Research may open the door to new drugs to fight ALS

Study describes a new pathway for the build up of the superoxide dismutase protein, which may be involved in the mechanism of amyotrophic lateral sclerosis.

By Karina Toledo

Agência FAPESP – Scientists do not yet fully understand all of the factors that lead to the development of amyotrophic lateral sclerosis (ALS), a progressive, fatal disease characterized by the degeneration of motor neurons. Data from the scientific literature, however, indicate that the aggregation of a protein known as superoxide dismutase plays a key role in the disease’s mechanism of action.

In an article recently published in the Journal of Biological Chemistry, researchers from the University of São Paulo (USP) described a new mechanism for aggregation of superoxide dismutase, associated with the oxidation of one of its amino acid residues: tryptophan 32.

The discovery may open the door to the development of new drugs to fight ALS, a rare disease (affecting one out of every 100,000 people every year) whose most famous sufferer is perhaps scientist Stephen Hawking of the University of Cambridge in the United Kingdom.

“Superoxide dismutase is one of the principal antioxidant defenses of the human body. Its primary function is to neutralize a free oxygen radical known as the superoxide anion, which, in excess, can be toxic to cells,” explained Ohara Augusto, a professor at the Chemistry Institute of the University of São Paulo and coordinator of the Center for Research on Redox Processes in Biomedicine (Redoxoma), a FAPESP Research, Innovation and Dissemination Center (RIDC).

According to Augusto, previous studies have shown that the medulla of patients with ALS and that of animals in models of the disease contains aggregates of superoxide dismutase and shows oxidative damage to proteins, lipids and DNA molecules.

“There is clearly oxidative damage, but we still do not know for sure whether or not it is a cause or a consequence of the disease,” Augusto said. The scientific literature also holds that nearly 10% of ALS cases are inherited, and in 20% of these cases, it is possible to find mutations in the superoxide dismutase gene. However, the other 90% of cases are considered sporadic, with an unknown etiology.

“Isolated enzyme mutations do not explain the disease, but because the symptoms and evolution of the familial and sporadic forms of ALS are similar, the same pathogenic mechanism is inferred for both. Oxidative modifications to superoxide dismutase could explain structural changes that would lead to protein aggregation,” Augusto noted.

Structural alteration

Certain oxidative modifications to the enzyme that are associated with ALS have already been described by other groups, but the study by the Redoxoma researchers was the first to describe a pathway that involves the oxidation of tryptophan 32 – an amino acid found in the superoxide dismutase enzyme of humans and simians but not in that of other mammals.

Coincidentally, ALS is described by scientists as a disease occurring almost entirely in simians, which suggests that the oxidation of tryptophan 32 is somehow involved in the pathologic mechanism.

“To induce the disease in rodents, for example, we needed to develop transgenic animals capable of expressing the human enzyme,” the researcher said.

In a previous study, Augusto described that in certain situations, the superoxide dismutase enzyme had a pro-oxidative activity, in effect forming a carbonate radical. In experiments performed in vitro, the researcher demonstrated that this carbonate radical is responsible for oxidizing the amino acid tryptophan 32.

“Once oxidized, this tryptophan 32 binds to another enzyme molecule – a tryptophanyl radical – and forms a tetramer (a molecule that consists of four units). We have shown through spectroscopic techniques that this causes the enzyme to uncoil (lose its functional form) and aggregate,” Augusto explained.

Step by step

To do the experiment, the researchers used human recombinant enzymes, or enzymes produced in the laboratory, utilizing genetically modified bacteria to express the superoxide dismutase gene.

They used both a normal enzyme (wild type)and one of the mutant forms associated with ALS known as G93A. To stimulate the pro-oxidant activity, the researchers incubated the two versions of the enzyme with hydrogen peroxide and bicarbonate.

“The enzyme uses the hydrogen peroxide to oxidize other molecules – in this case, the enzyme itself, which becomes inactive. The bicarbonate was used because it is an excellent physiological buffer; in other words, it is a substance found in all human body fluids and acts as a pH corrector,” Augusto explained.

The oxidation process of the two forms of the enzyme, the normal and the mutant, was monitored by the researchers over different time intervals for one week. All of the modifications made to the structure of the protein at the various times were identified with the help of mass spectrometry techniques.

“We observed that within one hour of oxidation, a 10 nanometer (nm) diameter tetramer had already begun to form and lasted 48 hours. After that, protein aggregates, measuring 800 nm in diameter, began to form,” the researcher noted.

According to Augusto, the mutant G93A aggregation process was slightly faster than that of the normal enzyme, but the difference was not statistically significant.

In one of the experiments, the researchers used phenylalanine in place of the tryptophan at amino acid position 32, and although the enzyme experienced oxidative activity in the presence of hydrogen peroxide, protein aggregation did not occur.

“These results show that oxidized tryptophan 32 must be present for aggregation to occur. It is something that had already been proposed in earlier studies but had never been examined in practice,” Augusto noted.

According to the scientist, in the event that additional studies find that the formation of the carbonate radical and the resultant oxidation of tryptophan also occur in vivo, and could thereby lead to protein aggregation, this amino acid could become a therapeutic target.

“One possibility would be to develop an antibody capable of recognizing the ditryptophan, which is the product of the tryptophan 32 oxidation, and preventing the proteins from aggregating,” August said.

The article Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme (doi: 10.1074/jbc.M114.586370 jbc.M114.586370) can be read at www.jbc.org/content/early/2014/09/18/jbc.M114.586370.