Aggregation Characterization of Wild-Type p53 and Six Common p53 Mutants

P53 is a tumor suppressor protein, which functions in maintaining the cell cycle. When p53 loses its function, cells may multiply at an uncontrolled rate and form tumors. This loss of function is linked to over fifty percent of human cancers. This investigation aims to explore the possible link between p53 aggregation and tumorigenesis. There is a possibility that p53, especially in mutant form, will aggregate beyond its normal tetrameric conformation and lose its function, leading to tumor formation. Wild-type p53 and six mutants, R175H, R175C, R248Q, R248W, R273C, and R273H (six of the most common mutations found in human cancers), were purified from E. coli using Ni-NTA agarose resin. Aggregation rates were monitored for the wild-type and each mutant by Thioflavin-T binding assays. Atomic force microscopy was used to visualize each of the p53 mutants pre-incubation and at time points that Thioflavin-T binding suggested the presence of aggregates. Utilizing this data, each mutant was characterized for its relative speed of aggregation; this suggests which mutants are more aggregation-prone. Combined with cell viability results, our data indicates that p53 aggregation of mutated variants is a contributing factor to tumorigensis. Those mutants with the greatest propensity to aggregate also appear to be those with the greatest ability to become immortal

Learning to Look through the Eyes of Our Students: action research as a tool of inquiry

The story we are about to tell occurred when Gayle was a middle school science teacher and graduate student in Joanne’s seminar on the study of teaching. Gayle was trying to make sense of her science students’ indifference toward the environment, an attitude that concerned her as an environmentalist. She turned her inquiry into an action research project that sought to answer the question, ‘What are the assumptions that my middle school students have about their relationship with the environment?’ Joanne was mentoring Gayle in her action research study, and at the same time exploring Gayle’s perspective as an action researcher. Now, several years later, we are both action researchers and teacher educators and understand that we have been looking through the eyes of our students in order to become scholars of our own teaching

Inhibition of Toxic IAPP Amyloid by Extracts of Common Fruits

The aggregation of the 37-amino acid polypeptide islet amyloid polypeptide (IAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type 2 diabetes. It is believed that inhibiting the aggregation of IAPP may slow down, if not prevent entirely, the progression of this disease. Extracts of thirteen different common fruits were analyzed for their ability to prevent the aggregation of amyloidogenic IAPP. Thioflavin T binding, immuno-detection and circular dichroism assays were performed to test the in vitro inhibitory potential of each extract. Atomic force microscopy was used to visualize the formation of amyloid fibrils with and without each fruit extract. Finally, extracts were tested for their ability to protect living mammalian cells from the toxic effects of amyloid IAPP. Several fruits showed substantial ability to inhibit IAPP aggregation and protect living cells from toxic IAPP amyloid

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease