic parameters of putative CobB interactors, and clearly showed that CobB could strongly interact with TopA and AccC. The novel CobB interactors that we identified could serve as a start point for further functional analysis.

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“…To further evaluate the binding of EFN-4 to LAD-2, we used biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance that can measure the association and disassociation of biomolecules (Abdiche et al, 2008;Wartchow et al, 2011;Wilson et al, 2010). In BLI assays, we immobilized EFN-4::Fc or the Fc control onto an anti-human Fc capture probe, after which we immersed the probe into conditioned tissue culture medium isolated from HEK293T cells transiently expressing the LAD-2 extracellular domain fused to alkaline phosphatase (LAD-2::AP).…”

Section: Lad-2 Biochemically Interacts With Efn-4mentioning

confidence: 99%

EFN-4/Ephrin functions in LAD-2/L1CAM-mediated axon guidance in Caenorhabditis elegans

et al. 2016

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During development of the nervous system, growing axons rely on guidance molecules to direct axon pathfinding. A well-characterized family of guidance molecules are the membrane-associated ephrins, which together with their cognate Eph receptors, direct axon navigation in a contact-mediated fashion. In C. elegans, the ephrin-Eph signaling system is conserved and is best characterized for their roles in neuroblast migration during early embryogenesis. This study demonstrates a role for the C. elegans ephrin EFN-4 in axon guidance. We provide both genetic and biochemical evidence that is consistent with the C. elegans divergent L1 cell adhesion molecule LAD-2 acting as a non-canonical ephrin receptor to EFN-4 to promote axon guidance. We also show that EFN-4 probably functions as a diffusible factor because EFN-4 engineered to be soluble can promote LAD-2-mediated axon guidance. This study thus reveals a potential additional mechanism for ephrins in regulating axon guidance and expands the repertoire of receptors by which ephrins can signal.

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Optical biosensing: Kinetics of protein A‐IGG binding using biolayer interferometry

Cited by 29 publications

References 37 publications

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Global identification of CobB interactors by an <italic>Escherichia coli</italic> proteome microarray

EFN-4/Ephrin functions in LAD-2/L1CAM-mediated axon guidance in Caenorhabditis elegans

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Optical biosensing: Kinetics of protein A‐IGG binding using biolayer interferometry

Cited by 29 publications

References 37 publications

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria

Global identification of CobB interactors by an &lt;italic&gt;Escherichia coli&lt;/italic&gt; proteome microarray

EFN-4/Ephrin functions in LAD-2/L1CAM-mediated axon guidance in Caenorhabditis elegans

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Global identification of CobB interactors by an <italic>Escherichia coli</italic> proteome microarray