he AT-rich interacting domain-containing protein 1a (ARID1a), also named BAF250a, p270, or hOSA1, is a vital component of the Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex. ARID1a, as part of the SWI/SNF complex, is responsible for crucial nuclear activities, including regulating transcription, DNA synthesis, DNA damage repair, and cell proliferation. Consequently, ARID1a is classified as a tumor suppressor gene and it is frequently mutated in solid tumor malignancies cancers, amounting to ~6% of every cancer and ~45% of all ovarian cancers. Loss of function mutations in ARID1a due to frameshift, stop-gain, or missense mutations leads to dysregulation of many gene pathways, including the prevention of tumor suppressor activities. Although the loss of function is evident in stop-gain and frameshift mutations, the impact of pathogenic missense mutations is subtler and more difficult to understand or predict. We hypothesize that pathogenic missense mutations impact the protein stability, DNA binding affinity, and structural dynamics, perturbing its function. Specifically, we seek to understand the effects of pathogenic missense mutations in the ARID domain of ARID1a, which is responsible for its direct interaction with DNA. Therefore, the broad goal of this research program is to understand the effects of pathogenic missense mutations in the ARID domain of ARID1a. The long-term implications of this program are the development of a unique framework that will pave the way for directly probing identified pathogenic missense mutations for future guided patient-specific screening therapeutic approaches. Our proposed study integrates biophysical studies, computational approaches, and single-molecule spectroscopy to characterize the stability, DNA binding, and structural dynamics of the ARID domain. Our studies will give insights into the correlation between missense pathogenic mutations and the structure-dynamics- function relationship. The PI and our research team are uniquely positioned to pursue the following specific aims: (1) to determine the impact of pathogenic missense mutations on the stability of the ARID domain; (2) to determine the impact of pathogenic missense mutations in the binding affinity of ARID domain to DNA; and (3) to determine the changes in the structure and dynamics of ARID domain as perturbed by pathogenic missense mutations. This interdisciplinary project will engage undergraduates in research to foster their interest and career development in both physical sciences and cancer research. The expected outcome will be a structural model of ARID1a’s ARID domain interaction with DNA, a library of pathogenic mutations ranked by the impact on stability and affinity, a system to improve pathogenic predictors, and the foundation for developing novel therapeutics and personalized medicine in the fight against cancer. Award: NCI 1R15CA280699.
Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology (Subaward)
Resolving the intoxication mechanism of botulinum neurotoxins using single molecule structural biology. The toxins produced by Clostridium botulinum are some of the deadliest known yet are also revered for their pharmaceutical utility. C. botulinum is classified into seven serotypes (A-G) based on the neurotoxins that they produce. Currently, pharmaceutical development has relied on botulinum neurotoxin type A1 (BoNT/A). However, botulinum neurotoxin type E (BoNT/E) is currently in clinical trials because it provides different pharmacokinetics, faster onset and shorter duration, which enable new treatment regimes. The BoNT proteins are members of the two-component, “AB toxin” family (e.g. tetanus, cholera, and diphtheria toxins), which inject a toxic cargo enzyme (part A) using a proteinaceous transmembrane delivery system (part B). As such, their structure and activity has been well studied. However, several fundamental open questions remain regarding the BoNT delivery mechanism, such as the number of toxins required to deliver the cargo. Additionally, while numerous structures have been solved of the dormant toxins, there is little structural information on the active delivery state(s). AB toxins deliver their cargo across cellular membranes, typically triggered by low pH, which causes structural changes of both parts A and B along with insertion into the membranes. The presence of aggregation at high protein concentrations and membranes provide many experimental challenges for techniques that rely on ensemble averaging. In contrast, single molecule fluorescence can observe individual proteins on single liposomes to revist these classic problems in AB toxin structural biology. These novel approaches will answer long-standing questions in the field and lead to new understanding of the differences between two clinically relevant isoforms. Award given to PI: Mark Bowen 1R01GM151334.
REU: Nature’s machinery trhough the prism of Physics, Biology, Chemistry, and Engineering
This REU Site award to Clemson University, located in Clemson, SC, will support the training of 10 students for 10 weeks during the summers of 2019-2021. Program participants work with faculty, postdocs, graduate students, and other undergraduates on collaborative research exploring biology through the prism of physics, biology, chemistry and engineering (e.g., computational and experimental approaches for molecular insights into amyloid aggregation, studies of how missense mutations affect protein-DNA binding affinity, and quantifying properties of active polymer networks and gels from cytoskeletal proteins). The focus is on cross-disciplinary training; while each participant has a specific project with their own mentor, participants are paired to work together within a larger collaboration. The program includes a biophysics boot-camp, seminar series, workshops on research tools and professional development, journal club, off-campus field trips, ethics training, and cohort-building activities. Participants regularly present their contributions to collaborators and hear about the research of their peers. Each participant presents a poster of their own work adjacent to their collaborator’s poster at the final summer research symposium, writes their own research manuscript, and is encouraged to present their work at scientific conferences. Participants also engage in outreach activities throughout the summer.
Funding: NSF BIO/DBI 1757658, 2349368
CAREER: Structural dynamics of post-translationally modified Calmodulin and its role in Target Recognition
The project is geared towards identifying the fundamental principles that allow the protein Calmodulin to regulate vital functions such as heart beating, muscle contraction, and learning and memory. Interestingly, Calmodulin can activate or deactivate these functions, but the process by which Calmodulin selects this action is not understood. The project will study modifications to the protein that likely alter the protein’s flexibility and three-dimensional structure. The obtained knowledge would explain how certain modifications regulate the way Calmodulin interacts with intended target molecules. The broader impact of this project includes the incorporation of a thorough educational and training program to prepare a pool of highly qualified undergraduate, graduate, and postdoctoral researchers across various disciplines. In particular, the program will create enriched curricular programs that provide new opportunities for hands-on experience using state-of-the-art instrumentation and train a globally competent workforce with the creation of new study abroad opportunities.
Funding: NSF BIO/MCB 1749778
Single Molecule Analysis of MAGUK Structure and Ligand Binding
The MAGuK scaffold proteins organize glutamate neurotransmission through multiprotein interactions and have been implicated in diseases such as neurodegeneration following stroke, autism, epilepsy and schizophrenia. This proposal investigates the post- translational regulation of MAGuKs by phosphorylation, palmitoylation and phase separation using a novel reconstitution of the postsynapse along with state-of-the-art single molecule methods. Understanding the allosteric regulation of MAGuKs through physiologically-relevant reconstitution may provide a new avenue for the treatment of neurological and neuropsychiatric disorders.
Funding: NIMH National Institute of Mental Health R01MH0 81923-11A1