Abstract v. 2

My original project this summer was to focus on analyzing mitochondrial DNA of human remains. However, due to unanticipated difficulties in obtaining new samples for study, I was unable to work on that project this summer. Fortunately, I was able to work on another project in Dr. Allison’s lab this summer.

The general focus of research in Dr. Allison’s lab is on the thyroid hormone receptor (TR) and how it is transported between the cytoplasm and the nucleus. The project I am working on focuses on the roles of certain proteins, called importins and exportins, in transport across the nuclear membrane. They do so by binding to TR in response to certain amino acid sequences or structures. We aim to explore and characterize which importins and exportins play a role in TR transport, as well as determine how and where they bind to TR. This summer, we focused on synthesizing fusion proteins of TR with polypeptide tags (that is, adding peptide sequences to the basic protein). These fusion proteins can be more easily detected and can be used in experiments to explore how importins and exportins bind to TR.

TR is a protein and transcription factor, affecting the expression of certain genes; it can either promote or repress transcription, and thereby gene expression. It can bind to both thyroid hormone (T3) and to DNA. In response to T3 binding to it, TR will promote expression of certain genes. T3 is an essential hormone found all over the body, and is important in many body systems, including the musculoskeletal system and central nervous system. It is also essential in maintaining proper metabolic balance and in cell differentiation.

There are two isoforms of TR: TRα and TRβ. They are encoded by different genes and are found in different areas of the body. TRα, which is the form we are studying, is found in the central nervous system. TRα protein can be divided into four sections, or domains, each with unique properties and functions. The A/B domain plays a role in gene activation and is where most of the differences between TRα and TRβ are located. It also contains 1 nuclear localization sequence (NLS); that is, one sequence in particular that signals for TR to be transported into the nucleus. The DNA binding domain (DBD) binds to the DNA itself, while the ligand binding domain (LBD) binds to T3. Between the DBD and LBD is the short hinge domain; it joins to two domains together and also contains another NLS.

While at any given moment, TR is located almost entirely in the nucleus, it has been established that TR is shuttling constantly between the nucleus and cytoplasm. Importins and exportins transport TR, as well as other molecular cargo, across the nuclear membrane. To do this, they bind to cargo (in this case, TR) in response to specific sequences on amino acids; after docking at the nuclear pore complex, the importin/exportin, with cargo, is translocated across the nuclear membrane.

What we wish to determine, in the long term, is which importins and exportins bind to TRα, and where they bind on TRα. We are still early in the project; synthesizing fusion proteins has proven difficult, and was the main focus of our work this summer.