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Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution.

ABSTRACT: Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an intramolecular interaction between TEM1 ?-lactamase and its inhibitor protein BLIP. Using the HIV1-p17 antibody as an initial target, the interaction between enzyme and inhibitor was carefully tuned to yield a reporter enzyme whose activity increased 10-fold in the presence of pM antibody concentrations. Reporter enzymes for two other antibodies (HA-tag and Dengue virus type I) were obtained by simply replacing the epitope sequences. This new sensor design represents a modular and generic approach to construct antibody reporter enzymes without the cumbersome optimization required by previous engineering strategies.

SUBMITTER: Banala S

PROVIDER: S-EPMC3800467 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution.

Banala Sambashiva S Aper Stijn J A SJ Schalk Werner W Merkx Maarten M

ACS chemical biology 20130813 10

Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an i ...[more]

PMID: 23941162

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Project description:The posttranslational modification (PTM) in protein occurs in a regiospecific manner. In addition, the most commonly occurring PTMs involve similar residues in proteins such as acetylation, ubiquitylation, methylation and sumoylation at the lysine residue and phosphorylation and O-GlcNAc modification at serine/threonine residues. Thus, the possibility of modification sites where two such PTMs may occur in a mutually exclusive manner (ME-PTM) is much higher than known. A recent surge in the identification and the mapping of the commonly occurring PTMs in proteins has revealed that this is indeed the case. However, in what way such ME-PTM sites are regulated and what could be their relevance in the coordinated network of protein function remains to be known. To gain such potential insights in a biological context, we analyzed two most prevalent PTMs on the lysine residue by acetylation and ubiquitylation along with the most abundant PTM in proteins by phosphorylation among enzymes involved in glucose metabolism, a fundamental process in biology. The analysis of the PTM data sets has revealed two important clues that may be intrinsically associated with their regulation and function. First, the most commonly occurring PTMs by phosphorylation, acetylation and ubiquitylation are widespread and clustered in most of the enzymes involved in glucose metabolism; and the prevalence of phosphorylation sites correlates with the number of acetylation and ubiquitylation sites including the ME-modification sites. Second, the prevalence of ME-acetylation/ubiquitylation sites is exceptionally high among enzymes involved in glucose metabolism and have distinct pattern among the subset of enzymes of glucose metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, glycogen synthesis, and the irreversible steps of gluconeogenesis. We hypothesize that phosphorylation including tyrosine phosphorylation plays an important role in the regulation of ME-acetylation/ubiquitylation sites and their similar pattern among the subset of functionally related proteins allows their coordinated regulation in the normal physiology. Similarly their coordinated dysregulation may underlie the disease processes such as reprogrammed metabolism in cancer, obesity, type 2 diabetes, and cardiovascular diseases. Our hypothesis provides an opportunity to understand the regulation of ME-PTMs in proteins and their relevance at the network level and is open for experimental validation.

Dataset Information

Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution.

Publications

Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution.

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