Targeted protein degradation of phospholamban for PLN treatment

Abstract

Phospholamban is a protein in heart cells. The protein has a regulatory function in the working of the heart. Some people have a mistake in the genetic code (also called mutation) of the phospholamban protein. Due to this mutation the protein cannot function properly, this can result in a heart disease. People with this specific heart disease get standard heart failure care. The aim of this treatment is to reduce symptoms. However, at this moment it is not possible to cure the disease. To try to make it possible to cure this disease we have set up this research project. During this project we tried to make a so-called fusion protein, which is a protein which is fused to something else. Our fusion protein consists of an E3 ligase (protein part) and a nanobody against the phospholamban protein. The E3 ligase is a protein which has a role in the protein degradation of the cell. A nanobody is a special kind of antibody which can be derived from llamas. Llamas produce the nanobody if they are injected with the protein wherefore you want nanobodies. This injected protein will be recognized as foreign which result in the production of nanobodies. We first tried to let a llama produce the nanobody for us but that failed. We think that it failed due to the fact that the phospholamban protein of the lama looks very much the same as that of the human, and that the llama did not recognize it as a foreign protein. Therefore, another approach was needed to make the nanobody. In the literature, a synthetically (in the lab) produced nanobody, called VHHB4, is described. This nanobody is able to bind to the healthy phospholamban protein, and we wanted to know if this nanobody is also able to bind to the phospholamban protein with the mutation. We were able to confirm that this is indeed the case, moreover we showed that VHHB4 did not bind better to the healthy or mutated version of the protein. Because the VHHB4 nanobody was able to bind the mutated phospholamban, we decided to use this VHHB4 nanobody for the production of our fusion protein. After we produced our fusion protein, we tested it in cells if the fusion protein is able to reduce the phospholamban levels and can improve the function of the cells. This data showed inconclusiveness and therefore it is hard to draw a conclusion. Because the used nanobody binds to the mutated phospholamban as good as the healthy phospholamban protein, it cannot be used to treat the genetic disease. So, to cure the heart disease another approach is needed.