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Research
Ubiquitination is a prevalent posttranslational modification in which the small protein ubiquitin covalently attaches to a target protein, thereby modifying the target’s biochemical properties. Additional protein-modification systems such as neddylation and autophagy play important roles in cell survival. During neddylation, proteins are conjugated to NEDD8, a protein that is highly conserved in most eukaryotes. Autophagy, or “self-eating,” is a catabolic process in which long-lived proteins and organelles are sequestered, degraded, and recycled. Many distinct autophagy-related (Atg) proteins are required for this process to occur; among these is a ubiquitin-like protein (UBL) called Atg8. Atg8 is unique among UBLs in that it is conjugated to a lipid, phosphatidylethanolamine. Because deregulation of neddylation or autophagy can contribute to neurodegenerative diseases and cancer, it is important that we understand the mechanism(s) by which these processes are catalyzed and regulated.
Attaching ubiquitin or a UBL to its target is a multistep process. First, the ubiquitin or UBL is activated by a ubiquitin activating enzyme (E1). Then, the ubiquitin or UBL is transferred to a ubiquitin-conjugation protein (E2). Finally, the E2 complex is bound to a ubiquitin protein ligase (E3). The UBL NEDD8 activates a family of approximately 300 ubiquitin E3s, thus triggering the ubiquitin-mediated turnover of many proteins. As such, NEDD8 functions as a major regulator of protein homeostasis, and the NEDD8 pathway has become a promising new target for anticancer therapies.
Like most ubiquitin-conjugation proteins, Ubc12, the E2 involved in neddylation, binds both E1 and E3. Ubc12 dictates a distinctive E3-dependent ligation of Nedd8 to Cul1, which involves the RING E3 ligase Rbx1 and the co-E3 ligase Dcn1. Brenda A. Schulman, PhD (Structural Biology, Tumor Cell Biology), and colleagues previously demonstrated that in yeast the interaction between Ubc12 and Dcn1 facilitates the transfer of Nedd8 from Ubc12 to cullin. Because this step accelerates the activity of the cullin–RING complex, which tags proteins to modify their function or promote their degradation, the investigators wanted to understand how human DCN1 and UBC12 interact. A major breakthrough was made when they realized that UBC12 is among the 35% to 50% of proteins in which an acetyl group is attached to methionine at the N-terminal end of the protein.
In a study published in Science1, Dr. Schulman’s team demonstrated that the acetylated methionine of Ubc12 is essential to the Ubc12-Dcn1 interaction. This research offers the first view of how N-terminal acetylation brings proteins together, the implications of which are potentially far reaching, given the prevalence of this modification. “The work presents a major new concept in protein-protein interactions,” said Dr. Schulman. “This raises the question of whether similar ‘keys’ on thousands of different proteins also unlock doors to allow them to function.” The group’s evidence included an X-ray crystallographic image showing Ubc12’s acetyl-methionine buried in a pocket on the surface of Dcn1 and lays the groundwork for the generation of small-molecule inhibitors to target the acetyl-methionine–binding pocket of Dcn1, a potent oncogene.
RING E3 ligases such as Rbx1 bind both a UBL-loaded E2 and a protein substrate targeted for UBL transfer. Earlier structural studies demonstrated how RING E3s bind E2s and recruit substrates; however, the mechanism through which the UBL transfer occurs was not known. Dr. Schulman’s team undertook studies to address how Rbx1 positions Ubc12 (E2) and Cul1 (substrate). In their report in Nature Structural & Molecular Biology2, the investigators discovered a new conformation of Rbx1 that places Ubc12 next to Cul1, where it is poised and ready for the UBL transfer. Thus, RING domain rotation may be a general mechanism for UBL transfer for the largest family of E3s.
The findings from this research provide the much-needed basic insight into the precise molecular events necessary for proper UBL transfer to target proteins. Given the importance of the NEDD8 pathway in maintaining protein homeostasis and the number of disease processes that result from its deregulation, findings such as these may facilitate the development of novel therapies that target this pathway. “I believe that determining the mechanisms by which enzymes transfer UBLs will be of broad importance, much like studies of protein kinases have influenced our knowledge of signaling pathways and their roles in diseases,” said Dr. Schulman.
1Scott DC, Monda JK, Bennett EJ, Harper JW, Schulman BA. N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex. Science Nov 4;334(6056):674-8, 2011. Epub 2011 Sep 22. PubMed PMID: 21940857; PubMed Central PMCID: PMC3214010. Abstract | Full Text
2Calabrese MF, Scott DC, Duda DM, Grace CR, Kurinov I, Kriwacki RW, Schulman BA. A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases. Nat Struct Mol Biol Jul 17;18(8):947-9, 2011. doi: 10.1038/nsmb.2086. PubMed PMID: 21765416; PubMed Central PMCID: PMC3245743. Abstract | Full Text
Photo: Daniel Scott, PhD
