The world is a beautifully complex place. There is hardly ever just one way to complete a task. Take tying shoes for example: the battle still rages on whether they most effective way is utilizing the bunny ears method or just the one loop technique. Either way, the job gets done. Biochemistry is no exception to this rule. Many biological systems have more than one way to accomplish the same process. Thermogenesis, the process of the body to produce heat, has more than one mechanism for this to happen. The human body does this in efforts to maintain its internal temperature homeostasis. In their recent article, authors Jonathan Long et al. uncovers PM20D1, a new enzyme that leads to another method of undergoing thermogenesis. Before understanding how and why PM20D1 becomes involved with this process, its predecessor needs to be discussed first.
The most researched thermogenesis pathway deals with uncoupling protein 1 (UCP1). This protein is present in two types of adipose tissues, brown and beige fats2. UCP1 disrupts the natural proton gradient established by oxidative phosphorylation. The protein pumps protons back into the mitochondrial matrix without producing ATP, but producing heat in the process1. This process is known as uncoupled respiration (Fig. 1). In addition to expression of UCP1, scientists continually test the many different roles these specific adipose tissues may be involved in. The first step of this study investigates other possible pathways of undergoing uncoupled respiration. Long et al. suspect co-expression of another gene along with Ucp1. The research team proceeds to compile a list of what they refer to as “core thermogenesis”2 genes along with what genes are expressed in the same conditions as UCP1. This is how peptidase M20 domain containing 1 (PM20D1) becomes the focus of this study. With a signal peptide and no transmembrane domains, PM20D1 is seen as a secreted enzyme. To confirm this, tagged Pm20d1 gene is transfected into cells and found to be expressed in both the cell cultures as well as the in the media. With that data supporting the idea PM20D1 is a secreted enzyme, the authors move on to determining the effects of the enzyme on a live biological system and transfect mice test subjects. While on a high fat diet, the mice expressing the PM20D1 show a decrease in weight gain compared to the mice transfected with GFP as a control. The difference in weight is due to different percentage of fat mass compared to lean mass. Complementary to the weigh results, a calorimetry measurement show that mice with PM20D1 has a higher energy spending despite the same exact diet between the PM20D1 mice subjects and control GFP mice subjects. Both findings suggests that this enzyme at fault.
After seeing that PM20D1 is likely to influence biomass regulation and energy exertion, the authors now have to determine what exactly is occurring with the enzyme. With knowing that PM20D1 is part of the mammalian M20 peptidase family and how this family tends to conduct activity on small molecule substrates2, the authors use liquid chromatography mass spectrometry (LC-MS) to determine PM20D1 molecular effects in the blood. Mice injected with the PM20D1 vectors show more MS peaks with a m/z of 428, which corresponds to the molecular ion of N-oleoyl phenylalanine. Tandem mass spectroscopy supports that peak’s identity by showing a m/z peak of 164 that corresponds to a phenylalanine ion. In addition to N-oleoyl phenylalanine, there are other N-acyl amino acids found in the blood samples of the PM20D1 mice. Mass spectroscopy and its derivatives are able determine the presence of certain metabolites and help compare what is found in PM20D1 mice and the GFP mice.
With the high levels of N-acyl amino acids in PM20D1 mice compared to GFP mice, it is likely that PM20D1 is the enzyme responsible in the compounds’ formations in vivo. In vitro techniques are required to controllably examine any products that would be made, if any, with purified mouse PM20D1, fatty acids, and amino acids. LC-MS shows how N-oleoyl phenylalanine was made after using oleate and phenylalanine as reactants. The synthesis reaction uses other amino acids, apart from glutamate and ethanolamine, but the phenylalanine is the most successful amino acid. The hydrolytic reaction occurs by the enzyme breaking the N-acyl amino acid back into the fatty acid and amino acid components. By mutating PM20D1, the authors can determine which residues are essential for catalytic activity. Only the wildtype of the enzyme produces any activity. Experiments with recombinant human PM20D1 determines if the enzyme functions similarly to the mouse version. The human version displays the same conserved catalytic residues and still acts as a bidirectional enzyme.
After determining what products are created and how via PM20D1, the authors can focus on how these metabolites contribute to uncoupled respiration. After disabling the ATP synthase in adipose tissue, the addition of N-oleoyl phenylalanine increases cellular oxygen consumption, meaning uncoupled respiration is occurring. Other N-acyl amino acids also increase the cellular oxidation consumption. Experiments that utilize cells that do not express UCP1 shows that addition of N-acyl amino acids increases oxygen consumption rates, meaning that UCP1 is not needed for PM20D1 to activate uncoupled respiration. More experiments show by adding modifications to the amino group of the N-acyl amino acids halts the respiration mechanism. The amino group is necessary for the reaction to occur. Isolating the mitochondria from the cells and treating it with the N-acyl amino acids reveals that respiration still happens, which suggests no organelles are needed to assist with uncoupled respiration.
Long et al. discussed significant findings utilizing common techniques such as LC-MS to purify and identify metabolites, western blot to determine if the enzyme of interest was present, and in vitro assays to test reaction parameters. Finding and characterizing this enzyme holds real promise for the future. When testing PM20D1 functions in vivo, mouse test subjects expressing the enzyme was shown to lose fat mass despite being on a high fat diet. The authors note that the weight loss was most likely a combination of both burning the fat and triggering a decrease in food consumption. Past studies have shown synthetic chemical uncouplers can lead to lethal side effects2. By uncovering both the enzyme and N-acyl amino acid metabolites, new targets for drug therapy has been brought to the table. Type 2 diabetes has been linked to obesity. If new therapies can be developed around PM20D1 that deal with weight loss, it could help diminish a risk factor of developing diabetes. This newly studied uncoupled respiration pathway holds potential to helping create a healthier environment. This could be a step forward in the right direction.
- Brondani L.A., Assmann T.S., Duarte G.C., Gross J.L., Canani L.H., Crispim D. The role of the uncoupling protein 1 (UCP1) on the development of obesity and type 2 diabetes mellitus. Arg Bras Endocrinol Metabol. 2012 Jun; 56(4):215-25.
- Long J.Z., Svensson K.J., Bateman L.A., Lin H., Kamenecka T., Lokurkar I.A., Lou J., . . . Spiegelman B.M. 2016 Jul 14; 166(2):424-35. doi: 10.1016/j.cell.2016.05.071.