Generation and characterization of a laforin nanobody inhibitor

Simmons, Zoe R.; Sharma, Sanjeev; Wayne, Jeremiah; Sheng, Li; Kooi, Craig W. Vander; Gentry, Matthew S.

doi:10.1016/j.clinbiochem.2021.03.017

Cited by 3 publications

(3 citation statements)

References 62 publications

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“…The Core 120 library was panned for three rounds using solid-phase plates ( 50 ) coated with PI3Kα (100 µg/mL in 100 mM NaHCO 3 , pH 8.2). The enrichment of antigen-specific phages was assessed by ELISA screening after each round of panning by comparing the number of phagemid particles eluted from antigen-coated wells with that eluted from negative (uncoated and blocked) control wells ( 51 ). This resulted in about 11-fold, 750-fold, and 2,300-fold enrichment after the first, second, and third rounds, respectively.…”

Section: Methodsmentioning

confidence: 99%

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

Hart

Liu

Pan

et al. 2022

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

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Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

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Section: Methodsmentioning

confidence: 99%

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

Hart

Liu

Pan

et al. 2022

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

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“…S2A) and utilized the recombinant protein in hydrogen-deuterium exchange (HDX) mass spectrometry (MS) experiments. This technique has previously been used to illuminate conformational dynamics of proteins by demonstrating areas of high and low solvent accessibility (44,48). We first determined deuterium uptake across the entire protein from 15 to 15,000 s (Fig.…”

Section: Resultsmentioning

confidence: 99%

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors

Murphy¹,

Chen²,

Lin³

et al. 2022

Journal of Biological Chemistry

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“…Similarly, Gross and collaborators (Fernandez et al, 2018;Adhikari et al, 2021) characterized epitopes for five antibodies, and Simmons et al (Simmons et al, 2021) In contrast to running multiple comparative HDX studies, Marshall and collaborators (Zhang Q. et al, 2013) used polyclonal antibodies (pool of antibodies extracted from serum) to characterize the immune response of a nut allergen. Subsequently, others described polyclonal antibodies for study of immune response against tumor necrosis factor α (TNF-α) (Abbott et al, 2013) and factor H-binding protein (fHbp) (Ständer et al, 2021).…”

Section: Using Automation and New Hardwarementioning

confidence: 99%

Hydrogen deuterium exchange and other mass spectrometry- based approaches for epitope mapping

Jethva

Gross

2023

Front. Anal. Sci.

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Antigen-antibody interactions are a fundamental subset of protein-protein interactions responsible for the “survival of the fittest.” Determining the interacting interface of the antigen, called an epitope, and that on the antibody, called a paratope, is crucial to antibody development. Because each antigen presents multiple epitopes (unique footprints), sophisticated approaches are required to determine the target region for a given antibody. Although X-ray crystallography, Cryo-EM, and nuclear magnetic resonance can provide atomic details of an epitope, they are often laborious, poor in throughput, and insensitive. Mass spectrometry-based approaches offer rapid turnaround, intermediate structural resolution, and virtually no size limit for the antigen, making them a vital approach for epitope mapping. In this review, we describe in detail the principles of hydrogen deuterium exchange mass spectrometry in application to epitope mapping. We also show that a combination of MS-based approaches can assist or complement epitope mapping and push the limit of structural resolution to the residue level. We describe in detail the MS methods used in epitope mapping, provide our perspective about the approaches, and focus on elucidating the role that HDX-MS is playing now and in the future by organizing a discussion centered around several improvements in prototype instrument/applications used for epitope mapping. At the end, we provide a tabular summary of the current literature on HDX-MS-based epitope mapping.

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Generation and characterization of a laforin nanobody inhibitor

Cited by 3 publications

References 62 publications

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors

Hydrogen deuterium exchange and other mass spectrometry- based approaches for epitope mapping

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