bims-lowpac 2023-12-31 papers

Issue of 2023‒12‒31
three papers selected by
Elodie Busch, University of Strasbourg

Methods Mol Biol. 2024 ;2743 123-133

Identification of Protein Tyrosine Phosphatase (PTP) Substrates.

Sravan Perla, Bin Qiu, Sam Dorry, Jae-Sung Yi, Anton M Bennett.

Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. The combinatorial action of both PTKs and PTPs is essential for properly maintaining cellular functions. In this unit, we discuss different novel methods to identify PTP substrates. PTPs depend on specific invariant residues that enable binding to tyrosine-phosphorylated substrates and aid catalytic activity. Identifying PTP substrates has paved the way to understanding their role in distinct intracellular signaling pathways. Due to their high specific activity, the interaction between PTPs and their substrates is transient; therefore, identifying the physiological substrates of PTPs has been challenging. To identify the physiological substrates of PTPs, various PTP mutants have been generated. These PTP mutants, named "substrate-trapping mutants," lack catalytic activity but bind tightly to their tyrosine-phosphorylated substrates. Identifying the substrates for the PTPs will provide critical insight into the function of physiological and pathophysiological signal transduction. In this chapter, we describe interaction assays used to identify the PTP substrates.

Keywords: Interacting proteins; Protein tyrosine phosphatases; Substrate identification; Substrate-trapping mutants

DOI: https://doi.org/10.1007/978-1-0716-3569-8_8

Methods Mol Biol. 2024 ;2743 301-316

High-Throughput Discovery and Characterization of Covalent Inhibitors for Protein Tyrosine Phosphatases.

Zihan Qu, Aaron D Krabill, Zhong-Yin Zhang.

Covalent inhibition has gained increasing interest in targeting the undruggable protein tyrosine phosphatases (PTPs). However, a systematic method for discovering and characterizing covalent PTP inhibitors has yet to be established. Here, we describe a workflow involving high-throughput screening of covalent fragment libraries and a novel biochemical assay that enables the acquisition of kinetics parameters of PTP inhibition by covalent inhibitors with higher throughput.

Keywords: Covalent inhibition; High-throughput screening; Kinetics of covalent inhibition; Protein tyrosine phosphatase; pNPP assay

DOI: https://doi.org/10.1007/978-1-0716-3569-8_19

Methods Mol Biol. 2024 ;2743 111-122

Examining Phosphatases Through Immunofluorescent Microscopy.

Caroline N Smith, Jessica S Blackburn.

Immunofluorescent microscopy enables the examination of cellular expression and localization of proteins. Cellular localization can often impact protein function, as certain molecular interactions occur in specific cellular compartments. Here we describe in detail the processes necessary for identifying phosphatases in the cell through immunofluorescent microscopy. Identification of phosphatase expression and localization could lead to the discovery of protein function in disease states along with potential substrates and binding partners.

Keywords: Antibody; Confocal; Immunofluorescence; Localization; Microscopy; Protein expression

DOI: https://doi.org/10.1007/978-1-0716-3569-8_7