The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.

The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.