Ubiquitin

It all starts with ubiquitin

Ubiquitin – as the name suggests – is found in all eukaryotic organisms from yeast to humans and although it is one of the smallest proteins in the cell it has one of the largest roles.  Nature attaches ubiquitin to lysine residues on other proteins, often growing ubiquitin chains via subsequent attachment of more ubiquitin to the lysines found in ubiquitin itself. ubquitin2

These chains act as “tags”, directing the modified protein to the proteasome for degradation or acting to recruit other proteins for propagation of intracellular signals. The DEGRONIMIDTM technology developed by C4T utilizes the cell’s intrinsic machinery to facilitate degradation of target proteins by targeting the disease relevant protein for ubiquitination.  This ubiquitination directs the target to the proteasome for subsequent degradation and the desired therapeutic effect.

The proteasome is the cell’s recycling machinery.

The proteasome is often been called the “garbage disposal” of the cell.  In reality, it is better described as the cell’s recycling plant.  Proteins that are delivered to the proteasome are threaded into the inner core particle in which they are disassembled into small peptides which ultimately degraded to amino acids which re-used by the cell to create new proteins. The main process through which proteins are delivered to the proteasome is through the attachment of ubiquitin chains, acting much like a shipping label on a package.

A depiction of the proteasome. The core particle – responsible for the main degradation process – is flanked by regulatory particles that include the base and the lid.  A cut-away of the model, illustrating the barrel-like architecture of the core particle, is shown on the right.

Once the ubiquitin tagged protein is brought to the proteasome, it interacts with the regulatory particle composed of the base and the lid. Proteins in the lid then remove the ubiquitin tag, allowing ubiquitin to be recycled and not degraded, and then other proteins in the regulatory particle begin to unravel and thread the protein into the core particle for digestion. C4T’s DEGROMINIDTM technology is used to ensure that disease causing proteins are tagged with the proteasome delivery label (a poly-ubiquitin chain) after which time the cell’s natural processes take over and eliminate the malfunctioning protein, leading to amelioration of the disease.

Normal protein degradation involves a cast of other proteins.

The process through which proteins are tagged for degradation and recycling is called the ubiquitin proteasome system, or UPS for short. Because this process is critical to maintaining a healthy, normal cell, it is highly regulated and involves multiple different components. The UPS begins with the proper formation of a poly-ubiquitin chain on a target protein, the labeling of the protein for proteasome delivery. This occurs through 3 different types of proteins called an E1 (ubiquitin activating enzyme), an E2 (ubiquitin conjugating enzyme), and finally an E3 (ubiquitin ligase). The ubiquitin protein is first activated, meaning it is now primed for attachment to another protein, through the activity of the E1 enzyme. Next, the E1 passes the activated ubiquitin to an E2 protein which acts to carry the ubiquitin to an E3 enzyme. There are hundreds of E3 enzymes in the cell, each of which interact with a set of target, or “substrate”, proteins, thus providing the specificity of the UPS. C4T’s DEGRONIMIDTM technology utilizes a class of E3 enzymes called “Cullin RING Ligases” (CRL’s) which form one of the largest groups of E3 enzymes in the cell. CRL’s are composed of a central cullin protein (in this example, Cullin 4 abbreviated as CUL4), a cullin adapter protein, in this case DDB1, and an E2 binding protein of the “really interesting new gene” (RING) family of proteins called RBX1. A fourth protein, known as a substrate adapter (in the example of CUL4 these are called DCAF proteins), interacts with the cullin adapter and acts to provide the specificity of the CRL for substrate proteins.

degradation

The overall cycle of protein degradation. Once ubiquitin is activated by the E1 enzyme it is transferred to the E2 enzyme, which often works in concert with an E3 protein to ensure the right protein gets ubiquitinated.
When the E2 binds to the E3, which holds the substrate protein, the E2 then transfers its ubiquitin onto a lysine amino acid of the substrate protein, resulting in mono-ubiquitination of the substrate. Next, another E2 carrying an activated ubiquitin arrives and then transfers its ubiquitin; this time to a lysine amino acid on the ubiquitin previously attached to the substrate protein. This process is repeated several more times, culminating is a poly-ubiquitin chain. In order to build this large poly-ubiquitin chain, the Cullin E3 enzyme itself is modified with a protein called Nedd8 through an analogous E1-E2 cascade. Nedd8 modification of the Cullin E3 releases constraints on the E2 binding platform (RBX1) giving it a wider mobility range and allowing the E2 protein improved access to build the poly-ubiquitin chain.

Once the poly-ubiquitin chain is built on the substrate protein, the ubiquitin-tagged substrate is released from the E3 enzyme, recognized for transport to the proteasome via its poly-ubiquitin chain, and is finally delivered to the regulatory particle of the proteasome to begin the degradation process. Once at the proteasome, enzymes in the regulatory particle removed the ubiquitin tag (allowing ubiquitin to be recycled) and the unravel the substrate and begin to thread it into the proteasome core particle. Once the protein is in the core particle, it is broken down into constituent parts which are in turn released back into the cell for use in the manufacture of new proteins which will eventually undergo the same process via the UPS.

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