5.2.4 Protein Regulators

1. Purpose

So far we have some pretty powerful systems to be able to regulate the genetic production of proteins. Transcription factors and gene controllers allow us to amplify or repress gene expression, or to completely turn genes on and off. However, on the time scale of cellular processes gene expression is too slow. It takes a long time to produce a large quantity of a protein and ship it to where it is needed. Cells typically use gene regulation only for longer term processes. For instance in learning in a neural network, gene expression is one of the last steps used to alter the connection strengths over a long period. It is local regulation of the proteins in the synapses and dendrites that eventually cause the gene regulation to produce long term effects. So in order to emulate this process what is needed is a way to regulate protein activity. Two forms of protein regulation were implemented in this system: allosteric and kinase regulation.

2. Allosteric Regulation

Mechanisms of Allosteric Inhibition and Activation

Figure 1. An allosteric enzyme consists of one or more catalytic subunits (C) and one or more regulatory subunits (R), each with an active site or an allosteric site, respectively. The enzyme exists in two forms, one with a high affinity for its substrate (and therefore a high likelihood of product formation) and the other with a low affinity (and a correspondingly low likelihood of product formation). Which form an enzyme is in depends on the concentration of the allosteric effector(s) for that enzyme. 5.2.4.1

Allosteric regulators perform a similar kind of regulation for proteins as transcription factors perform for genes. Their effects are only felt while the regulator protein is present in sufficient quantity in the cell. A given regulator can either activate or inhibit the activity of a protein. In nature the regulator functions by binding into the allosteric site and causing a conformational change in the regulated protein. This change in shape can open up the active site to activate the protein, or it can close off the active site to inhibit the activity of the protein. The same regulator can activate one protein and inhibit a different protein. It is the property of the regulated proteins allosteric binding site and the nature of the conformational change that determines the action that is taken when then regulator binds to the site. If there are no regulator proteins then they can not bind to the site and regulate the protein.

3. Kinase Regulation

The Regulation of Glycogen Phosphorylase by Phosphorylation.

Figure 2. (a) Glycogen phosphorylase is a dimeric enzyme in muscle cells that releases glucose molecules as glucose-1-phosphate, which can then be used by the muscle cells as an energy source. (b) Glycogen phosphorylase is regulated in part by a phosphorylation/ dephosphorylation mechanism. The inactive form of the enzyme, phosphorylase b, can be converted to the active form, phosphorylase a, but the transfer of phosphate groups from ATP to a particular series on each of the two subunits of the two enzyme. This phosphorylation reaction is catalyzed by the enzyme phosphorylase kinase. Removal of the phosphate groups by phosphorylase phosphatase returns the phosphorylase molecule to the inactive b form. 5.2.4.2

Kinase regulators are similar to genetic controller proteins. They actually switch proteins on and off just like controllers switch genes on and off. In nature proteins are typically regulated in this manner by adding or removing phosphate groups onto them. Like the allosteric regulators, the new phosphate groups cause a conformational change in the protein that opens or closes the active site of the regulated protein. The big difference between this and allosteric regulation is that once the phosphate groups are attached they stay on there until they are removed. The kinase protein is no longer needed at that point. Once the regulated protein has been activated it stays activated until something comes along and rips the phosphates off and deactivates it. Again though it is the shape of the regulated protein that determines what affect adding and removing the phosphate group will have. Sometimes adding the group actually shuts the active site and inhibits the protein, and for other proteins adding the group will open the active site. Unlike in allosteric regulation there is also one other factor that must be taken into consideration. That is what the state of the protein is when it is first created. Allosteric regulators are always created in their default state. But kinase regulated proteins can be created active or inactive, and then later flipped at will.

3. Regulator Properties

Regulator Properties

Protein Type: All proteins have this property. It is used to determine the type of protein to load. Binding ID: All proteins have this property. For GC's it matches up with control sites on genes to determine which genes that it can flip. Degrade Rate: All proteins have this property. This determines the rate at which this protein is degraded in the cell. On Switch: This is only on kinase regulators. It tells whether this regulator is trying to flip the switch on the regulated protein on or off.

Table 1.These are the different properties of regulator proteins that are defined in the digital genes.

Regulatory Site Properties

Binding ID: This value matches up to the BindingID of the gene control protein. Activator: This determines whether this site will activate the protein or inhibit it. Switch Default: This is only used for kinase regulation. This determines the default state of the switch on the protein. Rate: This basically tells how many regulatory units are needed to cause a regulation of this protein.

Table 2.These are the different properties of a regulatory site that is defined on the proteins.

Kinase Regulation Chart

Switch Activator Final State On True Activate Off True Inhibit On False Inhibit Off False Activate

Table 3.This table shows how the switch and activator values interact to activate or inhibit the protein.

Allosteric regulation is the easier of the two and has less properties. If the activator property on the site is false and their are regulatory units bound to the site then the protein is inhibited, and vice versa. If the rate is two then that means it takes two regulator units to effect one protein. The mechanism behind kinase regulation is more difficult to understand. I like to imagine the regulated protein as having a big light switch on it. The light switch can be turned on or off by the regulatory units, but turning it on can either activate the protein or inhibit it. Also, The regulated protein can be created with the switch on or off. Table 3 shows the different states that can occur. Lets say that we have a protein with a site that has a switch that is on by default. The switch deactivates the protein, and the regulator flips the switch off. When this protein is first created it the switch is on, but since the switch deactivates the protein it is actually created in an inhibited state. Now the regulator protein comes along and flips the switch off. The switch is no longer deactivating the protein, so it is now active and can perform its function. We can also have a regulatory unit that comes along and flips the switch back on and deactivate it again. This is the same principle as adding and removing phosphate groups. In both allosteric and kinase regulation if there is more than one site then all sites have to be active for the protein to be active. If even one site is inhibiting the site then the protein is inhibited.

5. Regulating Regulators

Regulatory Tree

Figure 3. This figure shows a regulatory tree where regulators regulate other regulators.

Can a regulator be regulated? The answer is yes it can. This happens very often in nature. Often there will be a long list of regulators that can activate and suppress each other before some final regulator effects the a transcription factor or receptor to cause a real action. This allows the cell to have several systems that can all come together to actual control a process. Figure 3 shows a simple regulation tree where one allosteric unit regulates another and so on until a number of different units come together to regulate a ligand. One limitation of the simulator system is that it does not allow loops. This is a situation like A regulates B regulates C which then regulates A. This does not cause an error but the link between C and A would be eliminated so that C no longer regulates A. This is perfectly valid in nature, but it is disallowed in this system in order to remove a ton of hassles and make the algorithm for regulation simpler and faster.

5. Allosteric Regulation Example

Figure 4. This graph demonstrates allosteric regulation.

This graph demonstrates some of the basics of allosteric regulation. The red line gives the total quantity of the regulated protein in the cell. The green line shows the quantity of that ligand that is active. If the protein is not active then it can not take place in other reactions. In this case the inactive ligand will not be able to bind to any receptors. At one second an activating regulator is injected into the cell. This can be seen in the blue line. Also, the green line is not visible at this point because it is directly underneath the blue line. Then at two seconds an inhibiting regulator is injected into the cell. This is shown using the purple line. Immediately the amount of active ligand dips down proportionally to the inhibitor quantity and as the inhibitor degrades away the active quantity increases back up and actually merges with the raw quantity trace. This is because the ligand is degrading faster than the activating regulator and it actually dips below the regulator at around 2.5 seconds. From that point on all of the raw ligands have activator units bound to them and the two lines will be identical. It is only the inhibitors that change this temporarily.

6. Kinase Regulation Example

Figure 5. This graph demonstrates kinase regulation.

Figure 5 shows kinase regulators in action. The red line shows the raw quantity of a ligand in the cell. This ligand is created in the inactive state by default. So the green line that shows the active quantity of the ligand begins at zero. The regulatory site is an activator so that if the switch is flipped the protein will become active. At one second a small quantity of kinase regulator is injected into the cell, this can be seen in the blue line. This regulator flips the switch on and begins activating the ligand so the green line climbs till it matches the raw quantity. The activating regulator then degrades to nothing, but even though it is soon completely gone the ligand remains activated. This is one of the key features of kinase regulation. Once the state is changed it will remain in that state until another regulator comes along to change it. This is exactly what happens when we inject an inhibiting regulator at five seconds. This regulator flips the switch off and thus deactivates the ligand. As soon as it is injected into the cell the green line begins dropping again until it is once again at zero and then stays there for the remainder of the graph.

7. Regulation Overview

Allosteric and kinase regulation provides a powerful mechanism for proteins to be able to control the activity of other proteins. This is used extensively in natural systems to quickly and locally control enzyme activity. It is true that the current system does not have a concept of locality. In other words, in the current cell description all proteins are in the same space and can interact with each other. However, once synapses and dendrites are added to the system this will no longer be true and it will be possible to have protein regulation occurring locally in a synapse. This regulation will be used to control short term changes in synapse strength and to trigger the production of transcription factors and gene controllers that will then affect long term changes in the synapse strength in the soma. So while this is still a useful concept to have even with the current interpretation of the cell, it will really be valuable once the system becomes a little more complicated and more like real neurons.