Aspartic acid substitutions in monoamine oxidase-A reveal both catalytic-dependent and -independent influences on cell viability and proliferation

Abstract

Post-translational influences could underlie the ambiguous roles of monoamine oxidase-A (MAO-A) in pathologies such as depression, cancer and Alzheimer disease. In support of this, we recently demonstrated that the Ca²⁺-sensitive component of MAO-A catalytic activity is inhibited by a pro-survival p38 (MAPK)-dependent mechanism. We substituted three aspartic acid (D) residues in human MAO-A that reside in putative Ca²⁺-binding motifs and overexpressed the individual proteins in the human HEK293 cell line. We assayed the overexpressed proteins for catalytic activity and for their influence on cell viability (using MTT conversion and trypan blue exclusion) and proliferation/DNA synthesis [using bromodeoxyuridine (BrdU) incorporation]. Innate MAO-A catalytic activity (and the capacity for generating hydrogen peroxide) was unaffected by the D61A substitution, but inhibited moderately or completely by the D248A and D328G substitutions, respectively. The Ca²⁺-sensitive activities of wild-type and D248A MAO-A proteins were enhanced by treatment with the selective p38(MAPK) inhibitor, SB203580, but was completely abrogated by the D61A substitution. Monoamine oxidase-A(D61A) was toxic to cells and exerted no effect on cell proliferation, while MAO-A(D248A) was generally comparable to wild-type MAO-A. As expected, the catalytic-dead MAO-A(D328G) was not cytotoxic, but unexpectedly enhanced both MTT conversion and BrdU staining. Variant-dependent changes in Bax and Bcl-2/Bcl-XL protein expression were observed. A different pattern of effects in N2-a cells suggests cell line-dependent roles for MAO-A. A catalytic-dependent mechanism influences MAO-A-mediated cytotoxicity, whereas a catalytic-independent mechanism contributes to proliferation. Context-dependent inputs by either mechanism could underlie the ambiguous pathological contributions of MAO-A.

Recent Developments in the Regulation of Monoamine Oxidase Form and Function: Is the Current Model Restricting Our Understanding of the Breadth of Contribution of Monoamine Oxidase to Brain [dys]Function?

“…It is interesting that these reports base their conclusions on MAO-A protein expression and do not include any estimate of MAO-A activity. Without any evidence to the contrary, the MAO-A protein detected in these reports could be in an inactive form and, as our work with the MAO-A(Asp328Gln) catalytic-dead variant shows [85], could be promoting proliferative phenotypes via non-catalytic-based de novo DNA synthesis and/or induction of anti-apoptotic Bcl-2-related proteins.…”

“…In fact, there is a reasonable evidence that the N-propargyl moiety (found in many MAO inhibitors, e,g, pargyline, clorgyline, selegiline, rasagiline, ladostigil) can exert effects and activate cellular signalling cascades (particularly those associated with neuroprotection/rescue) independent of any catalytic inhibition of the targeted MAO enzyme [5,44,134,135]. Although it remains to be determined whether non-catalytic properties of an off-target inhibition of MAO-A might be responsible for any Parkinson's disease-modifying effects of rasagiline or selegiline, our work on the catalytic-dead MAO-A(Asp 328Gln) variant and its ability to induce Bcl-2-related proteins [85] certainly support this possibility.…”

“…This seemingly minor observation has a significant impact on the interpretation of MAO-A function. Indeed, we have recently shown that overexpression of an MAO-A protein bearing the Asp328Gln substitution (already known to inhibit MAO-A activity [84]) was able to alter cell proliferation and de novo DNA synthesis in the human HEK293 cell line [85]. Furthermore, this same catalytic-dead variant was able to induce the expression of Bcl-2 and Bcl-XL, two anti-apoptotic/pro-survival molecules that also have been associated with pharmacological inhibition of MAO-A [86,87].…”

“…Yet one could also argue that it is MAO-A in its inactive state that is also partly responsible for these effects. While such a pool is present in the PS-1(M146V) mice [124] and an inactive human MAO-A protein does exert phenotypic changes in vitro [85], how would this mechanistically relate to the original Mao-A-deficient mouse generated by Cases and colleagues [80]? In fact, this is where it is important to recall that these mice were generated by insertional mutagenesis (i.e.…”

Dual inhibition of survivin and MAOA synergistically impairs growth of PTEN-negative prostate cancer

“…Although the molecular mechanisms and functions of MAOA in PCa are not clear, amine metabolism is associated with cellular processes including cell growth and differentiation ( Pietrangeli and Mondovi, 2004 ). Substitutions of aspartic acid residues in MAOA affected cell viability and proliferation ( Wei et al , 2012 ). Clorgyline, a selective and irreversible MAOA inhibitor previously used as an anti-depression medicine, showed efficacy in in vitro and in vivo PCa models ( Zhao et al , 2009 ; Flamand et al , 2010 ).…”