Likewise, each of the purified human MICAL proteins, similar to Drosophila Mical, has a UV-visible light absorption spectra with peaks at ~360 nm and ~450 nm (black lines) – and denaturation of the MICAL proteins releases FAD, which underlies this absorption spectra (green line). The isoalloxazine ring system within flavins generates the yellow/orange color of FAD and FMN and is also responsible for light absorption in the UV and visible spectral range such that the oxidized form of FAD has two peaks at ~360 nm and ~450 nm.
( c– f) Each of the human MICALs binds FAD.
The proline (*) in the FAD fingerprint 2 is also likely to be conserved. light blue = hydrophobic and cysteine residues, purple = acids, green = bases, yellow = proline, and orange = glycine. Note, however, that MICAL-1 has a “naturally” occurring substitution of an alanine residue instead of the important aspartate residue in the DG motif. The MICALs also have well-conserved FAD Fingerprint 2 (GD) and DG motifs, which are additional distinguishing features of flavoprotein monooxygenases. Each MICAL contains an exact match with each of the 11 residues of the ADP binding region of FAD binding proteins (GxGxxG motif), where (+) indicates that MICALs match the consensus, (*) indicates that MICALs match the highly important conserved residues, and (.) indicates the conserved spacing of these residues within these motifs. ( b) Amino acid sequence alignments show that each of the human MICALs (M1, M2, M3), similar to Drosophila Mical (dM), contains three sequence motifs that define them as flavoprotein monooxygenases. Multiple different splice forms of the MICALs have also been identified – some without the C terminus – as detailed in a recent review. ( a) Although variable in length (depicted with the white dashed lines), each of the human (h) MICAL protein family members contains the same core domains as Drosophila (d) Mical including a flavoprotein monooxygenase (FM) domain (also called the redox or MO domain), a single calponin homology domain, and a single LIM domain. Collectively, our results therefore define the MICALs as an important phylogenetically-conserved family of catalytically-acting F-actin disassembly factors.Įach human MICAL family member (MICAL-1, MICAL-2, and MICAL-3) is a flavoenzyme that consumes NADPH. Our results go on to reveal that MsrB/SelR reductase enzymes counteract each MICAL's effect on F-actin in vitro and in vivo. Genetic experiments also demonstrate that each human MICAL drives F-actin disassembly in vivo, reshaping cells and their membranous extensions.
We also find that each human MICAL uses an NADPH-dependent Redox activity to post-translationally oxidize actin's methionine (M) M44/M47 residues, directly dismantling filaments and limiting new polymerization. Specifically, each human MICAL selectively associates with F-actin, which directly induces MICALs catalytic activity. We now find that each human MICAL family member, MICAL-1, MICAL-2, and MICAL-3, directly induces F-actin dismantling and controls F-actin-mediated cellular remodeling. Mical proteins are also present in mammals, but their actin regulatory properties, including comparisons among different family members, remain poorly defined. Using the Drosophila model, we have recently identified an unusual actin regulatory enzyme, Mical, which is directly activated by F-actin to selectively post-translationally oxidize and destabilize filaments - regulating numerous cellular behaviors. Cellular form and function - and thus normal development and physiology - are specified via proteins that control the organization and dynamic properties of the actin cytoskeleton.
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