Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays.

Jimok Yoon,Ruei-Jiun Hung,Heng Wu,Jonathan R Terman

doi:10.3390/ijms22041991

Abstract

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)—thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs’ Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical’s Redox enzymatic and F-actin disassembly activity.

Highlights

Departments of Neuroscience and Pharmacology, The University of Texas Southwestern Medical Center, Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
3 conserved motifs for binding flavin adenine dinucleotide (FAD), MICALs have multiple other domains including a calponin homology (CH) domain, a LIM domain, a stretch of proline residues that serve as ligands for SH3-domain containing proteins and a coiledcoil C-terminal region that binds the Semaphorin receptor Plexin (Plexin-interacting Region, orange), as well as Rab family small GTPases. (C) MICALs provide a means by which one of the largest protein families of extracellular cues, the Semaphorins (Sema) and their Plexin receptors, disassemble F-actin
The MICALs are oxidoreductase (Redox) enzymes that bind and utilize F-actin as a substrate—such that F-actin triggers the MICALs Redox enzymatic activity and MICALs oxidize two specific methionine residues on actin to destabilize and disassemble actin filaments (Figure 1D,E; [13,14,25]; reviewed in [9,10,11,12]). In addition to their Redox enzymatic portion, MICALs contain multiple other domains—such that proceeding N-terminally to C-terminally is a calponin homology (CH) domain, a LIM domain, a stretch of proline residues that serve as ligands for SH3-domain containing proteins and a coiled-coil region that binds the Semaphorin receptor Plexin, as well as Rab family small GTPases (Figure 1B; [8]; reviewed in [9,10,11,12])

Summary

Introduction

Departments of Neuroscience and Pharmacology, The University of Texas Southwestern Medical Center, Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical’s Redox enzymatic and F-actin disassembly activity. Less-well appreciated, F-actin is regulated through the covalent alteration of its amino acids and characterizing these modifications constitutes a critical direction for further study [3,4]

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Conclusion