MICALs are large, multidomain flavin-dependent monooxygenases that use redox chemistry to cause actin to depolymerize. Little enzymology has been reported for MICALs, and none has been reported for MICAL-2, an enzyme vital for the proliferation of prostate cancer. The monooxygenase domains of MICALs resemble aromatic hydroxylases, but their substrate is the sulfur of a methionine of actin. In order to determine how closely MICAL-2 conforms to the aromatic hydroxylase paradigm, we studied its reaction with NAD(P)H. The enzyme has a strong preference for NADPH over NADH caused by a large difference in binding NADPH. A comparison of the reduction kinetics using protio-NADPH and [4R-(2)H]-NADPH showed that MICAL-2 is specific for the proR hydride of NADPH, as evidenced by a 4.8-fold kinetic isotope effect. The reductive half-reaction of the MICAL-2 hydroxylase domain is stimulated by f-actin. In the absence of actin, NADPH reduces the flavin relatively slowly; actin speeds that reaction significantly. The separate monooxygenase domain of MICAL-2 has the classic regulatory behavior of flavin-dependent aromatic hydroxylases (Class A monooxygenases): slow reduction of the flavin when the substrate to be oxygenated is absent. This prevents the wasteful consumption of reduced pyridine nucleotide and the production of harmful H2O2. Our results show that this strategy is used by MICAL-2. Thus, our data suggest that MICAL-2 could regulate catalysis through the monooxygenase domain alone; control by interactions with other domains of MICAL in the full-length enzyme may not be needed.

AntioxidantsQ2 · MEDICINE

Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures

Q2 · MEDICINE

ArticleOA

Author: Schmitt, Sebastian ; Kikuti, Carlos ; Mazur, Antonina J ; Mannherz, Hans Georg ; Houdusse, Anne ; Mügge, Andreas ; Jaquet, Kornelia ; Hassoun, Roua ; Hamdani, Nazha ; Erdmann, Constanze ; Geyer, Matthias

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

100 Deals associated with Mical-1(Crescita Therapeutics, Inc.)

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Indication	Highest Phase	Country/Location	Organization	Date
Psoriasis	Phase 2	-	Crescita Therapeutics, Inc.	-
Psoriasis	Phase 2	-	Ferndale Pharma Group, Inc.	-
Skin Diseases	Phase 1	CA	Nuvo Pharmaceuticals, Inc.	12 Mar 2020

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