Ning Zheng studies the molecular and structural mechanisms by which protein ubiquitination controls eukaryotic biology and human diseases.
Ubiquitin is a small protein ubiquitously found in all eukaryotes. It is used by the cell to covalently modify many other proteins in order to control their activities. Such modification, called ubiquitination, can signal proteins for rapid degradation or alter their biochemical functions and cellular destinations. Protein ubiquitination, which is extremely widespread, regulates essentially all biological functions, ranging from how fungi divide to how plants grow. It is also critical for many normal cellular processes in human cells. Its malfunction is closely linked to numerous human diseases, such as cancers, viral infections, and neurological disorders.
Our group is interested in understanding how protein ubiquitination and other biological processes are mediated by proteins and protein complexes that work together to achieve biochemical precision and temporal control. Protein x-ray crystallography, a technique we use, enables us to visualize the atomic structural details of how proteins function. Such detailed information will help us understand the underpinnings of living cells, the molecular basis of human diseases, and the working mechanism of potential therapeutic drugs. Our current research is focused on three overlapping areas: enzymatic complexes in protein ubiquitination, protein ubiquitination in plant biology, and the structural biology of membrane proteins.
Enzymatic Complexes in Protein Ubiquitination
Our group has a long history of studying how protein ubiquitination is promoted by a large family of cellular enzymes and enzymatic complexes known as ubiquitin ligases (E3s). By combining structural biology and proteomic approaches, we have identified a new family of human ubiquitin ligase complexes, the CUL4-DDB1 E3s, which play an essential role in cellular processes such as DNA replication and repair. These protein complexes are frequently hijacked by viral proteins to block cellular antiviral pathways. Our studies have not only demystified their actions but also suggested new strategies for antagonizing these viral proteins.
Like other modifications, protein ubiquitination is often reversible. Ubiquitin conjugated to a protein substrate can be cleaved or trimmed by a superfamily of enzymes known as deubiquitinases (DUBs). We have recently embarked on a series of studies of DUBs aimed at analyzing how these enzymes are regulated. By determining the crystal structure of the heterotetrameric histone H2B DUB module in the yeast SAGA complex, we were able to reveal the mechanism by which the yeast DUB protein Ubp8 assembles into an enzymatic complex. We unraveled the central roles of three Ubp8-binding proteins—Sgf11, Sgf73, and Sus1—in controlling the DUB activity of the deubiquitinase. This DUB module, remarkably, is also found in human cells, and its activity has been implicated in cancer.
Protein Ubiquitination in Plant Biology
Our studies on ubiquitin ligases have led us to the fascinating world of plants, which are the masters of using ubiquitin to control their physiology. Our journey started with the plant F-box protein TIR1 (transport inhibitor response 1), which was recently identified as the long-sought receptor for the plant hormone auxin. Chemically known as indole-3-acetic acid, auxin regulates almost every aspect of plant physiology. Upon sensing the hormone, the plant SCFTIR1 (Skp1-Cul1-F-box protein) ubiquitin ligase complex ubiquitinates and promotes the degradation of a family of transcription repressor proteins, thereby altering gene expression in plants. By determining a series of crystal structures of TIR1 in complexes with auxin compounds and a substrate peptide, we have revealed a striking "molecular glue" mechanism undertaken by the plant hormone to enhance ubiquitin ligase-substrate interaction. Our structure analysis of the TIR1 F-box protein serendipitously uncovered an inositol hexakisphosphate cofactor, which might regulate the SCFTIR1 E3 for auxin perception. Together, these results establish a new paradigm of protein ubiquitination and signal transduction, in which a ubiquitin ligase complex directly functions as a hub, perceiving not one but two small-molecule signals.
Since the mechanism of auxin sensing was elucidated, this new scheme of hormone perception by ubiquitin ligases has rapidly expanded in plant biology. The receptors of three other important plant hormones—jasmonate, gibberellin, and salicylic acid—have now been identified as multisubunit ubiquitin ligases. In 2010, we reported the crystal structure of the jasmonate receptor COI1 in complex with its substrate protein JAZ1 and jasmonate-isoleucine, the active form of the hormone. Based on our pharmacological, structural, and biochemical analyses, we found that COI1 and JAZ1 function as a coreceptor of the hormone, a mechanism also true for TIR1 and its substrate proteins. Moreover, our studies identified yet another new small molecule, inositol pentakisphosphate, which binds to and potentiates COI1 for hormone perception. By revealing how plants use these hormones and metabolic compounds to control their ubiquitin ligases, these studies point to an entirely new avenue for developing a novel class of protein-interaction-enhancing drugs targeting defective ubiquitin ligases associated with human diseases.
Just as our ancestors extracted numerous medicines from plants, we expect to learn many lessons from plants that will advance biomedical sciences. We also envision that the 21st century will witness a second Green Revolution, where modern biology approaches will be used on plants and microbes to develop new technologies for protecting the deteriorating environment, resolving the emerging food crisis, and exploring alternative energy sources. Our research program has now entered new territories in plant biology beyond protein ubiquitination.
Structural Biology of Membrane Proteins
Besides undertaking emerging challenges in the protein ubiquitination field, our group has also begun to explore the new frontier in structural biology—membrane proteins. Although many fundamental cellular functions are mediated by proteins embedded in the lipid bilayer, structural insights into the mechanism of their action are largely lacking.
In collaboration with William Catterall's group (University of Washington), we have recently determined the first crystal structure of a voltage-gated sodium channel that initiates action potentials in excitable cells and represents a family of important pharmacological targets. By revealing the atomic architecture of the selectivity filter, capturing the channel in a closed-pore conformation, and discovering a large central cavity of the pore module as a potential drug-binding site, our structure is a breakthrough in the ion channel field. We are continuing this line of research and extending our efforts to other membrane protein targets with high biomedical and agronomical relevance.
These studies are also supported by the National Institutes of Health, the Pew Scholar Program, the National Science Foundation, and the Burroughs Wellcome Fund.
As of March 11, 2016