Case study: The regulatory mechanism of Rac
Biological interactions defined using the Bio-Logic builder are described by Boolean expressions that users build by using qualitative descriptives (or “bio-logic” components) generally used by laboratory scientists to explain the interaction from experimental studies. Leveraging the qualitative nature in which many biochemical interactions are discovered, Bio-Logic Builder provides users with building blocks of two types. First, users can define modules corresponding to positive and/or negative regulators that are involved in a given biological interaction (e.g., kinase X phosphorylates and activates protein Y, as is the case in studies of biochemical signal transduction). Because only few biological interactions can be represented as simple positive and/or negative regulators, users can specify a second type of bio-logic modules. These modules – “conditions” and “subconditions” – allow users to describe regulatory mechanism in which the effects of one or more positive and/or negative regulators depend on an additional regulators step (e.g., localization, priming, co-factors etc.), and hence the activation state or presence (or absence) of an additional regulator (or group of regulators). As a result, the users can define complex positive and negative regulatory modules much in the same way biological data and knowledge are discovered in the laboratory. To demonstrate how Bio-Logic Builder is used to build biological regulatory mechanisms, in this section is presented a case study which centers around the construction of a relatively complex regulatory system of the signaling protein Rac. Note that a simpler example of how the tool can be used can be viewed in a tutorial video at http://www.thecellcollective.org
Rac is an important player in the regulation of many cellular processes such as cell migration, cytoskeletal reorganization, DNA synthesis, etc. Rac belongs to the Rho family of small guanosine triphosphatases (GTPases), a subgroup of the Ras superfamily. Rac becomes activated when bound to GTP, a process mediated by guanine nucleotide exchange factors (GEFs). The hydrolysis of GTP to GDP results in the inactive state of Rac. This conversion occurs via Rac's intrinsic GTPase activity and is further accelerated by GTPase-activating proteins (GAPs). However, in addition to GAPs and GEFs, Rac's activity also depends on its proper localization as well as the activity state of components of other signaling pathways. A summary of the intricacies involved in the (de-)activation mechanism of Rac as reported in the biochemical literature so far follows. (Note that the following regulatory mechanism of Rac reflects the optimized mechanism published as part of a validated large-scale model of signal transduction in a generic fibroblast cell 
In the aforementioned fibroblast model, Rac is defined as ON when it is GTP-bound and localized in the plasma membrane. (See for a graphical summary of the mechanism and the involved species.) RalBP1 
and p190RhoGAP 
are GAPs, and hence negative regulators of Rac when GTP-bound (i.e., active). RhoGDI is also a negative regulator of Rac 
because it sequesters GDP/GTP bound Rac. PAK is be able to break up Rac-RhoGDI complex and stop the negative regulation of Rac by RhoGDI 
. While Akt appears to also be a negative regulator of Rac 
, based on the context of the overall network, it was made not dominant over any of the following positive regulators (and hence does not effect the activity state of Rac). The activation of Rac is mediated by RasGRF 
, Tiam 
, Pix/Cool 
, or DOCK180 
. The effects of these activators is dependent on cell attachment which is represented in the model by the activity of ECM and integrins. However, despite the fact that many of the details haven't been fully discovered, the activation mechanism of Rac by Pix/Cool appears to be relatively complex. In addition to the requirement of cell attachment, there are three different scenarios under which Pix/Cool modulates the activity of Rac. First, when the G protein subunits
(represented by a single species G
AND PAK are ‘ON ’ Pix/Cool only activates Rac if both Cdc42 AND Rac are ‘Off’. Second, when G
is inactive, Pix/Cool activates Rac only if Rac was previously inactive. In addition, this step also requires the activity of Cdc42. Finally, when PAK is inactive, Pix/Cool activates Rac only when Cdc42 is active, Rac was previously inactive, and RhoGDI, as well all other positive regulators are also inactive 
. (Note that due to missing information and/or inconsistencies in biological data, some of the logic of the mechanism might have been adjusted in the context of the whole model.)
Graphical representation of Rac regulatory mechanism.
As one can see, the regulatory mechanism of Rac is intricate and involves a large number of upstream regulators. Specifically, the activation states of 13 upstream regulators, in addition to the activation state of Rac in the previous time point have to be considered, resulting in 14 regulating inputs of Rac. Thus the truth table representation of the function would require the scientist to manually fill out
(or 16,384) lines of the species' corresponding table. The complexity of the regulatory mechanism is also matched by the underlying Boolean expression representing the function:
Manual creation of the logical expression for a regulatory mechanism of this size and complexity would be difficult and error prone. Using Bio-Logic Builder, users can capture the complex activation mechanism of Rac in non-mathematical fashion by using the published qualitative information (as described above) and building the regulatory mechanism in a modular fashion as detailed below.
The Bio-Logic Builder tool is part of The Cell Collective modeling suite 
, which can be freely accessed via a web browser by visiting http://www.thecellcollective.org
. From the Models page, users can either create a new model, or access any of the existing (e.g., Published) models. New species can be added or existing species modified under the Model Bio-Logic page. The Rac species can be found under the Model Bio-Logic of the “Fibroblast” model (under Published models). Clicking the green “gear wheel” icon next to Rac in the species table takes the user to the Bio-Logic Builder tool where the regulatory mechanism of the (Rac) species can be defined/modified. The first screen allows the user to start building/modifying the regulatory mechanism of Rac by specifying either the positive or negative regulation modules. In this study case, we will start with the negative regulation modules in the Negative Regulation Center. (Note that the order in which the user starts does not result in a different output. Also note that the species in the Published model are read-only for curation purposes; to be able to modify the regulatory mechanisms of the model's species, a “private” copy of the model can be made from the Models page by clicking the “Copy to My Models” icon.)
Negative regulation center
As the name suggests, Negative Regulation Center is where users designate upstream regulation modules that have a negative (i.e., inhibitory) effect on the species of interest (Rac in this example). From the regulatory mechanism described above, the negative regulators of Rac include Akt, RalBP1, p190RhoGAP, and RhoGDI. As shown in , the left panel of the page displays the “Species Palette” which is responsible for the management of all upstream regulators of Rac. The Species Palette is available to the user throughout the entire building process so that new species can be added/edited as needed. By default, the species for which the regulatory mechanism is being built (i.e., Rac) is automatically added to the palette. Before the user can designate species as negative regulators, they first need to be added to the species palette. Specifying a species as a negative regulator is as simple as drag-and-dropping a species from the palette into the box in the main window ().
Once Akt, RalBP1, p190RhoGAP, and RhoGDI are designated as negative regulation modules, conditions can be specified. As discussed in the previous sections, conditions allow biologists to specify regulatory scenarios under which a particular upstream regulator is dependent on the activity state of another species (e.g., a co-factor). In our Rac example, RalBP1 and p190RhoGAP are responsible for removing GTP from a GTP-bound (i.e., active) Rac and replacing it with GDP, hence inactivating Rac. Therefore, the effects of these negative regulators are dependent on the activation state of Rac itself which can be represented as a condition for the two upstream regulators. Based on the context of the whole network in 
, the effect of Akt as a negative regulator is also dependent on the previous activity level of Rac. In the case of Rho-GDI, PAK can break up the Rac-RhoGDI complex, hence a condition also needs to be specified for the RhoGDI module (). Conditions for each of the negative regulation modules are defined under their respective “Centers” as discussed below.
The conditions page is accessed from the Negative (Positive) Regulation Center page by clicking on the Center of the negative (positive) regulation module for which conditions need to be added/modified. For example, the conditions page for RalBP1 can be accessed under the “RalBP1 Center”. Users can define conditions as IF/WHEN and UNLESS statements to define scenarios when the effects of the regulator for which a condition is being specified depend on the activity state of another biological species. As mentioned above, the condition associated with Akt, RalBP1, and p190RhoGAP is that these species are negative regulators IF/WHEN Rac is ON. In addition, for users' convenience, each condition can be annotated to reflect its biological meaning and context. In this case, the condition was named “Rac activity”, but any annotation can be used. In the case of RhoGDI, its effect on the activity of Rac depends on the presence/absence of PAK; specifically, RhoGDI acts as a negative regulator UNLESS PAK is ON. Note that any number of conditions (and subconditions) can be associated with any regulator, allowing for the definition of the most complex regulatory mechanisms. (An example of multi-condition scenario is presented in the next section.) See for the final Negative Regulation Center page which summarizes the complete negative regulation modules of Rac. The “Done with Negative Regulation Center” returns the user to the Center home page where the Positive Regulation Center can be selected.
Positive regulation center
Positive regulation modules of the species of interest (e.g., Rac) are specified in the Positive Regulation Center. As discussed at the beginning of the Case Study section, the activating species of Rac include RasGRF, Tiam, Pix/Cool, and DOCK180. Once these species have been added to the Species Palette, they can be defined as positive regulation modules in a similar fashion as was done with the negative regulation modules, and was demonstrated in the Negative Regulation Center section. As the regulatory mechanism suggests, all positive regulators are dependent on cell attachment, and hence the activity of ECM and Integrins. Therefore the positive regulation modules RasGRF, Tiam, and DOCK180 the condition (named Cell attachment) “IF/WHEN ECM AND Integrins are ON”. However, the conditions associated with the positive module Pix/Cool are more complicated. As discussed at the beginning of this case study, there are three nontrivial scenarios describing the role of Pix/Cool in the regulation of Rac activity. To capture this complex regulatory mechanism, both condition and subcondition bio-logic gates are necessary. Subconditions can be specified after clicking the “Subconditions” button on the condition page of the regulation module center.
The three scenarios differ based on the presence and absence of G
and PAK. First, when G
AND PAK are ‘ON’ Pix/Cool only activates Rac if both Cdc42 AND Rac are ‘Off’.In Bio-Logic Builder, the first scenario is represented as a condition “IF/WHEN PAK AND G
are ON”, followed by subcondition defining the requirement for the absence of Cdc42 and Rac as: “IF/WHEN Cdc42 AND Rac are OFF”. Second, when G
is inactive/absent, Pix/Cool activates Rac only if Rac was previously inactive, and in the presence of Cdc42. This scenario can be represented as a condition “IF/WHEN G
is OFF” which has a subcondition defined as “IF/WHEN Cdc42 is ON”. The third scenario – where contains Pix/Cool activates Rac when PAK is inactive only if Cdc42 is ON, Rac was previously inactive, and RhoGDI and all other positive regulators are OFF – is also defined as a combination of a condition with subconditions. As done in a similar fashion above, to indicate the dependence of Pix/Cool on the absence of PAK, a condition of “IF/WHEN PAK is OFF” is defined. To add the dependence on Cdc42, RhoGDI, DOCK180, RasGRF, Tiam and Rac's previous activation state, the following subconditions are defined in a co-operative manner: “IF/WHEN Cdc42 is ON” (for the dependence on Cdc42 activity), “IF/WHEN RhoGDI is OFF” (for the dependence on the absence of RhoGDI), “IF/WHEN DOCK180 AND RasGRF AND Tiam are OFF”, and “IF/WHEN Rac is OFF” (for the dependence on Rac's previous activation state). In addition, because the activation of Rac by Pix/Cool is also dependent on cell attachment (similar to the other positive regulators), all three conditions have the Cell attachment subcondition (specified above) associated with them. Screen shots in and show the Pix/Cool condition page and the summary page of all positive regulation modules, respectively.
Positive Regulation Center of Rac with all regulation modules fully defined.
Once all negative and positive regulation modules are defined, the user is led to the next screen, the Dominance Page. On this page, users can define the “strength” of each negative module in terms of how dominant it is over the individual positive regulation modules. A negative module dominant over all positive regulators (pre-selected by default) has the largest (negative) effect on the state of the species of interest, whereas a negative module dominant over none of the positive modules will have no effect on the activity of the species.
Once the strength of the negative regulation modules is selected, the user needs to specify the final component of the regulatory mechanism building process – the state (active/inactive) of the species in the case where none of the positive nor negative modules are active or present in the cell (model). Upon the last page and component of the Bio-Logic Builder tool, the user can navigate to the Summary Page (). This page displays all regulatory modules involved in the regulatory mechanism of Rac. The program builds the mathematical function based on the regulatory modules specified by the user, and constructs the appropriate truth table in the background. The generated truth table can then be plugged into a larger model and simulated/analyzed by one of the software tools mentioned in the Introduction
section. Specifically, ChemChains, as described in 
, directly supports logical models represented as truth tables and can be used to easily simulate models created with Bio-Logic Builder. The truth table and logical expression for individual species can be downloaded from the model species page, or a set of all Boolean expressions and truth tables, as well as the SBML file for the entire model can be downloaded from the main Models page in The Cell Collective.
A screen shot of the Summary Page of the Rac regulatory mechanism.
Defining the “head regulator” of a positive/negative regulation module
In Bio-Logic Builder, the head regulators represent the main positive/negative regulation modules, within which conditions and subconditions are subsequently added. In the Rac case study presented in this section, the head regulators of the negative regulation modules included Akt, RalBP1, p190RhoGAP, and RhoGDI, whereas the head regulators of the positive regulation modules constituted RasGRF, Tiam, Pix/Cool, and DOCK180 (). All of these head regulators had one or more conditions (and subconditions in the case of Pix/Cool for example), and hence forming the corresponding regulation modules. However, what if it is not clear as to which species should be considered the head regulator and which species should be the condition building block of the regulation module? How does one decide which way it be depicted? Does it matter (in terms of the mathematical representation) which way the module is represented?
While for many biological interactions it is clear (based on the available published data) which of the species is considered the “head regulator”, there are many instances in which regulatory mechanisms can be ambiguous and hence become confusing to the user of Bio-Logic Builder. These few regulatory mechanisms can even be relatively simple in terms of the number of species involved in the interaction. For example, consider a hypothetical biochemical signaling protein X with two phosphorylation sites in its regulatory region. Let's assume that, in order to be fully activated, both of the phosphorylation sites of X need to be phosphorylated, one by kinase Y and the other one by kinase Z. From this described situation, one could consider both kinases as “equal” rather than as a “head regulator”/“condition” relationship. However, based on the Rac case study and the previous discussions of the Bio-Logic Builder algorithm, one of the kinases (Y or Z) has to be considered the head regulator, whereas the other one is represented as a condition (IF/WHEN ON) as part of the regulation module. Which way this scenario should be defined, however, is not clear in this example. Nonetheless, it is important to note that when a number of regulation species appear to be conceptually equal, Bio-Logic Builder requires one of these species be selected as the head regulator whereas the others be considered as a (sub)condition(s). Fortunately, because of the mathematical relationship between the head regulators and the conditions, the mathematical representation of the interaction will be the same in both cases (as detailed in Supporting Information S1). If such a scenario arises, the user will need to use their discretion and decide how to represent the regulatory mechanism.