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- All Subjects: Particulate Guanylyl Cyclase Receptor A
- Creators: Johnston, Stephen
Description
Currently, treatment for multiple myeloma (MM), a hematological cancer, is limited to post-symptomatic chemotherapy combined with other pharmaceuticals and steroids. Even so, the immuno-depressing cancer can continue to proliferate, leading to a median survival period of two to five years. B cells in the bone marrow are responsible for generating antigen-specific antibodies, but in MM the B cells express mutated, non-specific monoclonal antibodies. Therefore, it is hypothesized that antibody-based assay and therapy may be feasible for detecting and treating the disease. In this project, 330k peptide microarrays were used to ascertain the binding affinity of sera antibodies for MM patients with random sequence peptides; these results were then contrasted with normal donor assays to determine the "immunosignatures" for MM. From this data, high-binding peptides with target-specificity (high fluorescent intensity for one patient, low in all other patients and normal donors) were selected for two MM patients. These peptides were narrowed down to two lists of five (10 total peptides) to analyze in a synthetic antibody study. The rationale behind this originates from the idea that antibodies present specific binding sites on either of their branches, thus relating high binding peptides from the arrays to potential binding targets of the monoclonal antibodies. Furthermore, these peptides may be synthesized on a synthetic antibody scaffold with the potential to induce targeted delivery of radioactive or chemotherapeutic molecular tags to only myelomic B cells. If successful, this would provide a novel alternative to current treatments that is less invasive, has fewer side effects, more specifically targets the cause of MM, and reliably diagnoses the cancer in the presymptomatic stage.
ContributorsBerry, Jameson (Co-author) / Buelt, Allison (Co-author) / Johnston, Stephen (Thesis director) / Diehnelt, Chris (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Division of Teacher Preparation (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Particulate Guanylyl Cyclase Receptor A (pGC-A) is an atrial natriuretic peptide receptor, which plays a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain of pGC-A interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop a method that can regulate pGC-A, structural information regarding its intact form is necessary. Currently, only the extracellular domain structure of rat pGC-A has been determined. However, structural data regarding the transmembrane domain, as well as functional intracellular domain regions, need to be elucidated.This dissertation presents detailed information regarding pGC-A expression and optimization in the baculovirus expression vector system, along with the first purification method for purifying functional intact human pGC-A. The first in vitro evidence of a purified intact human pGC-A tetramer was detected in detergent micellar solution. Intact pGC-A is currently proposed to function as a homodimer. Upon analyzing my findings and acknowledging that dimer formation is required for pGC-A functionality, I proposed the first tetramer complex model composed of two functional subunits (homodimer). Forming tetramer complexes on the cell membrane increases pGC-A binding efficiency and ligand sensitivity.
Currently, a two-step mechanism has been proposed for ATP-dependent pGC-A signal transduction. Based on cGMP functional assay results, it can be suggested that the binding ligand also moderately activates pGC-A, and that ATP is not crucial for the activation of guanylyl cyclase. Instead, three modulators can regulate different activation levels in intact pGC-A.
Crystallization of purified intact pGC-A was performed to determine its structure. During the crystallization condition screening process, I successfully selected seven promising initial crystallization conditions for intact human pGC-A crystallization. One selected condition led to the formation of excellent needle-shaped crystals. During the serial crystallography diffraction experiment, five diffraction patterns were detected. The highest diffraction resolution spot reached 3 Å.
This work will allow the determination of the intact human pGC-A structure while also providing structural information on the protein signal transduction mechanism. Further structural knowledge may potentially lead to improved drug design. More precise mutation experiments could help verify the current pGC-A signal transduction and activation mechanism.
ContributorsZhang, Shangji (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021