Project

Overview

Proteins are molecules composed of hundreds of thousands of amino acids arranged in a sequence.  From the 20 different amino acids, countless combinations of amino acids are created to produce proteins with a very wide variety of properties. Proteins are also important molecules in biological activities, and are involved in the maintenance of cell structure, movement, and transport of substances.

For example, actin and myosin are proteins that enable muscle contraction. Actin forms the cellular cytoskeleton and is involved in muscle cell contraction and extension, as well as in cell movement. Muscle contraction occurs as actin arranges itself into filamentous structures, allowing myosin to slide along these filaments. Myosin generates contractile force by pulling the actin filaments, enabling movement and actions such as heartbeats. Additionally, hemoglobin is essential for transportation within the body. Hemoglobin, found in red blood cells, transports oxygen from the lungs to cells throughout the body. By efficiently carrying oxygen, it supports each cell’s ability to produce energy using oxygen. In this way, functions like movement enabled by actin and myosin and oxygen transport facilitated by hemoglobin allow the body to move flexibly and efficiently deliver necessary substances. Proteins play an indispensable role in maintaining life through these diverse functions. 

Problem

Reaction systems found in nature, such as those involving actin, myosin, and hemoglobin, are highly compatible with biological systems and coexist without interfering with other reaction systems. While numerous other proteins capable of self-organization are also known, it is well recognized that these reaction systems are often highly complex. Repurposing naturally occurring self-organizing systems for other applications presents numerous challenges and inconveniences for both the system itself and its intended application, and significantly modifying existing self-organizing systems is not easily achievable.

However, this raises an intriguing question: could we extract the principles of these complex natural reaction systems and recreate them in a simplified form? While it may be difficult to artificially replicate every element of a natural reaction system, if we focus solely on reproducing its core functions, it would not be an exaggeration to say that we have successfully recreated the natural system. Achieving a simplified version of the reaction system could facilitate integration with other systems, making it relatively easy to incorporate into new reaction frameworks.

With this in mind, we have set a goal to extract principles from natural reaction systems and explore whether we can recreate their functions using a minimal set of components.

Solution

We hypothesize that Actin, a type of molecular motor, can realize this idea. (For details, please refer to the Design page.) Actin is a protein that self-assembles into fibrous structures, fulfilling the requirement to connect two points: referred to as TARGET and BASE. Furthermore, Actin is well-studied, and both Polymerization Activators and polymerization inhibitors are known. By polymerizing Actin extending from TARGET and BASE into fibrous structures and connecting the fibrous Actin strands extending from both directions, TARGET and BASE can be connected via Actin.

However, a significant issue arises when Actin is simply present in solution. Actin spontaneously polymerizes into fibrous structures in solution. If this spontaneous polymerization occurs, fibrous Actin will form without properly connecting TARGET and BASE; this is ineffective for the intended objective.

To control Actin polymerization, it is necessary to introduce polymerization inhibitors. Several types of Actin polymerization inhibitors are known, including CapZ, tropomodulin, and Thymosin β4: 

1. CapZ is a protein that suppresses Actin polymerization by gathering around fibrous Actin. 

2. Tropomodulin is a protein that binds to the minus (-) end of fibrous Actin to inhibit Actin polymerization. 

3. Thymosin β4 is a polypeptide that binds to Actin monomers and inhibits the addition of new Actin to the plus (+) end. 

To prevent Actin in solution from spontaneously polymerizing, it is necessary to inhibit the polymerization of Actin monomers. Additionally, a simpler structure makes design more straightforward and easier to handle. For these reasons, we selected Thymosin β4, a polypeptide, as the polymerization inhibitor for fibrous Actin.

In solution, Actin monomers can maintain their monomeric state without spontaneous polymerization by binding to Thymosin β4. At the start of polymerization, we introduce a molecule that functions as a Polymerization Activator, strongly binding to Thymosin β4 and removing it from Actin. Actin released from Thymosin β4 binding then polymerizes with Actin on TARGET and BASE, self-organizing into fibrous structures. The fibrous Actin extending from both TARGET and BASE connects, thereby linking TARGET and BASE.

Our GOAL

To summarize the reaction system we aim to achieve using Actin: numerous Actin fibers extend from both TARGET and BASE, connect together, and subsequently shorten in length. Consequently, the distance between TARGET and BASE gradually decreases, pulling TARGET closer.

Like other innovative and groundbreaking ideas, establishing many novel aspects of this system requires precise scientific validation, which in turn demands significant funding, time, and advanced research facilities. However, our activities in BIOMOD are time-constrained. Therefore, we focused on designing the key protein consisting of Thymosin and Actin, while simultaneously addressing foundational elements. Achieving the objectives outlined below will constitute success for our BIOMOD project.

The functionality of the proposed reaction system is composed of five main elements. For these five functions, we set milestones in the following order to progressively approach their implementation: extracting principles, reconstructing functions, selecting molecules for application, simulation and analysis, and Experiment. By setting detailed goals for each step, we believe that it may clear up the overall progress of the system.

Feasibility

Many newly designed elements are required to realize the entire reaction system, which demands significant time and cost. While achieving each individual element might be possible with enough resources, connecting these elements and driving them as a unified reaction system requires careful design of the interfaces. Without such integrative design, the reaction system may not function properly, even if the individual components are successfully designed.

What we focused on designing this time was the most critical part of the reaction system. By simplifying the structure, we aimed to demonstrate our concept. However, even with a simplified structure, time and cost constraints remained, requiring us to achieve as much as possible within these limitations. Additionally, there are multiple uncharted aspects in our proposed reaction system, making it challenging to thoroughly evaluate what is feasible and what is not within the limited time available. To bring the reaction system closer to realization, we extensively utilized simulation software for protein design. By using simulation software, we could reduce the time spent on protein synthesis experiments and focus only on experiments with promising results predicted by the simulations, enabling a more efficient protein design process.

We also leveraged generative AI for tasks such as literature research and generating simulation software code. With AI assistance for suggesting relevant literature and generating initial code, we could concentrate on understanding the content of the papers and refining and improving the code, thus enabling higher-quality project progress.

To ensure efficient project advancement, we not only assigned specific roles to each team member to allow them to focus on their tasks but also held regular meetings–both offline and online–to ensure transparency about the work being done in other areas. This allowed all members to stay updated on project progress. Additionally, we conducted frequent meetings with our mentors to keep them informed about our progress and receive advice based on scientific insights regarding feasibility, costs, experimental facilities, and the time required for experiments and validations.

REFERENCE

[1] Sin-Ichi Odagaki, Haruko Kumanogoh, Shun Nakamura & Shohei Maekawa (2009) "Biochemical interaction of an actin-capping protein, CapZ, with NAP-22" Journal of Neuroscience Research, 87:1980-1985

[2] Annemarie Weber, Cynthia R. Pennise, Gary G. Babcock, and Velia M. Fowler (1994) "Tropomodulin caps the pointed ends of actin filaments" Journal of call biology, 127:1627-1635