Global analysis of protein turnover

A central focus of the lab is the development of novel methodologies for system-wide analysis of protein homeostasis. Specifically, we are developing proteomic techniques that can measure the kinetics of protein formation and degradation on proteome-wide scales. We are using these approaches to assess the impact of protein aggregate accumulation on proteome dynamics in models of aging and neurodegenerative diseases. It is hoped that these proteomic screens will identify novel molecular pathways involved in age-dependent neurodegenerative diseases.

Substrate selectivity in macroautophagy

In eukaryotic cells, macroautophagy is the principal catabolic pathway for the degradation of long-lived proteins. The mechanism of macroautophagy involves the sequestration of a portion of the cytoplasm in double-membraned vesicles (autophagosomes) and subsequent degradation upon fusion with the lysosome. Macroautophagy is constitutively active under basal (nutrient rich) conditions and upregulated during periods of starvation. Although macroautophagy has historically been considered a non-selective degradation pathway, recent evidence indicates that organelles and proteins can be specifically targeted for autophagic degradation. Notably, macroautophagy appears to play a key role in the degradation of damaged and misfolded proteins and its inhibition has been associated with a number of neurodegenerative disorders. We are seeking to develop novel proteomic approaches to investigate two features of the selectivity of macroautophagy. First, we are identifying and characterizing subsets of the proteome that are preferentially targeted by macroautophagy as part of their constitutive turnover cycle. Second, we are determining the mechanism by which damaged proteins are selectively targeted by macroautophagy.

Global analysis of protein folding efficiencies

Most research on the folding behavior of proteins is conducted within cell-free systems. These in vitro experiments can outline the basic physical principles of protein folding but often fail to capture the inherent complexity of the cellular environment where natural proteins must attain their native conformations. In the crowded environment of the cell, a number of molecular chaperone families play vital roles in promoting efficient protein folding and minimizing protein aggregation. However, despite the sophistication of the cellular chaperone machinery, in vivo protein folding is not perfectly efficient. A significant percentage of native proteins (estimated to be between 5% and 30%) never attain their native conformation and are instead targeted for degradation by the cellular quality control pathways immediately following synthesis. Little is known about variations in folding efficiencies of individual proteins and how biological and physical properties of proteins influences their propensity to fold successfully within a cell. We are developing novel quantitative methodologies for proteome-wide analyses of in vivo protein folding. These approaches utilize in vivo stable isotope labeling and mass spectrometry to quantitate relative levels and rates of flux for nascent and native proteins on a global scale. Using these methodologies, we are investigating the relationship between in vitro folding properties of proteins and in vivo folding efficiencies.

The mechanism of prion propagation and pathogenesis

Most proteins populate a single, ordered three-dimensional structure in solution. However, a subset of proteins, termed prions, have the capacity to fold into alternative self-replicating conformations, enabling a single polypeptide sequence to be associated with multiple phenotypes. The ability of prions to fold into diverse meta-stable, self-seeding conformations facilitates two protein-based functionalities that were previously thought to require the involvement of nucleic acid-based genetic elements: infectivity and heritability. Although historically associated with a group of neurodegenerative disorders in mammals, prions have now been discovered in a number of organisms and have been implicated in the regulation of non-pathogenic biological activities including translation, transcription, mating compatibility and memory. Current projects in the lab are seeking to understand the cellular mechanisms of prion propagation and pathogenesis - and to identify effective therapeutics against prion diseases.

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