Our passion is to understand the basic biology of protein homeostasis and how it relates to major human diseases. Three questions motivate our research: (1) How do cells maintain protein homeostasis? (2) How do changes in protein homeostasis lead to pathology? (3) Can modulation of protein homeostasis be used to treat disease?
Protein homeostasis generally refers to the posttranslational mechanisms that maintain a normal cellular repertoire of functional proteins. It has become increasingly clear over the past decade that protein homeostasis is critical to the health of cells and organisms. Defects in protein homeostasis underlie diseases that afflict millions of people, including cancer and neurodegenerative diseases. Gaining a deeper understanding of protein homeostasis will shed light on how these diseases develop, which in turn may lead to new methods of diagnosis and therapy.
The major effectors of protein homeostasis include factors that mediate protein folding, assembly, and degradation. We are particularly interested in mechanisms that mediate protein degradation. Cells are constantly turning over proteins, making room for new ones. Within cells, the vast majority of protein degradation is carried out by the ubiquitin-proteasome system (UPS) (Figure 1). Proteins slated for degradation by the UPS are first tagged with the protein ubiquitin by enzymes referred to as ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). The ubiquitin tag is subsequently recognized by the proteasome, which is a large proteolytic complex that binds ubiquitin tags and degrades the protein to which the tag is attached.
Protein degradation via the UPS serves two general functions (Figure 1), both of which we are studying. The first function is to mediate protein quality control. Proteins that fail to fold or assemble are degraded by the UPS shortly after their synthesis. Five to 15 percent of newly synthesized proteins fail to attain a mature conformation; their degradation is initiated during or shortly following synthesis. This represents a major load on the UPS, and mutations that perturb this process lead to neurodegeneration. The second major function of the UPS is to mediate the degradation of regulatory proteins that control crucial cellular processes. This includes degradation of cell cycle control proteins (e.g., cyclins and Cdk inhibitors), transcription factors (e.g., Myc), and checkpoint control proteins (e.g., p53). Hundreds of proteins that control almost all aspects of cellular and organismal biology are controlled by the UPS, and multiple mutations that perturb this regulatory function have been identified as root causes of cancer.
The breadth of action of the UPS in regulating protein homeostasis and eukaryotic biology is enabled by the sheer complexity of the system. More than 1,000 genes encode proteins that mediate the conjugation, perception, or removal of ubiquitin signals. Of these, ubiquitin ligases comprise the largest group, with more than 500 encoded in the human genome (Figure 1). One of our major efforts is to investigate the biggest family of ubiquitin ligases, known as cullin-RING ubiquitin ligases (CRLs), which we codiscovered more than 15 years ago. CRLs are key regulatory enzymes and are the target both of anticancer drugs and of mutations that predispose to cancer. Our broad range of approaches draws on biochemistry, mechanistic enzymology, biophysics, chemical biology, quantitative proteomics, molecular genetics, and systems biology to study how members of the CRL family are assembled, how they work, how their activity is controlled (Figure 2), and what they do. Given the major regulatory impact of CRL enzymes, achieving a deep understanding of this family will have a broad impact on our knowledge of basic cell biology of both normal and diseased cells.
Once ubiquitin tags are attached to a protein by CRLs and other ubiquitin ligases, ubiquitin receptors interpret the signal to effect a specific outcome. A prominent (but not the only) outcome is the degradation of the modified protein by the proteasome. Ubiquitin receptors that act between the CRLs and the proteasome include the ATPase p97/VCP (Figure 3) and its extensive network of adaptor proteins. The p97-adaptor complexes bind directly to ubiquitin ligases and to ubiquitin-modified substrates and can carry out further processing of the ubiquitin modification. For reasons that remain unknown, p97 is essential for the degradation of some but not all proteasome substrates, including both quality control and regulatory substrates. One hypothesis is that p97 assists the proteasome by extracting ubiquitin-modified proteins from larger structures and unraveling them, so that they can be fed into the proteasome (Figure 3). Using the range of approaches mentioned above for CRLs, we seek to understand what p97 does, how its activity is regulated, and how it specifically selects its substrates. To assist our studies, we have developed small molecules that inhibit the activity of p97. In 2014, a derivative of one of these molecules entered human clinical trials for cancer therapy, which illustrates how our fundamental investigations of the UPS and its enzymes can be translated directly into medicine.
Once p97 has acted upon a substrate, it can be degraded by the proteasome. There is much we do not understand about the mechanics of this process. We seek to develop new assays, methodologies, and tools—including novel small-molecule inhibitors—that will enable us to dissect the mechanism of proteasome activity and its regulation.
Grants from the National Institutes of Health provide support for the work on the mechanism and regulation of cullin–RING ubiquitin ligases.
As of October 3, 2014