The EMBO Meeting 2013

University of Washington, Seattle

How RING Ubiquitin Ligases Work

The attachment of one or more ubiquitin (Ub) molecules onto cellular proteins constitutes one of the major regulatory processes in eukaryotic cells. Three enzyme activities known as E1 (Ub-activating), E2 (Ub-conjugating), and E3 (Ub ligase) are required. A given genome encodes for only one or several E1s, dozens of E2s, and hundreds to thousands of E3s. RING and RING-like ubiquitin ligases (E3s) constitute the vast majority of known E3s and are implicated in numerous cellular processes. In light of their importance in human physiology and disease, an understanding of how E3s work is paramount. Lacking an active site, the mechanism by which RING-type E3s activate transfer of ubiquitin has been enigmatic. Our recent structural and biochemical studies on the RING E3 BRCA1/BARD1 and the U-box E3, E4B, provide insights that are generalizable to the entire family of enzymes. In addition, our discovery of an unexpected property of the E2 UbcH7 has led to new insights regarding a sub-class of RING E3s known as the RING-Between-RINGs (RBRs). Comparison of canonical RING E3s and RBR E3s reveal new understanding into how each class of enzyme works.

Biography

Rachel Klevit is Professor of Biochemistry, Adjunct Prof. of Pharmacology and Chemistry, and Director of the Molecular Biophysics Program at University of Washington. She earned a D.Phil. in Chemistry from Oxford University studying with Prof. R. J. P. Williams astext one of the earliest female Rhodes Scholars. She was an American Cancer Society post-doctoral fellow at Duke University. Her early use of 2DNMR to study proteins was recognized by awards such as The Biophysical Society's Dayhoff Award and The Protein Society's DuPont Young Investigator Award. Dr. Klevit was elected as an AAAS Fellow in 1998. She leads a research team that uses a combination of structural biology and biochemical approaches to study fundamental questions of protein ubiquitination. Her group has solved important structures including the RING E3 BRCA1/BARD1, the UbcH5/Ub complex, E2~Ub conjugates, and a ternary E3/E2~Ub complex. Dr. Klevit also studies human small heat shock protein structure and function.

The Gurdon Institute & University of Cambridge, Cambridge

Assembly and disassembly of protein complexes at sites of DNA damage

Despite its inherent stability, the DNA double-helix is subject to various forms of damage, with each human cell sustaining tens of thousands of DNA lesions per day. To combat this threat to genome stability, cells have evolved elaborate ways to detect, signal the presence of and repair DNA damage. The importance of such processes is highlighted by inherited or acquired defects in them being associated with various human pathologies, including immune-deficiencies, neurodegenerative diseases and various forms of cancer1. Moreover, our increasing knowledge of cellular DNA-damage responses is providing exciting opportunities for developing novel classes of drugs to treat cancer and other age-related diseases1,2. Work in my laboratory aims to decipher the mechanisms by which cells respond to the most toxic of all DNA lesions – DNA double-strand breaks (DSBs) – with much of our work addressing the generation and molecular functions of post-translational modifications (PTMs) on specific DSB-responsive proteins3,4. In this talk, I will highlight some of our recent work showing how the PTMs sumoylation, ubiquitylation and phosphorylation control important molecular transitions in DDR-protein complex assembly and disassembly, and how such events promote genome stability and thus guard against cancer and other age-related diseases.

Biography

Steve Jackson is the University of Cambridge's Frederick James Quick Chair of Biology, and is Senior Group Leader and Head of Cancer Research UK Labs at the Gurdon Institute in Cambridge, UK. His research – which involves mammalian cell and molecular biology, biochemistry and yeast genetics – has been instrumental in shaping our understanding of how cells respond to DNA damage and how defects in these responses contribute to human disease. Much of Steve's current work focuses on understanding how DNA-damage responses are controlled by protein phosphorylation, ubiquitylation and sumoylation. Steve has received various prizes, including most recently the Royal Society Buchanan Medal (2011). He is an elected member of EMBO, the Academy of Medical Sciences (UK) and the Fellowship of the Royal Society. In 1997, Steve founded KuDOS Pharmaceuticals Ltd to develop drugs to interfere with DNA repair in ways such that they kill cancer cells but not normal cells by "synthetic-lethality" mechanisms. Recently, he established a new company, MISSION Therapeutics to exploit additional drug-discovery and therapeutic opportunities arising from his work.

Genentech & University of California, San Francisco

Tumor Suppressor role for the deubiquitinase, BAP1

Deubiquitinating enzyme BAP1 (BRCA1-associated protein is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. While the Drosophila ortholog Calypso deubiquitinates histone H2A as part of the polycomb complex that represses transcription of developmental regulators, only mammalian BAP1 has the binding motif that engages the transcriptional co-factor HCF-1 (Host Cell Factor 1) and therefore might form additional complexes. Here we use knock-in mice expressing BAP1 with a C-terminal 3xFlag epitope tag and mass spectrometry to define the endogenous BAP1 interactome in various tissues. We also investigate the normal physiological role of BAP1 using Bap1-deficient mice. Bap1 gene deletion was lethal during embryo development, but systemic deletion in adult mice caused splenomegaly and many of the features of human myelodysplastic syndrome (MDS), including neutrophilia, monocytosis, thrombocytopenia, anemia, and an expanded hematopoietic stem cell-enriched lineage- ScaI+ Kit+ (LSK) population. Subsequent sequence analysis of bone marrow aspirates from de novo human MDS patients identified a novel somatic, mutation in Bap1 within the catalytic domain, implying that Bap1 loss of function has similar consequences in mouse and man. By comparing ChIP-sequencing data, obtained using BAP1.3xFlag KI cells to map BAP1 complex binding sites, and RNA sequencing data that identified genes dysregulated in BAP1-deficient cells, we identified a list of potentially critical BAP1-regulated genes that included the Il7r gene, a known regulator of hematopoietic cell survival that is decreased in clinical MDS samples.

Biography

Vishva Dixit has conducted pioneering studies defining the biochemical framework of the cell death and pro-inflammatory pathways. His laboratory was the first to: i) show that caspases are components of the death receptor-induced apoptotic pathway; ii) demonstrate that death receptors signal by an entirely novel mechanism of recruiting and activating a death protease (FLICE/caspase-8) by an induced proximity mechanism; iii) identify the mammalian death protease equivalent to the CED3 protein in worms (YAMA/caspase-3) as well as other pro-apoptotic caspases including caspase-6,-7 and -9; iv) identify receptors and signaling pathways engaged by TRAIL, a cytokine that preferentially kills transformed cells; v) show that the death domain-containing molecule MyD88 is a key adaptor in IL-1 signaling; vi) discover paracaspases and metacaspases: two ancient families of caspase-related proteins, one of which plays a key role in MALT lymphoma; vii) confirm that NOD proteins that possess a death-fold are critical components of the inflammasome complex.