This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports the development of novel tools that will aid researchers in the discovery of enhancers of degradation of misfolded proteins that are processed by cells. The degradation of misfolded proteins is important both in bioprocessing and in medicine, as one of the biggest problems in expression of certain recombinant therapeutic proteins is the intracellular aggregation of these proteins. Proteins which are targeted for secretion that accumulate as aggregated or misfolded proteins inside cells cause cellular stress and eventually cell death via apoptosis, leading to loss of productivity in bioprocessing applications. In diseases such as Alzheimer's or Parkinson's, the presence of an aggregated protein appears to lead to neurodegeneration. In protein misfolding diseases, endoplasmic reticulum or ER stress may play a role in cell death. The enhancement of mechanisms which would lead to degradation of those proteins might slow down the progression of disease. In this project, the investigators propose to develop a novel fluorescence based assay for the detection of the activation of a specific protein degradation pathway (ER-degradation). This assay is the first step in being able to develop high throughput screens for molecules that will enhance intracellular degradation of misfolded proteins. The assay will have applications both in the bioprocessing industry and in biomedicine. The PI will integrate research and education at the graduate and undergraduate levels, and work to increase the diversity of the biotechnology field.
This NSF-EAGER award by the Biotechnology, Biochemical and Biomass Engineering program supported the development of novel molecular probes of protein degradation that will aid researchers in the discovery of enhancers of degradation of misfolded proteins that are processed by cells. Protein misfolding and aggregation are currently one of the main challenges of the fields of bioengineering and biomedicine. The aberrant accumulation of misfolded proteins often leads to the formation of insoluble aggregates in both prokaryotic and eukaryotic organisms, limiting the high-yield production of recombinant proteins for research, industrial and diagnostic applications. Protein misfolding is also a common theme in the cellular pathogenesis of protein misfolding diseases, such as Parkinsonâ€™s and Alzheimerâ€™s, which in turn provide excellent model systems to study protein aggregation. The degradation of misfolded proteins in eukaryotic cells is catalyzed by the ubiquitin proteasome system. High expression rates of proteins (both native and recombinantly expressed) can overload and impair the capacity of the ubiquitin proteasome system to dispose of misfolded intermediates, leading to enhanced aggregation of newly synthesized proteins. The cell typically responds to misfolding stress with the activation of a cascade of events to restore proteins homeostasis. However, when sustained, this cascade culminates in apoptosis. The enhancement of mechanisms that mediate degradation of misfolded proteins is likely to ameliorate the phenotypes associated with accumulation of misfolded proteins, thus providing important tools to engineer cell lines for high-yield production of recombinant proteins as well as to understand and treat misfolding diseases. About a third of cellular proteins are processed through the secretory pathway and destined to the extracytoplasmic space. Misfolding and aggregation of secretory proteins (which fold in the endoplasmic reticulum, ER) is a common theme in the production of recombinant proteins, particularly antibodies, as well as in the cellular pathogenesis of human diseases. Secretory proteins are targeted to the proteasome for degradation through the ERAD (ER-associated degradation) pathway. Intellectual merits: The outcome of this study is the development of fluorescent reporters engineered to function as ERAD sensors. Specifically, the research team rationally designed a series of artificial proteins that are expressed in the ER and that are targeted to ERAD. These engineered sensors of ERAD provide molecular tools to quantify protein degradation and are thus the first step in the development of high throughput screens for molecules that will enhance the intracellular degradation of misfolded proteins. Broader Impacts: This project provided a platform to integrate research and education at the graduate and undergraduate levels and to increase the diversity of the biotechnology field by providing mentoring and research opportunities to undergraduate and high school students.