BIOCHEMICAL STUDIES OF HUMAN MICAL1, THE FLAVOENZYME CONTROLLING CYTOSKELETON DYNAMICS
Tesi di Dottorato
Data di Pubblicazione:
2015
Citazione:
BIOCHEMICAL STUDIES OF HUMAN MICAL1, THE FLAVOENZYME CONTROLLING CYTOSKELETON DYNAMICS / T. Vitali ; tutor: M.A. Vanoni. DIPARTIMENTO DI BIOSCIENZE, 2015 Dec 09. 28. ciclo, Anno Accademico 2015. [10.13130/vitali-teresa_phd2015-12-09].
Abstract:
MICAL from the “Molecule Interacting with CasL” indicates a family of cytoplasmic multidomain proteins conserved from insects to humans, which participates in the control of cytoskeleton dynamics. A unique feature of MICAL proteins is the presence of a catalytic N-terminal flavoprotein monoonoxygenase-like (MO) domain that is followed by several protein interaction domains, namely: a calponin homology (CH) domain, a LIM domain and a C-terminal region containing potential coiled-coil motifs.
In neurons MICAL1 is an essential component for the transduction of the semaphorin signaling downstream of plexin by catalyzing a NADPH-dependent F-actin depolymerization through the N-terminal flavoprotein domain. But it may also control integrin pathway and microtubules assembly through interaction with CasL and CRMP, respectively and apoptosis through interaction with NDR1. The MICAL2 and MICAL3 isoforms are implicated in vesicles trafficking and gene transcription.
The aim of this project is to contribute to define the function of human MICAL1 by characterizing the catalytic properties of the MO domain and how they are modulated by its CH, LIM and C-terminal domains and, eventually, by its interacting proteins.
The human MICAL1 form containing the isolated MO domain (55.1 kDa; pI 9) and the form comprising both the MO and the CH domains (MOCH; 68.5 kDa; pI 7.7) have been produced according to the procedures available in the laboratory, which were further optimized. The full-length MICAL1 (MICAL; 119 kDa; pI, 6.2) and the form lacking the C-terminal region (MOCHLIM; 86.4 kDa; pI, 6.7) were produced in E.coli cells and their purification protocols were set-up exploiting the engineered C-terminal His6-tag. All the purified MICAL forms are stable and greater than 95% homogeneous.
MICAL forms are isolated with the correct complement of FAD bound to the MO domain and zinc ions bound to the LIM domain, which predicts the formation of two zinc fingers. The absorption spectra of all MICAL forms are similar to each other, with an extinction coefficient at 458 nm of ≈8.1 mM-1cm-1 similar to that previously determined for the isolated MO domain, indicating that the CH, LIM and C-terminal regions do not alter the conformation of the catalytic domain. However, the LIM domain causes MOCHLIM to oligomerize to yield dimers, trimers and higher order aggregates, while the full-length protein yielded stable dimers as opposed to the monomeric state of MO and MOCH forms.
All MICAL forms catalyze a NADPH oxidase (H2O2-producing) activity, which is associated with the MO domain. By combining steady-state kinetic measurements of the reaction as a function of pH and of solvent viscosity we concluded that the CH, LIM and C-terminal domains lead to a progressive lowering of the catalytic efficiency (MO, ≈165 s-1mM-1; MOCH, ≈18.5 s-1mM-1; MOCHLIM, ≈15 s-1mM-1; MICAL, ≈0.75 s-1mM-1) of the reaction due to an increase of Km for NADPH from ≈20 μM (MO), to ≈130 μM (MOCH), ≈230 μM (MOCHLIM) and ≈370 μM (MICAL). The 200-fold drop of the catalytic efficiency of the full-length MICAL compared to that of MO is also due to a ≈10-fold decrease of kcat. The study of the pH and viscosity dependence of the NADPH oxidase reaction of MICAL forms led us to conclude that the observed changes in the values of the kinetic parameters are not due to changes in rate determining steps of the reaction taking place within MO. The increase of KNADPH correlates with a decrease of the positive charge of the protein due to the acidity of the CH, LIM and C-terminal domains. The 10-fold drop of kcat observed with full-length MICAL is consistent with the proposal of an autoinhibitory role of the C-terminal region on MICAL catalytic activity. Our experiments reveal that
Tipologia IRIS:
Tesi di dottorato
Keywords:
MICAL; flavoproteins; FAD-containing monooxygenase/oxidase; F-actin depolymerization; semaphorin signaling
Elenco autori:
T. Vitali
Link alla scheda completa:
Link al Full Text: