Michael Rape is interested in understanding principles of ubiquitin-dependent signaling and the roles of this posttranslational modification during cell division and differentiation. His work bridges the identification of critical ubiquitylation enzymes in cancer cells and embryonic stem cells with a biochemical dissection of the mechanisms that underlie the modification of important substrates. His studies provide insight into reactions that are required for normal development and whose misregulation causes several diseases, most notably cancer.
Posttranslational modification of proteins with the highly conserved ubiquitin, a reaction referred to as ubiquitylation, is essential for cell division, differentiation, and survival in all eukaryotes (Figure 1). During ubiquitylation, the carboxy-terminus of ubiquitin is attached to a substrate lysine, resulting in the formation of a covalent isopeptide bond. Transfer of a single ubiquitin often results in changes in substrate interactions, localization, or activity. The substrate-attached ubiquitin is, however, frequently modified with additional molecules to generate polymeric ubiquitin chains. Dependent on the attachment site used for chain formation, these chains adopt different structures and result in distinct consequences for the modified protein: K11- and K48-linked ubiquitin chains trigger substrate degradation by the proteasome, whereas K63-linked chains act as molecular glue, holding together large complexes that mediate DNA repair or NF-kB transcription factor activation. In my laboratory, we are interrogating the assembly and function of specific ubiquitin marks, dissecting their roles in proliferation and differentiation, and devising methods to alter ubiquitin-dependent signaling by small molecules. In short, we are attempting to decipher the ubiquitin code and reveal its roles in proliferation and differentiation.
Rape Research Abstract Slideshow
Figure 1: Posttranslational modification with ubiquitin is essential for cell division and differentiation in all eukaryotes. Our lab focuses on these examples of different cell cycle reactions that are controlled by ubiquitin.
From the Rape lab
Figure 2: The Cul3 ubiquitin ligase is an essential regulator of stem cell morphology.
(A) Depletion of Cul3 results in striking morphological changes in mouse embryonic stem cells. (B) Cul3 and its substrate adaptor Klhl12 regulate the size of emerging COPII coats. An immunofluorescence analysis of COPII structures with or without an increase in the expression levels of the Cul3-Klhl12 ubiquitin ligase is shown. Cul3-Klhl12 promotes the monoubiquitylation of Sec31, a component of the COPII-coat. This reaction allows an increase in the size of COPII structures and enables stem cells to export collagen, a large cargo molecule that is important for the maintenance of stem cell morphology.
Achim Werner and Lingyan Jin. See also Jin L. et al. 2012 Nature 482:495–500.
Interrogating the Code: Assembly and Function of Specific Ubiquitin Modifications
Ubiquitylation requires a cascade of at least three enzymes, referred to as E1, E2, and E3. The human genome encodes two E1 enzymes with specificity for ubiquitin, ~40 E2s, and more than 600 E3s. E3s recruit specific substrates and activated ubiquitin, thereby promoting the modification of substrates with a particular ubiquitin mark. The biological outcome of ubiquitylation, therefore, depends on the substrate and linkage specificity of an E3-dependent ubiquitylation complex.
By analyzing products of an essential E3, the anaphase-promoting complex (APC/C), we discovered the function of an atypical chain linked through Lys11 of ubiquitin. We could show that K11-linked chains are specifically formed during cell division, consistent with their role in allowing cells to rapidly progress through mitosis. Using these novel chains as our model, we have uncovered key principles of linkage-specific chain formation, the reaction that allows cells to implement the ubiquitin code. We could show that rapid chain formation relies on substrate motifs, referred to as initiation motifs, which promote transfer of the first ubiquitin to a substrate lysine. Chain initiation is the rate-limiting step in K11-linked chain formation, and the responsible E2 Ube2C (also known as UbcH10) is tightly regulated. The short chains appended to a substrate by the APC/C and Ube2C are then rapidly extended by Ube2S, an APC/C-specific E2 that we discovered. By using an integrated approach combining structural, biochemical, and bioinformatic techniques, we could show that Ube2S achieves its linkage specificity by substrate-assisted catalysis, during which critical residues of the catalytic center are contributed by Ube2S and the ubiquitin molecule that contains the target lysine residue.
Our current work focuses on those aspects of the ubiquitin code that remain poorly understood, concentrating on three main questions:
- What are the mechanisms of ubiquitin chain elongation? We are particularly interested whether enzymatic requirements for assembling long chains are different from those leading to formation of the first linkage.
- What are functions of K11-linked chains? To answer this question, we have developed a novel technique, E3 reprogramming, that allows us to change the linkage specificity of a ubiquitylation enzyme in cells.
- What are functions of other uncharacterized linkages? To this end, we are searching for E3s with novel linkage specificity.
As of March 23, 2016