Mechanically transduced immunosorbent assay to measure protein-protein interactions

Petell, Christopher J.; Randene, Kathyrn; Pappas, Michael G.; Sandoval, Diego; Strahl, Brian D.; Harrison, Joseph S.; Steimel, Joshua

doi:10.7554/elife.67525

Cited by 4 publications

(3 citation statements)

References 54 publications

(70 reference statements)

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“…One can then fit a Log-Log plot of the measured rolling parameter for known K d values, including the biotin-streptavidin interaction which has a binding affinity of approximately 10 -15 M and a null interaction of DrrA WT -PI which was given an estimated K d of approximately 1M. This fitted function allows one to estimate binding affinity values for previously unknown interactions as previously demonstrated [1,2,21,24]. With other traditional optical readout based techniques determining the preference in binding affinity of Auxilin 1 to PI4P and Auxilin 2 to PI3P was either not possible or extremely difficult.…”

Section: Resultsmentioning

confidence: 96%

“…To overcome such limitations, here we study the interactions of several lipid binding domains with PIP lipids using a new mechanically transduced immunosorbent assay (METRIS) [1,2,21,24]. METRIS leverages the fundamental physical concept of friction and utilizes rolling protein-functionalized ferromagnetic beads of diameter D on a supported lipid bilayer (SLB), as seen in Fig.…”

Section: Introductionmentioning

confidence: 99%

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Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Steimel

Sarkis

Capraro

et al. 2022

Preprint

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Protein-lipid interactions constitute a very important class of biological interactions critical for multiple cell and tissue functions. It is believed that most lipid-protein interactions are very weak, with affinities in the 1mM-1μM range. Here we study the interactions of multiple protein lipid binding domains with lipid membranes containing signaling lipids known as phosphatidylinositol phosphates (PIPs) using a new mechanically transduced immunosorbent assay (METRIS). We demonstrate that this assay can measure extremely weak interactions at PIP bilayer concentrations below 1%, which is close to the biological lipid concentration regime. In particular, we have studied the interaction of DrrAWT, DrrAK568A, PH-δ, and 2XFYVE as well as previously unexplored lipid binding domains such as Auxilin 1 (PTEN) and Auxilin 2 (GAK) against a wide palette of PIPs. Our results confirm that each of these domains interacts specifically with a PIP partner. In the case of Auxilin 1 and Auxilin 2, both proteins in the Clathrin endocytotic pathway, we find that their PTEN-like domain interacts specifically yet with ultra low affinity with PI3P and PI4P respectively. We have also found a new unknown medium-high affinity interaction between GAK with PI34P2. Our work, thus, provides a direct and robust method to measure and catalog protein lipid interactions which are important in many processes such as signaling and membrane sculpting. Furthermore, this assay can be extended in a straightforward manner to study other interactions such as ligand-receptor or antibody-antigen.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Steimel

Sarkis

Capraro

et al. 2022

Preprint

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show abstract

“…5c). Second, we utilized a new technique termed the mechanically transduced immunosorbent (METRIS) assay 47 . In brief, this system measures weak protein interactions by utilizing a protein that is covalently conjugated to ferromagnetic particles placed on coverslips precoated with another protein of interest (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

et al. 2021

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The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

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Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C)

Bodrug,

Welsh,

Bolhuis

et al. 2023

Nat Struct Mol Biol

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Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3–E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

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Mechanically transduced immunosorbent assay to measure protein-protein interactions

Cited by 4 publications

References 54 publications

Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C)

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