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The Guardian Angel Enzyme

Zach Sekulich 
Department of Biology 
Lake Forest College 
Lake Forest, IL 60045

There are many people who have claimed that a guardian angel watches over them, looks out for their well-being and protects them from harm. There is now evidence that all of us have a guardian angel looking out for us, in fact we all have immense amounts of them inside our bodies regulating the way we feel pain. The deubiquitinating enzyme that regulates the amount of Voltage gated Calcium channels (Cav3.2) present on our cell membranes (USP5) is this guardian angel enzyme. There are numerous mechanisms researchers have shown that regulate how pain is felt, but now thanks to García-Caballero et al. (1) a new tool has been added to the repertoire of pain regulation.

The biological mechanisms by which pain operates have been well studied over the past decades. Neurons that specifically respond to external pain stimuli have been labeled nociceptors (2). From the ion channels within the nociceptors themselves (1, 3, 4) to the neurotransmitters released in the brain once the signals of pain have been transmitted (5) there are a suite of approaches to study how our bodies feel pain (6). This includes enzymes that operate on parts of the path, such as deubiquitinating enzymes. Deubiquitinating enzymes are enzymes that stop another protein from being degraded by removing a ubiquitin molecule from the protein that has marked it for degradation (7). It was known by Caballero et al. (1) that voltage gated calcium channels 3.2 (Cav3.2) played a primary role in the molecular pain pathway (2, 8) and that the calcium currents could be inhibited by inhibiting Cav3.2 (9). Caballero et al. (1) found that Cav3.2 cell surface density can regulate neuropathic and inflammatory pain by the concentration of deubiquitinating enzyme Ubiquitin carboxyl-terminal hydrolase 5 (USP5).

Caballero et al. (1) made one groundbreaking discovery after another outlined in their paper, The Deubiquitinating Enzyme USP5 Mod- ulates Neuropathic and Inflammatory Pain by Enhancing Cav3.2 Channel Activity. This study began by identifying that Cav3.2 channels are subject to ubiquitination in mouse dorsal root ganglia (DRG) neurons. Not only was it identified that the channels were subjected to destruction via ubiquitination, but the site where the ubiquitin molecules were attached was identified. USP5 was then identified as a key candidate as a regulator of the channel. This was confirmed because when this enzyme was stopped from being expressed in the cells, the amount of channels in the cell was five times as high as before. After this was confirmed, in pain-induced experiments involving mice it was confirmed that USP5 absence significantly increased inflammatory and neuropathic pain.

The techniques used to uncover these findings are vast and technical, but some explanation as to how these researchers established these novel findings must be addressed. By harvesting DRG cells from mice and adding reagents to stop certain known degradation systems from functioning, it was found that the channels were subject to the proteasome-ubiquitination system. By looking at the amino-acid sequence of the channel, the researchers were able to narrow down as to where the possible region where ubiquitination enzymes could bind to the channel. This method of homing in on possible candidates proved successful when the particular region in question was mutated in different ways and tested to see if the channels were still subject to ubiquitination. When the channels were not ubiquitinated in mutated cells, it was known that the region had been found!

The next step in this scientific inquiry had to identify which of the many specific ubiquitin enzymes were acting on this channel, so eight candidate ubiquitin enzymes were tested to see how tightly they bound to the channels in order to determine which candidate may regulate the channel surface density of the cells. This led to the suspicion that USP5 was the enzyme that Caballero et al. (1) had been searching for. This suspicion was confirmed using special RNA called short-hairpin RNA that are used to stop expression of specific proteins in a cell. When USP5 was stopped from being expressed, the amount of Cav3.2 channels expressed on the cell surface was greatly increased. Finally USP5 inhibitor was injected into living mice and the mice were given injections of specific chemicals that induced pain. The intensity of the pain was measured by how intensely the mice withdrew their paws. As the researchers suspected, the mice with no USP5 and therefore more calcium channels had a much lower pain tolerance than mice not given the USP5 inhibitor.

Now that this enzyme has been identified as the regulator of the quantity of Cav3.2 channels on the cell surface of nociceptors, new therapies and drugs can now be developed to target these molecular enzymes or channels. Thanks to Caballero et al. (1) for not only establishing the enzyme but additionally where the enzyme binds on the channel, there are multiple targets that can be utilized in order to treat pain disorders. These kinds of discoveries are in dire need right now, as one of the central issues with current pain medications being utilized is their addictive prop- erties (10). These new findings may additionally open up new avenues for research on treatment and function of Cav3.2 in idiopathic generalized epilepsy (IGE) (11) or even autism as this channel has been associated with these disorders.

Now that this function of USP5 has been uncovered, it is indeed worth the moniker of the “guardian angel enzyme” for its inhibition can lead to treatments in a wide range of disorders, but particularly those in which pain is a primary symptom. Perhaps new medication that target USP5 or the calcium channel won’t have the same addictive properties of current pain medications already on the market, making for a safer way in which to treat patients with pain disorders.

Cav3.2 channels: the new theuaputic target for pain? Cav3.2 channels are key in allowing calcium influx into nociceptors allowing for pain signals to be transmitted. If new drugs target the inhibition of USP5 there will be more Cav3.2 channels degraded and pain sensitivity should be greatly relieved.


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