Maintaining the proper three-dimensional structure, concentration, activity, and localization of proteins is a critical and constant challenge for all organisms. Dysregulated protein homeostasis is inextricably linked to disease states. Accordingly, the most prominent diseases of modern times—including neurodegenerative diseases like Alzheimer’s disease, diabetes, loss-of-function diseases like cystic fibrosis, many types of cancer, and even viral infections—are either caused directly by a failure to maintain protein homeostasis or reliant on innate cellular protein folding mechanisms. Proteome repair achieved by targeting the cellular mechanisms that regulate protein folding could transform the therapeutic options for broad swaths of protein folding-related disease. Critically, methods to intervene in a single important protein folding pathway could be applied to multiple, diverse pathologies.
The Shoulders Lab is developing and applying a chemical biology and small molecule-derived toolbox to probe the cell's protein folding network and manipulate it in defined ways. We employ a multi-disciplinary approach to understand how the cell remodels itself to address challenges to protein homeostasis, to elucidate the pathophysiology of protein folding-related diseases with poorly defined etiologies, and to target the biological processes we uncover for the development of first-in-class small molecule drugs. Brief overviews of a few of our projects are provided in the sections below.
Chemical Biology Method Development to Enable the Study of Metazoan Proteostasis
A major obstacle in our field is the paucity of small molecule-regulated techniques to achieve precise, researcher control of cellular proteostasis. To address this need, we have developed two strategies. In the first approach, we regulate the regulators in each subcellular compartment. For example, we developed a highly selective and potent small molecule-regulated genetic inhibitor of the transcription factor HSF1, which is the master regulator of cytosolic proteostasis. This method is opening doors to study how global modulation of cytosolic proteostasis network composition impacts a variety of protein folding issues, including protein aggregation in neurodegenerative disease.
In a second approach, we built on state-of-the-art Cas9-based genome engineering strategies to define the levels of any desired endogenous gene transcript across a wide dynamic range using small molecules (with Prof. Amit Choudhary’s group at Harvard Medical School). The resulting chemical control of Cas9 transcriptional activity enables selective perturbation of the levels of any one or more proteostasis network components, and is key to success in our other projects.
Collagen Folding, Misfolding, and Quality Control
Collagen is the molecular scaffold for multicellular life and by far the most abundant protein in the human body. Collagen presents a uniquely complex and still poorly elucidated proteostasis challenge to cells. Imbalances in collagen proteostasis are related to diverse, currently incurable diseases ranging from osteogenesis imperfecta (brittle bone disease) to fibrosis. Our approach focuses on illuminating molecular details of intracellular collagen folding and quality control, followed by using what we learn to identify potential new therapeutic strategies for the collagenopathies. We have built an array of biochemically amenable platforms to explore the collagen proteostasis problem. With new methods in hand, we have now: (1) Developed a customized proteomics workflow to map the proteostasis mechanisms that engage nascent collagen type-I, leading to the discovery of an unprecedented collagen-I post-translational modification (aspartyl hydroxylation) and providing key insights into collagen-I quality control pathways. (2) Pioneered a high-throughput, cell-based screen for modulators of collagen-I secretion that resulted in the identification of several drug classes that impact collagen-I proteostasis. (3) Demonstrated that the endoplasmic reticulum proteostasis network can be remodeled to resolve collagen-I production defects, a finding that may have translational implications for osteogenesis imperfecta, a skeletal disease that is prototypical of the collagenopathies.
Metazoan Glycobiology and New Proteostasis Network Functions
The unfolded protein response (UPR) is responsible for maintaining proteostasis in the endoplasmic reticulum (ER). Aside from its role as a protein folding and quality control factory, another important function of the ER is asparagine N-glycosylation, engendering diverse and important sugar modification patterns on proteins. Using our chemical genetic methods to probe proteostasis, we uncovered a previously unknown function of the UPR—regulating the molecular architecture of ectopically expressed secreted and endogenous cell-surface proteins by altering N-glycan maturation patterns. We are exploring the mechanistic origins of this observation, and its consequences for normal physiology, cancer, and innate immunity.
Protein Evolution, RNA Viruses, and Chaperones
Because the proteostasis network assists client proteins in attaining functional conformations, it is a primary determinant of the mutational landscape accessible to evolving proteins in biological settings (e.g., the work of Susan Lindquist, Dan Tawfik, and others). Of particular interest to our group, the rapid evolution of RNA viruses engenders high adaptability to environmental pressures (e.g., host immune systems). Understanding, predicting, and constraining RNA virus evolution requires a comprehensive picture of factors that define the accessible mutational landscape. Our data show that RNA viruses hijack host cell proteostasis mechanisms to assist the folding and trafficking of viral protein variants with sub-optimal biophysical properties, enabling more comprehensive exploration of the mutational landscape. The results are providing insight into systems beyond the HSP90 chaperone that is already known to have key roles in evolution.