Francisca E. Reyes-Turcu

Regulation and Cellular Roles of Ubiquitin-Specific Deubiquitinating Enzymes

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin(-like) protein, and remodel polyubiquitin(-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five gene families. Most DUB activity is cryptic, and conformational rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multiprotein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have led to new insights into the function of yeast and human DUBs. This review discusses ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes.

Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63‐linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48‐linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin‐binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF‐κB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.

RNA Elimination Machinery Targeting Meiotic mRNAs Promotes Facultative Heterochromatin Formation

Making Heterochromatin Heterochromatin is a particularly compact DNA-protein assembly that can repress gene expression. Constitutive heterochromatin is found, for example, at centromeres and the subtelomeric regions. Working with Schizosaccharomyces pombe, Zofall et al. (p. 96, published online 1 December) examined the heterochromatin islands often found near meiotic genes, which are maintained in a silenced state during vegetative growth. Heterochromatin formation at these loci did not generally involve the RNA interference machinery, as is observed at centromeres, but did require transcription. The exosome, an RNA-degrading machine, was involved in the formation of the heterochromatin islands, which could be remodeled in response to sexual differentiation. RNA processing factors regulate the assembly of heterochromatin at individual gene loci in fission yeast. Facultative heterochromatin that changes during cellular differentiation coordinates regulated gene expression, but its assembly is poorly understood. Here, we describe facultative heterochromatin islands in fission yeast and show that their formation at meiotic genes requires factors that eliminate meiotic messenger RNAs (mRNAs) during vegetative growth. Blocking production of meiotic mRNA or loss of RNA elimination factors, including Mmi1 and Red1 proteins, abolishes heterochromatin islands. RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. Heterochromatin islands disassemble in response to nutritional signals that induce sexual differentiation. This process involves the antisilencing factor Epe1, the loss of which causes dramatic increase in heterochromatic loci. Our analyses uncover unexpected regulatory roles for mRNA-processing factors that assemble dynamic heterochromatin to modulate gene expression.

Polyubiquitin binding and disassembly by deubiquitinating enzymes.

Ubiquitin (Ub) is a highly conserved protein of 76 amino acids that is covalently linked to target proteins altering their localization, function, or stability 1-3. Proteins can be modified with a large number of different isoforms of ubiquitin and these different ubiquitins are thought to signal different outcomes. The question of how these different forms of ubiquitin are recognized is central to understanding the specificity of various types of ubiquitination 4-6. Ubiquitin acts as a signal by being conjugated to proteins through three sequential steps. In the first step, ubiquitin is activated by the ATP-dependent formation of a thiolester bond between the C-terminus of ubiquitin and the active site cysteine of an ubiquitin activating enzyme or E1. The second step involves the transfer of the ubiquitin molecule from the E1 to the active site cysteine of an ubiquitin-conjugating enzyme or E2. Finally, the ubiquitin is transferred to a lysine residue of the target protein in a reaction catalyzed by an ubiquitin ligase or E3. This last step occurs in a substrate-specific manner and it is highly regulated 7-9. Several rounds of ubiquitination can occur on ubiquitin itself, leading to the formation of a polyubiquitin chain. Any of seven lysines, or the amino terminus, of ubiquitin can be used to polymerize ubiquitin and so there are a huge number of differently linked polyubiquitin signals that can be formed. Chains can be linked by the same lysine on each ubiquitin (K29, K48, K63, etc.) to yield homogeneous chains, or utilize different lysines on some ubiquitins to yield heterogeneous chains. In the latter case, the lysine used can vary from ubiquitin to ubiquitin, or chains can be formed that are branched at a single ubiquitin by linking two ubiquitins to two different lysines at the branch point. It is commonly assumed that different polyubiquitin chains are associated with different cellular fates. Receptors are thought to recognize the different ubiquitin modifications (mono- and polyubiquitin) attached to the target proteins and to mediate the different signaling outcomes 4,10. These receptors have ubiquitin binding domains that interact with ubiquitin or polyubiquitin, and may also have domains that can also interact with the modified target proteins or other macromolecules. Like most posttranslational modifications, ubiquitination is reversible 11 and its removal is carried out by enzymes collectively known as deubiquitinating enzymes (DUBs) 12. DUBs are proteases that have been implicated in a wide variety biological processes 12,13. They are responsible for the removal of ubiquitin or polyubiquitin from target proteins, the processing of ubiquitin precursors, and the disassembly of unanchored polyubiquitin (a polyubiquitin chain not attached to another protein) that is either synthesized de novo, or released by the action of other DUBs. Thus, like the cellular targeting receptors they recognize the different forms of ubiquitin and polyubiquitin. For instance, the tumor suppressor CYLD acts exclusively on K63-linked chains 14, yeast OTU1 prefers long K48-linked chains 15, and USP5cleaves both linkages 16. Nearly 100 DUBs in five different protein families are encoded by the human genome. Several DUBs have been shown to bind or process polyubiquitin or polyubiquitinated substrates in vivo, and many DUBs have been shown to cleave polyubiquitin in vitro. This review will discuss the specificity of ubiquitin and polyubiquitin binding by DUBs. The DUBs discussed will be limited to those where binding and specificity have been directly demonstrated, either through structure determination or direct binding and catalytic studies. It will focus on the current body of knowledge regarding structure of ubiquitin binding domains of DUBs and the mechanisms by which these DUBs recognize and selectively disassemble different polyubiquitin chains. In addition to clarifying the mechanisms of chain recognition by DUBs, the conclusions gleaned from these proteins may well serve as a model for the recognition of these chains by other receptors.

Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of ∼130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys48 linkages, having little or no activity on Lys63- and Lys29-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

Recognition of Polyubiquitin Isoforms by the Multiple Ubiquitin Binding Modules of Isopeptidase T

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.

Polyubiquitin binding and cross‐reactivity in the USP domain deubiquitinase USP21

Modification of proteins by ubiquitin (Ub) and Ub‐like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub‐specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross‐reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon‐stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub‐ and ISG15‐binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.

RNAi triggered by specialized machinery silences developmental genes and retrotransposons

RNA interference (RNAi) is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that, in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and a corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Notably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover an interaction between RNAi and the exosome that is conserved in Drosophila, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance

The regulation of protein-coding and noncoding RNAs is linked to nuclear processes, including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA-processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc-finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.