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Molecular Mechanisms of Gene Silencing and Polyubiquitin Assembly
Summary: Cynthia Wolberger's research is focused on the molecular mechanisms by which eukaryotic genes are silenced by Sir2 proteins. She also studies the assembly, disassembly, and recognition of linkage-specific polyubiquitin chains.
Transcriptional Regulation by Sir2 Enzymes
The DNA in eukaryotic cells is packaged by histone proteins, giving rise to a nucleoprotein complex called chromatin. Some regions of chromatin are relatively accessible to the transcriptional machinery and contain actively transcribed genes; other regions are silenced and are organized in a way that prevents transcription. We study the enzymes and protein complexes that establish and maintain transcriptional silencing. Our focus is on Sir2, an enzyme that deacetylates lysine side chains in an unusual reaction that requires NAD+.
Enzymes similar to Sir2 are found in virtually all organisms, where they are involved in a variety of critical biological processes, including transcriptional silencing, DNA repair, chromosome stability, and enzyme regulation. Sir2 proteins play an intriguing role in regulating life span, with high levels of Sir2 activity extending life span and low levels of Sir2 activity leading to shortening of life span in yeast, worms, and flies. Members of the Sir2 enzyme family, also known as sirtuins, also regulate important pathways in humans that are involved in protection of neurons from degeneration, fat mobilization, and cell death. The activity of Sir2 is linked to cellular concentrations of NAD+, a substrate of Sir2, as well as to levels of nicotinamide, a reaction product that inhibits Sir2 activity. Some Sir2 enzymes catalyze a somewhat different reaction in which NAD+ is cleaved to produce ADP-ribose, which is then attached to a substrate in a reaction known as mono-ADP ribosylation. Understanding the chemistry and regulation of Sir2 proteins, as well as the basis for the dual enzymatic activity, is central to unraveling how these enzymes help to regulate different cellular pathways.
We have determined structures of Sir2 enzymes bound to a variety of substrates and products, as well as in complex with reaction intermediates. Each of these structures has provided a snapshot of the reaction, beginning with the binding of sirtuins to acetylated peptide and NAD+, through the release of the products nicotinamide, O-acetyl ADP-ribose, and the deacetylated peptide. Structures with a variety of peptide substrates have shown how interactions between the enzyme and the substrate residues flanking the acetyl-lysine influence the peptide substrate specificity of the enzyme. We have also determined structures of Sir2 enzymes bound simultaneously to both peptide and NAD+ before catalysis, as well as after the enzymatic reaction has occurred in the crystal.
These structures have shed light on the unique mechanism by which Sir2 enzymes catalyze the cleavage of NAD+ and the transfer of an acetyl group from the peptide lysine to ADP-ribose, as well as how these enzymes are regulated by the metabolite nicotinamide. Recently we succeeded in trapping a covalent intermediate bound to a sirtuin. The structure of this complex revealed how side chains within the active site promote the initial cleavage of NAD+ and rearrange to promote subsequent steps in the reaction. Together these structures have provided insights into the unusual NAD+-dependent deacetylation reaction of sirtuins. These mechanistic studies are also shedding light on how a subset of sirtuins catalyze a second reaction: the transfer of the ADP-ribose portion of NAD+ to a protein substrate, a reaction that is known as ADP ribosylation. Using mass spectrometry and proteomics approaches, we have pinpointed the determinants for ADP ribosylation and identified the modified side chains. Our ongoing studies of sirtuins are focused on identifying in vivo substrates for both deacetylation and ADP ribosylation.
In some organisms, the transcription of genes that lie near the ends of chromosomes—known as telomeres—is subject to silencing mediated by Sir2 enzymes. This type of transcriptional repression, known as telomeric silencing, requires additional proteins that help recruit Sir2 enzymes. We have determined the structure of the yeast protein Rap1, which recruits Sir2 and other silencing proteins to telomeres, and we have carried out extensive genetic studies of Rap1 mutants to investigate the role of Rap1 in the different types of silencing in yeast and in telomere length maintenance. These studies have revealed unexpected differences between telomeric silencing and mating-type silencing, where Rap1 was previously thought to play a role identical to that at telomeres.
Linkage Specificity of Polyubiquitin Chains
Ubiquitin is a small protein that serves as the building block for chains of polyubiquitin, which are attached to other proteins and thereby mediate a variety of biological processes. Polyubiquitin is formed when the C terminus of one ubiquitin is joined to one of the seven surface lysines of ubiquitin. Polyubiquitin chains with different types of lysine linkages play distinct biological roles. For example, Lys48-linked polyubiquitin chains target proteins for destruction by the proteasome, whereas Lys63-linked polyubiquitin chains are nondegradative signals that play a role in DNA damage tolerance and NFκB activation.
We study the molecular basis for Lys63-linked polyubiquitin chain assembly and how these chains are attached to substrates. Lys63-linked chains are assembled by a specialized heterodimer that helps to catalyze formation of a covalent bond between Lys63 of one ubiquitin and the C terminus of the next. We determined the structure of the yeast enzyme Mms2/Ubc13, as well as a complex containing ubiquitin covalently joined to the active-site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Ub~Ubc13/Mms2 complex to the acceptor-binding site of Mms2/Ubc13 in an adjacent complex allowed us to visualize the molecular determinants of acceptor-ubiquitin binding and explain how Mms2 helps to orient ubiquitin to give rise to a Lys63-linked polyubiquitin chain. We continue to study how Mms2/Ubc13 cooperates with the ubiquitin ligase Rad5 to attach Lys63-linked polyubiquitin chains to PCNA (proliferating cell nuclear antigen), which is a required step in the switch to error-free DNA repair in yeast.
Since linkage type determines the functional consequence of modification with polyubiquitin, there must be structural differences between different chain types that can be distinguished by the cell. We determined the structure of Lys48-linked tetraubiquitin, which is the first to show the full, ordered structure of this chain, and have recently determined structures of Lys63-linked chains. These two chains adopt distinct conformations that could be distinguished by ubiquitin-binding proteins that are sensitive to linkage type. Our ongoing studies are aimed at determining structures of proteins that bind preferentially to one or the other type of polyubiquitin chain, including enzymes that only cleave one type of polyubiquitin linkage.
Portions of this work have been supported by the National Science Foundation and the National Institutes of Health.
Last updated: September 24, 2008
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