The proteasome: a magnificent example of cellular nanotechnology
Many intracellular processes are conducted by large multiprotein machines. These machines frequently consist of many unique yet highly similar polypeptide subunits. Yet, each subunit must typically occupy a distinct position within the final complex for its proper function. This is especially true of the proteasome, a 66+ polypeptide ATP-dependent protease complex responsible for most intracellular protein degradation. Many proteins fulfill their function and are no longer needed, and must be removed from the cell to maintain homeostasis; other proteins become damaged in the course of their lifetimes and need to be destroyed to prevent them from accumulating in the cell and becoming toxic.
These proteins to be degraded are typically marked with a chain of the small protein ubiquitin, which serves as a flag for delivery to the proteasome. The proteasome then recognizes the polyubiquitin signal, removes it, unfolds the protein substrate using mechanical force derived from ATP, and cleaves the protein into short peptides that are eventually recycled into new proteins. Protein homeostatis is frequently disrupted in human diseases, and can cause or exacerbate cancer, neurodegenerative disorders, and diabetes, among others. By better understanding the proteasome's structure and function, we aim to identify novel points of intervention in its assembly and function that can be modulated to improve human health.
Assembly and function of the 26S proteasome
With 66+ individual protein components, a practically iimitless number of possible assembly sequences could be utilized to arrive at the final complex. Yet, only a handful or fewer appear to ever be followed in the cell. How is this remarkable complexity reduced and managed to assure that a properly composed, functional complex is always the endpoint? We are interested in understanding the molecular and cellular mechanisms that enforce this ordered, hierarchical assembly of the proteasome in vivo. We use a combination of biochemistry, biophysics, protein engineering, and yeast genetics and cell biology to better understand this process. Understanding proteasome assembly provides basic insight into how large macromolecular structures are formed inside the cell, and could elucidate novel constriction points in assembly that could be targeted, either to disrupt or augment proteasome assembly, or perhaps even to alter the final subunit composition!
Modulation of the proteasome in human cells
Small molecule inhibitors of proteasomal proteolytic activity (bortezomib/Velcade, carfilzomib/Kyprolis) have been extremely successful in the treatment of several human cancers, such as multiple myeloma and mantle cell lymphoma, and have shown promise against several types of solid tumors. However, these inhibitors have a poor therapeutic window, unpleasant routes of administration, and can cause severe dose-limiting side effects. Further, mutations that cause resistance to these drugs are increasingly a problem in the clinic. We are interested in finding novel ways to perturb the assembly or function of the proteasome in human cells, with the hypothesis that alternative routes to modulate proteasome activity will be just as effective as or even synergistic with conventional inhibitors in cancers. In fact, these adjunct approaches may be useful in cases where resistance to bortezomib or carfilzomib have occurred.
Quality control of the proteasome, a model multisubunit complex
Cells have evolved highly effective mechanisms to identify and remove damaged or misfolded proteins. However, many proteins function in the context of multisubunit complexes. In addition to the usual risks of translational errors or misfolding, these proteins could potentially also misassemble into a higher-order complex. It only takes a single defective subunit to poison the entire complex, either stalling its formation or yielding a nonfunctional or even toxic end product. Yet, we know very little about how the cell enforces the quality of multisubunit complexes. This is all the more surprising, considering that multisubunit machines mediate many of the most basic, essential life processes.
We are interested in identifying the cellular pathways, mediators, and determinants of multisubunit complex quality control, using the proteasome as a model. The proteasome is an exceptionally good model for this process because it contains subunit architectures found in many other multiprotein machines, and its structure is known in near-atomic detail. This allows us to combine the power of targeted and high-throughput yeast genetics with structure-guided protein engineering approaches to rapidly elucidate how defective or damaged multisubunit complexes are recognized and processed in cells.