Intellectual merit. Most cellular events are controlled by precise regulation of protein expression. Termination of protein expression is commonly achieved by moving proteins into cellular recycling centers, where they are broken down into amino acids that can be reutilized in new protein synthesis. Entry into these recycling centers is tightly regulated. Proteins that are turned-over inside proteasomes, barrel-shaped recycling centers in the cytoplasm, need a special modification that serves as a key or signal for entry. This "key" is a small sequence of amino acids, named ubiquitin, which is linked or ligated to proteins by a class of regulatory proteins known as ubiquitin ligases. Ubiquitin ligases thus control protein expression by their ability to recognize and tag other proteins for destruction. Ubiquitin ligases have multiple substrates and thus commonly impact multiple key cellular pathways, including regulation of the cell cycle, programmed cell death, and the integration of signaling pathways during development. Uncovering the function of ubiquitin ligases is essential to our understanding of all cellular processes.
Most ubiquitin ligases are soluble proteins that ubiquitinate cytoplasmic proteins. This research will focus on identifying the proteins tagged for turnover by a specific ubiquitin ligase, an endosomal membrane protein that is stabilized and moves to the inner nuclear membrane when cell signaling pathways under the outer cell membrane are activated. To identify proteins tagged by this ligase, experiments will compare the protein composition of purified inner nuclear membrane when the ligase is active and when its expression is knocked down. Proteins that escape ubiquitin-mediated degradation and thus increase in quantity in the inner nuclear membrane fraction when expression of the ligase is knocked down will be identified by mass spectrometry. Identification of ligase substrates correlated with specific nuclear functions will indicate a role for the endosomal ubiquitin ligase in nuclear regulation.
Broader impacts: These studies will serve as a vehicle for educating undergraduates, graduate students, and postdoctoral fellows, including members of under-represented groups. The project is based on observations initially obtained by a graduate student. The studies were confirmed and expanded by a postdoctoral fellow. The principal investigator has and will continue to train undergraduate chemistry and biology majors, who either elect research for course credit or spend the summer in the lab with REU grant support. The undergraduates attend lab meetings, have their own projects, and are included on publications. All trainees not only master new techniques but, more importantly, learn to analyze data, to propose creative solutions to problems, and to summarize and present data in a professional format. Mentoring undergraduates is excellent training for the graduate students and postdocs in the lab, teaching them to teach, to organize and schedule, and to encourage others.
Cells that experience stress due to changes in their environment modify their behavior to ensure their survival. Rapid changes in cellular behavioral programs, such as migration or division, are achieved by altering the half-life of proteins that regulate the expression, or transcription, of genes. These regulators of gene expression are concentrated in the nucleus, under the nuclear membrane in a region of structural elements known as the nuclear lamina. One protein that can change the half-life of other proteins is Ring Finger Protein 13. RNF13 is a cellular assassin that targets proteins for destruction by marking them with the peptide ubiquitin, a tag that enables their entry into cytoplasmic recycling centers known as proteosomes. RNF13 normally resides in the outer membranes of cytoplasmic vesicles, but when cells experience stress, RNF13 is upregulated and recruited to the nuclear lamina. In order to understand how cells respond to stress, it is important to identify the "victims" of RNF13 in the nuclear lamina. The goal of our study was to learn which proteins are turned-over by RNF13-mediated ubiquitination when cells are stressed. Using mass spectrometry, we have identified almost 100 proteins that specifically decrease in concentration when RNF13 expression is increased and RNF13 is moved to the nuclear lamina. Many of these target proteins regulate transcription. We have validated the turnover of three model proteins using an alternative approach, polyacrylamide gel elecrophoresis, to determine target protein concentration. Our data suggest that in response to stress, RNF13 ubiquitinates multiple nuclear proteins, which leads to a decrease in their quantity within cells, and so modifies gene expression. Our findings direct future studies which will assay for direct binding of RNF13 that leads to ubiquitination of these putative substrates. Only by understanding how cells modulate gene expression on the molecular level will it become possible to intervene and change cellular programs, blocking, for example, the migration of a cancer cell to a new tissue during the process of metastasis.
