Ubiquitin Signaling: Chemistry Comes to the Rescue - Journal of the

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Ubiquitin Signalling: Chemistry Comes to Rescue Sachitanand M. Mali, Sumeet K. Singh, Emad Eid, and Ashraf Brik J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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Journal of the American Chemical Society

Ubiquitin Signalling: Chemistry Comes to Rescue

Sachitanand M. Mali, Sumeet K. Singh, Emad Eid, Ashraf Brik*

Schulich Faculty of Chemistry, Technion—Israel Institute of Technology, 3200008 Haifa, Israel

* Correspondence to Ashraf Brik [email protected]

1 Environment ACS Paragon Plus


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Abstract Posttranslational modification of proteins by ubiquitin, i.e. ubiquitination, mediates a variety of cellular processes ranging from protein homeostasis, cell cycle, DNA repair to viral infections. Understanding the molecular mechanism of ubiquitination in these events is the basis for unraveling its precise role in health and disease. However, the inherent complexity of ubiquitin signalling due to the high atomic complexity of ubiquitin conjugates, where ubiquitin is attached to other ubiquitin molecules and to protein substrates in various forms, imposes a major challenge for these studies. In this regard, the enzymatic approaches employed for the preparation of important ubiquitin conjugates have severe limitations to deliver them in high homogeneity and in adequate amounts for the desired study. Recent developments in the area of chemical and semisynthesis of proteins offer great solutions to the enzymatic limitations and enabling the preparation of various ubiquitin conjugates with precise control over the atomic structure. These conjugates significantly contribute in deciphering ubiquitin signalling at the molecular level and with the synthetic tools in hand, chemical biologists have become key players in efforts to understanding the complexity of the ubiquitin code. In this perspective, we highlight the key contributions of these synthetic approaches and how the development of novel ubiquitin based reagents is greatly assisting in uncovering unknown aspects of ubiquitin signalling. We also discuss future aspirations to address unresolved questions in this exciting area of research.


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Introduction Proteins are the main players in regulating nearly every cellular function. Apart from their native forms, most proteins during their life span are modified with different posttranslational modifications (PTMs), such as phosphorylation, acetylation, methylation, glycosylations, and ubiquitination. Such modifications have profound effects on the protein structure, function and cellular localization.1 Ubiquitination is considered as one of the key regulators in governing protein function and fate. It is one of the major PTMs where one protein (ubiquitin) is covalently attached to other proteins (substrate) to modulate its biological function in a spatiotemporal manner. Ubiquitin (Ub) is a 76 amino acids protein, which possesses a highly conserved sequence and is ubiquitous in nature. During ubiquitination, Ub or polyUb chain is attached via the C-terminus Gly of Ub to the ε-amine of Lys residue from a substrate protein.2 A cascade of three enzymes, known as the E1 activating enzyme, E2 conjugating enzyme and the E3 ligase, work together to assemble the Ub chain and to modify the target protein with Ub or a Ub chain (Figure 1).3 The simplest form of ubiquitination is the attachment of a single Ub to a substrate protein onto a lysine residue. The histone protein H2A was found to be the first ubiquitinated protein; a discovery which contributed to the early studies of this modification.4 The discovery of Ub mediated protein degradation by 26S proteasome system by Ciechanover, Rose and Hershko, highlighted the unprecedented importance of this small modifier protein.2 These findings overruled earlier believes that proteins are long lived and their destruction is mediated by lysosomal machinery.5-7 The groundbreaking discovery of involvement of 3 Environment ACS Paragon Plus


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ubiquitination in regulating protein homeostasis motivated several groups to study the role of this complex signal in different physiological processes. This has revealed the enormous role of the Ub proteasome system (UPS) in protein degradation and its significance in in many cellular events, such as in cell division,8 inflammation,9 DNA repair10 and endocytic trafficking.11 Though many studies have shown the importance of Ub signalling in general and UPS in particular in aforementioned physiological events, we still lack a comprehensive understanding of many molecular aspects of this system and thereby the mechanism of governing various cellular functions. Although early studies have focused on Lys48-linked chains as the main Ub signal in proteasomal degradation12 nowadays it is clear that Ub signalling is much more complex than ever thought before. The complexity starts at the Ub level since Ub bears seven Lys residues (Lys63, Lys48, Lys33, Lys29, Lys27, Lys11 and Lys6), where each can act as a site for ubiquitination to form different Ub chains (Figure 1). The modification through each one of the Lys residues may result in a distinct physiological signal.13,14 In addition, the Nterminus of Ub can also be ubiquitinated to form so called linear chains thus allowing for eight possible ubiquitination linkage types. Moreover, different types of linkages, i.e. mixed or branched, can also be formed. In the branched case, one Ub is linked with other Ub simultaneously through different Lys residues, while in the mixed chains the Ubs are linked together one at a time through different Lys residues from another Ub unit (Figure 1). The Ub chains linked through Lys48 are termed as typical chains whereas the other chains are classified as atypical chains.15 Ub chains with variable lengths and linkages possess distinct conformational topology, which are read out by


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different Ub binding domains (UBDs)16,17 of the interacting proteins. These interactions provide the specificity required for the desired Ub signal. Hence, the linkage type and length of the Ub chains determine the fate of the ubiquitinated proteins.

For example, Ub chains which are linked through

Lys48 triggers proteasomal degradation18 while the Lys63-linked chains play key role in endocytic trafficking DNA repair, and signal transduction.10,13 The heterotypic mixed chains exhibit various roles, mainly in non-proteolytic signaling and the branched chains appear to also play a role in protein degradation in the cell cycle where these chains show high affinity to the proteasome due to their unique structural features.19 Understanding the molecular basis of Ub signalling has become a major goal for many academic and drug discovery laboratories since the aberration in this signal plays a major role in various diseases like neurodisorders and cancer 20, 21. The complexity of the Ub signal has recently become even more apparent with the discovery that Ub itself can be posttranslationally modified.22 For example, it has been recently reported that PINK1 mediated S65 phosphorylation to recruit and activate Parkin (E3 ligase) to facilitate ubiquitination of mitochondrial outer membrane proteins for subsequent mitochondrial autophagy.22 These modifications lead to changes in the structure and dynamic of Ub and could contribute in regulating Ub signalling by affecting the UPS components such as deubiquitinases (DUBs), E2 and E3 enzymes. However, a complete picture of the enzymes, which are adding or removing these modifications, the interacting proteins with the modified Ub and the other physiological roles of these modifications are still lacking.



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O S

Ub

HS SH O Ub

E1

OH

SH

O Ub

E2

S

Ub

E2 E3

O

E3

S O

2 E1s

E1

30-40 E2s

E2 Ub

E3

S

NH2

E3

HS > 500 E3s

Ub

HN O

O NH2

N H

n

Ub

O NH Ub

m

D U B Deubiquitination ~100 DUBs

Figure 1: Enzymatic ubiquitination and deubiquitination. Deubiquitinases (DUBs) are the class of enzymes, which remove Ub or the polyUb chain from the substrate protein and are considered a key player in the Ub signalling.23 DUBs function at various stages of the Ub life cycle, starting from the generation of mature Ub from inactive fusion proteins to the control of the function and destiny of ubiquitinated proteins by removing or editing the Ub chain. For example, amputation of Ub chains by DUBs averts


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the ubiquitinated substrate from degradation. Some DUBs exhibit specificity towards certain Ub chain types due to their defined structural topology and dynamic.24 Vital roles of DUBs in cellular processes such as in cell cycle, protein localization, and in tumor suppressor proteins have been reported.23,24 The accumulated knowledge about DUBs supports their important roles in various diseases and is now clear that their regulation by novel inhibitors offer a promising direction in drug development.25 The advancement in the enzymatic approaches for constructing unanchored Ub chains or ubiquitinated proteins has assisted in the identification of new E2, E3 ligases 26, 27, DUBs 24 and specific interacting domains from associated proteins.16 These enzymes assisted to prepare unanchored polyUb chains of defined linkages (e.g. Lys48, Lys63 and Lys11) as well as ubiquitinated substrates, yet without controlling the chain lengths in these examples.27 These studies have assisted in our understanding the specific role of ubiquitination as well as how at the molecular level these Ub chains or ubiquitinated substrates interact with different proteins to achieve the specific function. However, obtaining such a molecular understanding for some linkage types or for a specific ubiquitinated substrate remains very challenging. The major impediment of the enzymatic approaches is the requirement for the identification of specific E2 and E3 enzymes, out of the known 30-40 E2 and over 500 E3, for a chain linkage type and a particular substrate. Even when these enzymes are identified, the lack of homogeneity in constructing ubiquitinated proteins with a desired Ub chain is a major limitation. Moreover,



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for certain studies modifying the Ub, Ub chains or the ubiquitinated protein with a specific handle to generate a particular probe, makes it even more challenging to prepare such a modified Ub conjugate enzymatically. To overcome these shortcomings, chemical and semi-synthesis of proteins have been applied to prepare Ub conjugates, which become extremely useful in the efforts to decode the Ub signal. Several non-enzymatic methods have been developed to generate various polyUb chains and polyubiquitinated proteins with native and non-native bonds.28-32 These strategies offer precise control over the Ub chain linkage type and length, which is difficult or impossible to obtain otherwise and have already demonstrated great value in the area of Ub signalling. 1. Chemical ubiquitination via native isopeptide bond: The isopeptide bond is an amide bond flanking the Lys side chain and is found in various natural conjugates, mainly linking two distinct proteins to mediate several critical biological processes. Ubiquitination, sumoylation, sortase-mediated cell surface protein anchoring and transglutamination are examples where the end product has an isopeptide bond. In recent years several organic chemists have developed different chemical methods to prepare homogenous Ub conjugates with a focus on constructing the isopeptide bond linkage in a highly specific manner.30-32 The seminal work by Muir and co-workers in preparing ubiquitinated short peptides utilizing a photo cleavable auxiliary-mediated ligation approach (Scheme 1a) has inspired the development of several chemical strategies for ubiquitination. The major limitation of this approach is the extremely slow


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ligation time and the difficulties, which could emerge when multiple isopeptide bonds are required to build a particular Ub conjugate with high atomic complexity.33

H2B (115-125)

H2B (115-125)

H2B (115-125)

H2B (115-125)

Scheme 1: Auxiliary mediated ubiquitination a) Photolabile auxiliary assisted ubiquitination of a peptide fragment derived from H2B. b) & c) Examples of other auxiliaries employed in protein ubiquitination. TCEP = tris(2carboxyethyl)phosphine, MES = 2-mercaptoethane sulfonate.

Recently two ubiquitination methods have been reported based on the auxiliary mediated isopeptide bond formation. In the first approach developed by Liu and coworkers (Scheme 1b), the TFA-labile 1-(2,4-dimethoxyphenyl)-2mercaptoethyl auxiliary was used to assist the synthesis of the Lys27-linked di- and tri-Ub chains.34 The crystal structures of these Ub conjugates were 9 Environment ACS Paragon Plus


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solved to shed light on their unique structural features. Notably, the authors prepared the D- and L-forms of these Ub chains for racemic crystallography35, which was essential in obtaining the X-Ray structures of these chains. Such an example highlights the power of chemistry in solving a longstanding question—how different linkage types in Ub chains affect their structures. Here, the crystal structures of Lys27-linked Ub chain provided the molecular basis for its distinctive function and revealed that the isopeptide linkage is confined in a buried conformation when compared to other Ub chains. In the second approach, Chatterjee and coworkers employed the 2aminooxyethanethiol auxiliary to mediate chemical ubiquitination (Scheme 1c).36 The milder conditions to remove the auxiliary by zinc in acidic conditions enabled to install the native isopeptide bond at the ligation junction. This strategy is advantageous over the earlier reported auxiliary

33

in terms of

ligation kinetics. Moreover, it can be used in presence of Cys residues in the protein, which could enable its use in combination with native chemical ligation (NCL). Notably, the presence of the auxiliary in the synthetic Ub conjugate was found to stabilize the isopeptide bond against several DUBs. This represents a distinct example of a chemical approach, which enables the preparation of Ub conjugates with native or non-native isopeptide bonds for various biophysical and biochemical studies. The strategy was further improved by performing ubiquitination of folded proteins and employing 4mercaptophenylacetic acid (MPAA) for post ligation cleavage of the N-O bond to remove the auxiliary.37


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A major breakthrough in the chemical protein synthesis area is the discovery of NCL approach by Kent and co-workers, which has been used in synthesis and semisynthesis of hundreds of proteins.38,

39

To employ a strategy that is

reminiscent to Cys-NCL for the synthesis of Ub conjugates with native isopeptide bond, this required the derivatization of Lys to install the thiol handle at gamma or the delta carbon. With this in mind, the Brik and Liu groups, independently, reported the synthesis of δ- and γ-mercaptolysine, respectively. Liu and co-worker used orthogonally protected γ-mercaptolysine, to enable sequential ligation reactions at the α- and ε-amines via a sixmembered ring intermediate.40 On the other hand, we used orthogonally protected δ-mercaptolysine to promote ligation to the ε-amine, via a fivemembered-ring intermediate (Figure 2a), to achieve native isopeptide linkage after a desulfurization step.41 In order to extend the use of the δmercaptolysine for Boc- and Fmoc-SPPS and in sequential ligations, we prepared several analogues of the δ-mercaptolysine bearing various orthogonal protecting groups (Figure 2b).42 These analogues, in particular, the thiazolidine-protected δ-mercaptolysine, were employed in the synthesis of various Ub chains and ubiquitinated substrates.40, 41, 43-49 All seven native diUb chains as well as the unanchored tetra-Ub chain were synthesized to shed light on the conformation and behavior of some of these chains with various DUBs.43,50 One limitation of the mercaptolysine is the lengthy synthesis of its analogues (average 14 steps), which offers an opportunity for employing more sophisticated synthetic approaches to shorten their syntheses.



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Figure 2: a) δ-mercaptolysine mediated chemical ubiquitination. b) List of δmercaptolysine derivatives and their applications in the synthesis of ubiquitinated peptides and proteins.


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Our group has also utilized the thiazolidine-protected analogue of δmercaptolysine in the synthesis of mono and polyubiquitinated α-synuclein (αSyn).44,51 This protein plays a major role in neurodegenerative diseases as evident by numerous biochemical, cellular and animal studies.52 In addition, it has been shown by several biochemical and cellular studies that mono or polyubiquitinated α-Syn are closely associated with Parkinson Disease (PD) and were identified in α-Syn fibrils, which are the key elements of Lewy bodies (LB) that define PD.52 However and despite extensive efforts to shed light on the role of ubiquitination in the pathophysiology of α-Syn, several fundamental questions remain unanswered. In order to dissect the role ubiquitination on the fate of α-Syn, the synthesis of di- and tetra-ubiquitinated α-Syn was carried out. First, α-Syn having δ-mercaptolysine at K12 (K12*) was obtained from two fragments.44 The chemically synthesized α-Syn(1-29)-thioester was ligated with the recombinantly obtained α-Syn(30-140) fragment harboring Nterminal Cys, followed by methoxylamine treatment to uncover the thiazolidine ring at position 12. In order to achieve tetraubiquitinated α-Syn, two different fragments of di-Ub–thioester were prepared, followed by two sequential ligation steps with α-Syn (Scheme 2). Finally, a desulfurization step was performed to give the desired conjugates in high homogeneity and workable quantities for the biochemical and biophysical analyses.



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Scheme 2: Synthesis of tetra-ubiquitinated α-Syn. We found

that

contrary

to

the

monoubiquitinated

form

of

α-Syn,

polyubiquitinated α-Syn is resistant to DUBs and was efficiently degraded by the proteasome. Our study also suggested that the ubiquitination, (mono and poly) is not required for α-Syn fibrillization and LBs formation. Finally, this study also enabled us to examine how ubiquitination can influence the kinases activities on α-Syn and how simultaneous phosphorylation and ubiquitination could affect the aggregation of α-Syn. These studies demonstrated

how

chemical

strategies

could

be

used

to

access

polyubiquitinated proteins to assist in understanding important aspects of ubiquitination in regulating pathophysiology of certain diseases such as PD.


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The impressive advancement in molecular biology approaches to incorporate unnatural amino acids into proteins enabled the Chin group to utilize the photo-protected form of δ-mercaptolysine to facilitate ubiquitination.53 The same group developed a strategy utilizing genetically encoded orthogonal protection and activated ligation method (GOPAL) to introduce the native isopeptide bond.54 They employed GOPAL to synthesize Lys6- and Lys29linked di-Ub chains for structural and functional analyses. In this strategy, the Lys(Boc) amino acid was recombinantly encoded at the desired position in Ub, while all other Lys residues were orthogonally protected with (N(benzyloxycarbonyloxy) succinimide; Cbz-OSu). This allowed for selective removal of the Boc-protecting group from designated Lys for direct coupling with Ub-thioester whose all Lys residues were Cbz protected (Scheme 3A). Selective acylation of Ub-thioester in presence of HOSu/AgI/ DIEA led to the desired isopeptide bond. Finally, global deprotection of all amines was performed to obtain native Lys6- and Lys29-linked di-Ub chains. These di-Ub analogues were crystallized and their X-ray structures were solved for the first time. This study revealed a very interesting observation regarding the compact and asymmetric conformation of Lys6-linked chain in which the proximal and distal Ub moieties interact through distinct residues compared to the structures of Lys48- or Lys63-linked Ub chains. Fushman and co-workers have further optimized the 'GOPAL' strategy by replacing the Cbz protecting group with the allyloxycarbonyl (Alloc) group, which can be removed using milder conditions (Scheme 3A).50 These improvements helped the group to assemble Ub chains longer than di-Ub. Utilizing this improved method the group was able to generate the Lys11-



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linked di-, tri-, and tetra-Ub chains, the Lys33-linked di-Ub and a mixedlinkage Lys33, Lys11-linked tri-Ub chains. These analogues were subjected to NMR structural analyses to shed light on their conformations.

Peptide

Peptide

Peptide

Peptide

Peptide

Peptide

Scheme 3: Synthesis of ubiquitinated peptide/protein using an orthogonally protected Lys residue. A) Genetic encoding of Lys(Boc) (PG1= Boc), orthogonal protection (Cbz/Alloc) of rest of Lys residues, selective deprotection of PG1, selective acylation of Ub-thioester and global orthogonal deprotection to achieve native isopeptide linked Ub chains. B) Chemical insertion of Lys(ivDde), (PG2= ivDde) at a desired position and selective


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orthogonal deprotection of PG2 followed by SPPS, peptide cleavage, NCL and desulfurization to obtain the ubiquitinated peptides/proteins.

Similar to the methods, which relied on orthogonal protection to direct ubiquitination, we developed a chemical approach relying only on SPPS coupled with NCL for the synthesis of ubiquitinated peptides and proteins.55 In this approach orthogonally protected Lys with 1-[4,4-dimethyl-2,6-dioxocyclohexylidene]-3-methylbutyl (ivDde) was introduced in the peptide during SPPS. Subsequently, selective deprotection of the ivDde allowed the direct formation of the isopeptide bond followed by peptide elongation of part of the Ub sequence, Ub(47-76), having N-terminal Cys (Scheme 3B). To further extend it to the full ubiquitinated peptide, the purified Ub(47-76) fragment which also bears the peptide at the C-terminus, was ligated with Ub(1-46)thioester. The strategy was applied to prepare five ubiquitinated peptides derived from the histone H2B with varying lengths, which enabled examining the effect of the peptide length on the efficiency of the isopeptide bond cleavage by UCH-L3.55 2. Chemical approaches of ubiquitination via a non-native isopeptide bond Along with the synthetic efforts, which have been invested in preparing Ub conjugates with native isopeptide bond one has to bear in mind that several research aspects of Ub signaling are difficult to study when the native isopeptide bond is present. For example, to obtain structural information about DUBs using X-Ray crystallography it is important in some cases to have a



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non-cleavable isopeptide bond if mutation of the catalytic residues is not desired. In addition, in studies aiming to fish out proteins that interact with a specific Ub conjugate, it is desirable to stabilize the isopeptide linkage against DUBs activities to keep the construct intact. Moreover, the synthesis of Ub conjugates with a non-native isopeptide bond is often easier compared to their native forms. Wilkinson and coworkers reported the first attempt in these directions and prepared Lys11-, Lys 29-, Lys 48-, and Lys 63 di-Ub conjugates having the non-native isopeptide bond (Figure 3a).56 The non-native isopeptide bond was introduced by the incorporation of a Cys residue at the desired position in the proximal Ub (e.g. 11, 29) and instead of Gly76 of the distal Ub. 1,3dichloroacetone was then used to link the two Ubs to form the acetone-based linkage. Although the resulted non-native isopeptide bond is different from the native one, the study revealed that these conjugates could inhibit the activity of several DUBs suggesting that these di-Ub analogues have similar conformations to their native forms. Inspired by this strategy, Pratt and coworkers further improved the method by introducing

1,3-dibromoacetone

to

link

Ub

bearing

C-terminal

aminoethanethiol to α-Syn in which its Lys6 was mutated to Cys (Figure 3b).57 This method proved advantageous over the Wilkinson approach in terms of the overall yield. In addition, the new linkage resembles the native isopeptide bond more. The group prepared four different ubiquitinated α-Syn conjugates with the bis-thio-acetone (BTA) linker at positions 6, 23, 43, and 96 and tested their aggregation and toxicity. Their results suggested that ubiquitination at


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23, 43, and 96 positions in α-Syn inhibited both aggregation and toxicity. However, ubiquitination at position 6 resulted in the formation of irregular small fibers. One key limitation of this approach is the requirement for a Cys residue in the desired position and the necessity to mutate other Cys residues in the target protein. Recently the Brik and Wolberger groups demonstrated the use of multiple approaches to shed light on how the deubiquitination of H2B is achieved at the molecular level.58 In this study, ubiquitinated H2B with both a native and non-native isopeptide bond was prepared and utilized to construct the ubiquitinated nucleosome to decipher the molecular basis of the interaction of SAGA DUB module with the ubiquitinated nucleosome.

A non-native

isopeptide bond between Ub and H2B was formed via the dichloroacetone approach (Scheme 4A), while the δ-mercaptolysine mediated native isopeptide bond formation strategy was employed to yield ubiquitinated H2B at Lys120 (Scheme 4B). Each type of bond was selected according to the questions that needed to be answered. There was a need for the stable analogue in the case of solving the crystal structure of the DUB module bound to ubiquitinated nucleosomes. To further confirm the role of the key residues in stabilizing the complex, ubiquitinated substrates with a native isopeptide bond was required. Together these studies provided a framework for understanding the global interactions between the SAGA complex and nucleosomes.



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Scheme 4: H2B ubiquitination via: A) A non-native linkage to obtain crystal structure of H2B-SAGA DUB complex (PDB: 4ZUX, DNA shown in orange red, histone proteins in cyan, Ub in green, SAGA complex in pink) B) A native linkage for better understanding the interactions of ubiquitinated H2B and the SAGA DUB module, also highlighting some of the conclusions that were made based on this study.

In another approach to prepare stable Ub conjugates, Cys was further employed to generate disulfide bond as a replacement of the isopeptide linkage. The research groups of Muir and Zhuang reported, independently, the use of the Cys residue of a protein to attach it to Ub via a disulfide bond (Figure 3c).59, 60 The Muir group tested the effect of the ubiquitination site of H2B on the stimulation of H3K79 methylation by hDot1L. The group found that ubiquitinated H2B at position 125 stimulates the activity of hDot1L by 85%, while substantially reduced when Ub was placed at positions 108 and 116.59 On the other hand, the Zhuang group tested the effect of ubiquitination on PCNA to reveal its role in DNA damage response and chemically prepared different analogues of ubiquitinated PCNA (K164, K127, K107 and R44).60


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These study revealed that the ubiquitination site does not have significant effect on PCNA function. Though disulfide strategy can be employed to construct non-native isopeptide linkages in a highly efficient manner, the presence of disulfide bond can be a limiting factor. The ease of reduction of the disulfide bond in a biological environment and the need of reducing agents in a variety of biological experiments limit the broader applicability of this approach. In addition, the strategy is limited to a protein with only single Cys residue as in the cases described above. In recent years, the azide-alkyne cycloaddition reaction (click reaction) catalyzed by Cu (CuAAC) has proven to be a great tool for modifying biomolecules with various functional groups. The combination of molecular biology approaches and the unique reactivity of click reaction was successfully applied to construct the seven Ub dimers.61 To achieve this, Gly76 of Ub was mutated with azidohomoalanine (Aha) using protein expression (Figure 3d). For the alkyne handle, the amber codon technology was employed to incorporate the propargyl protected Lys derivative (Plk) at the different Lys positions in Ub. Having decorating the Ub monomers with the azide and alkyne functionalities, CuAAc conditions were then applied to obtain the seven di-Ub analogues with the triazole linkage. Using similar CuAAC chemistry the Mootz group generated stable triazole linked SUMOylated peptides.62



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O Ubiquitin Y

O N H

Ubiquitin

X

+

Substrate

X

Substrate

Reagents and conditions

Y

O

a

SH

NH X Y

SH

Cl

SH

Br

Non-native Isopeptide Bond O S

Cl

S

O SH

b

c

S

S

S

O2N

56, 58

O Br

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SH

SH

References

S

57

59, 60

S

2 O

d

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S

Li+ OP O Me hv (365nm)

Me

e

SH Me

N N N

S

61, 62

O

O

S

64, 65

Figure 3: Selected strategies employed for the preparation of non-native isopeptide bond.

Encouraged by the high bimolecular rate constant (~106 M-1 s-1) for the addition of thiyl radicals to alkenes and with the opportunity to form a stable thioether linkage,63 the Strieter group explored the use of this chemistry to generate non-native isopeptide bond (Figure 3e).64 In this strategy, the desired Lys residue in the proximal Ub was replaced with Cys and the Cterminus of the distal Ub modified with allyl amine (AA). Employing thiol-ene reaction conditions generated the di-Ub conjugates. The utility of this method was demonstrated in the synthesis of 6 different dimeric Ub conjugates (Lys6, Lys 11-, Lys 27-, Lys 29-, Lys 33-, and Lys 63-linked di-Ub). In this approach, the formed non-isopeptide linkage has an extra bond compared to the native


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one. To verify the uniqueness of this bond as an excellent replacement to the native isopeptide form, the group compared the stability of these analogues with their native isoforms in the presence of two DUBs (A20-OTU which cleaves Lys48-linked Ub chain, and AMSH which cleaves Lys63-linked Ub chains). The study revealed that the non-native isopeptide bond was processed in a similar manner to the native isopeptide bond as A20-OTU cleaved the Lys48-linkage but not the Lys63-linkage and vice versa with AMSH. Furthermore, the group used small-angle X-ray scattering (SAXS) to study the structural similarities of the native and the non-native Lys48 and Lys63-linked di-Ub.65 The analysis revealed that both analogous have a close structural similarity. In addition, a steady-state kinetic study was carried out to analyze the behavior of the various DUBs towards the Ub dimers bearing the non-native linkage. These experiments demonstrated that the applied DUBs have a similar processivity towards the native and non-native conjugates. Such approaches could aid to prepare all types of Ub chains aiming to study different aspects of Ub biology. These include understanding the differential behavior of DUBs towards various Ub chains and their role in assisting specific physiological events. It remains to be tested if this can also be extended to the preparation of ubiquitinated proteins. Recently we reported a site-specific attachment of mono- or poly-Ub chains to a protein via its native Cys residue.66, 67 To achieve this, different electrophiles and nucleophiles have been introduced at the C- terminus of the proximal Ub in the chain. On the other hand, α-globin has been decorated with a complementary functional group to generate ubiquitinated α-globin in which the isopeptide bond is replaced by disulfide, thioether or oxime linkage.



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Employing the disulfide strategy, the synthesis of mono-, di-, tri-and tetra-Ub α-globin conjugates was achieved.66 However, as the disulfide bond is reducible in biochemical conditions, we aimed to develop an alternative strategy, which would provide a stable bond under reducing conditions. Hence, a thioether stable linkage was formed between the target protein and the Ub chains. However, the low efficiency of this strategy restricted us from performing such a step beyond di-ubiquitination of α-globin and prompted us to explore alternative methods.

Scheme 5: Synthesis of oxime linked polyubiquitinated α-globin analogues to study their proteasomal degradation behaviors. A solution to this obstacle emerged from the use of oxime-based ligation between aldehyde and aminooxy functionalities. We developed an approach where the Cys side chain of α-globin was converted to thio-acetaldehyde using chloroacetaldehyde as an alkylating reagent.67 Later, the modified αglobin was reacted with Ub-oxyamino to afford oxime linked mono-Ub-αglobin also bears free δ-mercaptolysine at Lys48 of Ub, which helped us to further elongate the Ub chain (Scheme 5). Utilizing this method the synthesis of mono-, di-, tri- and tetra-Ubs α-globin was achieved with the stable linkage


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between the Ub chain and substrate against DUBs activities. The same strategy was successfully applied to afford bis-ubiquitinated β-globin conjugates. With these reagents in hand, we analyzed how the Ub chain length affects the outcome of protein degradation and found that by overcoming trimming at the proteasome, tetra-Ub is a fundamentally different signal than the shorter chains. Our results also demonstrated that the cleavage of the proximal isopeptide bond is not a necessity for proteasomal degradation and the tetra-Ub chain can undergo degradation with the target protein. This work exemplifies how stabilizing the isopeptide bond between a given substrate and the proximal Ub to prevent removal of the chain during the degradation process could illuminate a specific feature of proteasome action. Such a study can be performed mainly by using chemical synthesis with its clear advantage in modifying the substrate as desired. The strategies discussed above have demonstrated the usefulness of each method in different biochemical, biophysical, structural and functional studies despite that each of these has its own limitations. It is clear that we further need improvement in these methods for synthesizing complex protein conjugates. One should always be careful about choosing the right chemistry for the desired study and should be open minded in using more than one synthetic strategy to achieve the desired target. For example, it is very important to consider using both native and non-native isopeptide bond synthetic strategies depending on the biochemical questions.58 By employing the most effective method according to the desired characteristics and functionalities of the target protein and the goal of the research, one could



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achieve the required protein conjugates in high homogeneity and in great amounts. 3. Expanding the chemical toolbox for studying and targeting DUBs Over the last decade, the development of different non-enzymatic approaches for the preparation of the various polyUb chains and polyubiquitinated proteins have greatly assisted in understanding several aspects of the Ub code. There are around 100 DUBs encoded in the human genome. Based on structure similarities and mode of actions, DUBs are classified into five families. Four of them including Ub C-terminal hydrolase (UCH), Ub specific proteases (USP), ovarian tumor proteases (OTU), Josephin/Machado–Joseph disease protease (MJD) are Cys proteases, while the fifth JAB1/MPN/MOV34 metalloenzyme (JAMM) belongs to the zinc metalloproteases.23 The involvement of DUBs in several physiological processes motivated several laboratories to study their biochemical, structural and functional features.24 Apart from the role in proteasome mediated protein degradation DUBs play vital roles in several cellular events. For example, USP1 plays an important role in DNA damage response by removing Ub from Fanconi anaemia complementation group D2 protein (FANCD2) and proliferating cell nuclear antigen (PCNA).68 CYLD and A20, which serve as tumor suppressors, play vital roles in the negative regulation of NF-κB activation.69,

70

USP7 and USP2 regulate the tumor

suppressor protein p53 and its ligase MDM2.71, 72 Though these discoveries advanced our understanding of the regulatory role of DUBs, the molecular basis of substrate specificity of various DUBs is still in its infancy. In the cell, regulations of DUBs are associated with conformational changes because of


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their interactions with different protein partners, their modifications by diverse PTMs, cellular localizations, and their associations in larger complexes.24 To study DUBs specificities and structures with their native substrates, these enzymes should be isolated and characterized. The recent progress in developing chemical tools for the preparation of various Ub conjugates has significantly aided in the discovery of new DUBs and studying their biochemical, structural and functional aspects.73 3.1 Ub Chains and Ubiquitinated Proteins Accessing homogeneous Ub conjugates with native and non-native isopeptide bond is one of the important requirements in order to dissect the mode of action and specificity of various DUBs. As described above various synthetic strategies have been employed for the preparation of Ub chains in their free and anchored forms. These achievements have been the basis for novel discoveries in the Ub field in general and in DUBs studies in particular.73 In order to study the preference of diverse DUBs towards different Ub chains, Chin and co-workers synthesized Lys6- and Lys29-linked di-Ub chains using the GOPAL strategy as described in section 1.54 These di-Ub conjugates were treated with a panel of eleven DUBs from USP, UCH, OTU, and JAMM/MPN+ DUBs families, which revealed that members from the USP family such as USP2, USP5 and USP21 have high protease efficiency towards these chains. In addition, it was also observed that TRABID from the OTU family exhibits 40-fold specificity towards the Lys29 over the Lys63-linkage. To examine the effect of DUBs regulatory domains from the USP family on their activities, Ovaa and co-workers prepared all eight di-Ub chains48 and 27 Environment ACS Paragon Plus


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used them to determine the kinetics of twelve DUBs from the USP family.74 This assisted in comprehending the role of intra and intermolecular domains in regulating DUBs activities. The study revealed the variable activities and preferences for all USPs towards different Ub linkages. In most cases, the catalytic turnover and Ub binding of these USPs were significantly influenced by the presence of these domains. These studies, which used Ub chains with different linkages for the characterization of DUBs have significantly enhanced our understanding about DUBs specificities. However, analyzing the behavior of DUBs towards Ub chains, which are linked to various substrates, would provide knowledge about the contribution of DUB-substrate interactions in the hydrolysis process.58 Such studies are still very limited in the Ub field, however with the current state of the art chemical methods to prepare ubiqutinated proteins, one should be able to advance in this particualr aspect. 3.2 Ub based probe development The field of activity based protein profiling has shown great potential to complement standard proteomic methods, which mainly provide information on the overall abundance of proteins.75 Since most DUBs contain an active Cys in their catalytic sites this has motivated researchers to develop thiolreactive functionalities positioned in Ub in such a manner that it would react covalently with active site Cys of the particular DUB. In order to visualize or recover the probe trapped DUBs, recognition or a retrieval element is incorporated in the Ub probe. These probes have greatly assisted in shedding light on unknown structural and functional aspects of DUBs in normal and


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disease states. This includes isolation and crystallization of DUBs to unravel the molecular interactions between the DUB and Ub or Ub chains, which dictates the specificity to a particular DUB.28,76 The pioneering work of the Plough group in preparing Ub based probes has ignited the development and use of several activity based probes to study DUBs in great detail. In the initial work, the C-terminus of Ub was modified with vinyl sulfone to trap and visualize various DUBs present in yeast and mammalian cell extracts (Figure 4i).77 To achieve more efficient Ub based probes, this has encouraged various groups to develop new probes. These probes were applied to identify new DUBs,78 profile their activities

79

and

examine the potency and selectivity of DUBs inhibitors.80 Recently, Ekkebus et al. found that propargyl moiety at the C-terminus of Ub, unexpectedly reacted with the catalytic Cys of UBCH13 and other DUBs (Figure 4ii).81 These alkynes showed no reactivity towards excess thiol or Cys residues present in non-targeted proteins confirming their high selectivity; which makes them advantageous over the previous probes.82 DUBs perform their activity on polyubiquitinated substrates in basically two modes. First is the en bloc removal in which the cleavage occurs between proximal Ub and substrate.83 The second mode is trimming, where cleavage occurs between two Ubs.84 In order to have activity based probes that take into consideration the specificity of DUBs, which recognize more than one Ub unit, di-Ub based probes are required. In these probes, the reactive group should be positioned between the two Ubs or the C-terminus of the proximal Ub in the chain.



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These efforts started in 2012 when Iphofer et al. reported the first probe that mimics di-Ub.85 In this study, the ε-amino group of Lys residue from a 13amino acids peptide derived from Ub was modified with the glycine vinyl formic acid (VA), which was further ligated to HA-tagged Ub-thioester (Figure 4iii). The Lys48- and Lys63-linked di-Ub mimics were prepared bearing reactive functionality and subjected to cell lysate to examine their selectivity. The results revealed that DUBs such as USP5, USP7, UCH-L3, and UCH-L5 showed a strong preference to the Lys48-linkage, whereas USP19 and USP38 favored the Lys63-linkage. To develop a full length di-Ub based probe, which would maximize the interactions with the specific DUBs, McGouran et al. synthesized a triazole linked di-Ub probe in which the reactive group was placed between two Ubs (Figure 4iv).86 Using this strategy all eight different di-Ub conjugates were prepared. Combining proteomics and quantitative mass analyses enabled the group to determine the specificities of 28 DUBs from HEK293T cell lysate for various di-Ub linkages. In an attempt to achieve a di-Ub based probe with a linker that closely resembles the native linkage, Zhuang and co-worker synthesized the Lys48and Lys63-linked di-Ub probes using a different linker.87 In order to achieve this, the desired Lys residue from the proximal HA-tagged Ub was mutated with Cys, while the C-terminus of distal Ub was modified with the reactive αbromo-vinylketone for the thiol group. Reacting these modified Ubs resulted in the desired di-Ub probe (Figure 4v). The activity of several DUBs was


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analyzed using these di-Ub probes, which represented a similar trend of reactivity reported earlier by McGouran.86 Our group developed a di-Ub based probe utilizing NCL approach and dehydroalanine (DHA) formation.88 Using SPPS we selectively incorporated a Cys residue at the side chain of Lys48 or Lys63 from the proximal Ub, which performed a dual function in the synthesis of the DHA probe. First, it assisted the ligation with distal Ub(1-75)-thioester for the synthesis of defined di-Ub chains. Subsequently, the generated thiol side chain of this Cys was eliminated to give the DHA functionality as a reactive warhead (Figure 4vi). Parallel to our work, Ovaa and co-workers developed another strategy where they synthesized and incorporated iso-Cys derivative at the proximal Ub.89 As in our approach, this has allowed the use of NCL to synthesize di-Ub followed by the subsequent elimination of the thiol handle to form a Michael acceptor positioned in the Ub backbone (Figure 4vii). More recently, our DHA approach to generate Ub probes has inspired the Chatterjee group to use selenocysteine instead of Cys to enable selective DHA formation in presence of other Cys residues in the Ub conjugate.90 To understand how different Ub binding sites in DUBs interact with particular Ub-linkage type to enforce their specificity91, 92 Flierman al. recently reported the synthesis of seven differently linked di-Ub based probes. These probes contained di-Ub connected through triazole linkage, in which the C-terminus of proximal Ub was also modified with a propargyl moiety as the reactive warhead (Figure 4viii).91 Analyzing a panel of purified DUBs, as well as DUBs in cell lysate towards these di-Ub probes, revealed the importance of S1 and



S2 Ub binding pockets in DUBs to confer the specificity towards di-Ub chain cleavage.

Pe pt id e

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Figure 4: Activity based probes based on mono-Ub and di-Ub. 3.3 DUBs Assays


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Studying Ub signaling revealed that DUBs are key components of this pathway and are involved in the regulation of all Ub-mediated cellular functions. Aberration in DUBs functions is associated with various diseases such as neurodegenerative diseases20 and cancer.21 Hence it is highly important to establish assays to monitor DUBs activities, which would provide the basis to design novel inhibitors. Reagents like Ub-based assays have been influential to map the reactivity of DUBs and in executing highthroughput screening to identify potent inhibitors.93 Several fluorescence Ubpeptide and di-Ub based assays have been developed with mimicry elements of DUBs substrates to assist in the identification of inhibitors for some class of DUBs such as USP and UCH.73 Ub-amino-4-methylcoumarin (Ub-AMC) is the first Ub-based assay that was developed (Figure 5A) and employed in understanding enzyme kinetics of several DUBs as well as used to identify DUBs inhibitors.94 However, this assay bears a peptide bond between the fluorophore and Ub and does not include a recognition element at the proximal end. Hence connecting the fluorophore with Ub through an isopeptide bond would provide more precise information on DUB activity.

To meet this requirement, a fluorescence

polarization (FP) assay bearing 5-carboxytetramethylrhodamine (TAMRA) linked to N-terminus of a short peptide derived from the nearby sequence of Lys48 in Ub was prepared (Figure 5B).49 An enzymatic approach was followed to conjugate the Ub monomer to the TAMRA linked short peptide. In this assay, upon cleavage of the short peptide linked to Ub, changes in the FP can be followed to reflect the activity of the employed DUB. Recently, an improved method to prepare the TAMRA based assay was reported where δ-



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mercaptolysine was used to conjugate Ub to the short peptides labeled with TAMRA.95 Our laboratory prepared a quenching pair assay for practical high-throughput screening (HTS).96

The assay is based on a synthetically labeled

ubiquitinated peptide with the Dnp (2,4 dinitrophenyl) and MCA (7methoxycoumarin-4-acetic acid) quenching pair (Figure 5C). The model peptide derived from p53 was synthesized using SPPS where its N-terminus was modified with MCA. Subsequent SPPS of Ub(46-76) was carried out through the Lys residue of the peptide and Asp52 was replaced with Asp(Dnp). Ligation with Ub(1-45) thioester gave the ubiquitinated substrate bearing the quenching pair in a good yield.97 3.4 DUBs Inhibitors The progress in understanding of Ub-mediated pathways revealed the importance of DUBs in various cellular events where their deregulations lead to various diseases.20,

21

Not surprisingly, DUBs are considered potential

therapeutic targets and have motivated several research groups from academia and industry to search for potent inhibitors for members of this family. However, targeting DUBs is challenging since DUBs recognize large protein surfaces and adopt different conformations in the active and inactive forms. In addition, most DUBs belong to the Cys proteases family where selective targeting of the specific DUB is challenging. DUBs inhibition could be achieved by targeting the Ub-binding domains using proteins or small molecule inhibitors.98,99 Up to date there has been no success in converting


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DUBs inhibitors into practical drugs. The lack of easily accessible assay limits the efforts for developing potent and selective inhibitors for certain DUBs.

A)

O

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Emission

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NH2

O

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Figure 5: Ub based assays A) Ub-AMC B) TAMRA-linked ubiquitinated peptide C) Ubiquitinated peptide with quenching pair.



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We have been interested in addressing these issues by taking advantage of our quenching pair assay for monitoring DUBs activities.96 Our initial efforts to screen chemical libraries of compounds against USP2 and other DUBs led to the discovery of quinone-based compounds with high potency.100 One example of such a compound is the known natural product β-lapachone (1), which is in phase II clinical trial against cancer.101 It has been reported that the catalytic activity of Cys based DUBs is hampered by the exogenous oxidizing agents such as H2O2, which could convert the catalytic Cys to sulfenic acid (-SOH) or sulfinic acid (-SO2H) and sulfonic acid (-SO3H).102 Analyzing mechanistic aspect of β-lapachone mediated inhibition of USP2 revealed a similar mechanism of inhibition through irreversible oxidation of catalytic Cys by the reactive oxygen species generated by β-lapachone (Figure 6). This activity of β-lapachone against DUBs was never reported before despite the large effort to investigate its mechanism of action.103 Inspired by this finding we further turned our attention to understand the basis of quinones reactivity and their ROS generating capabilities for DUBs inhibitions. This resulted into the identification of 4-methoxy-substituted 1, 2naphthoquinones (2, 3) as a potent inhibitor for USP2. Electrochemical study of various quinones correlated nicely their structures, ROS generating capabilities and the inhibition profile against UPS2.104 Despite the current progress in small molecule based inhibitors,105 we are still lacking novel inhibitors with diverse scaffolds that have high selectivity and strong affinity towards DUBs. Protein based inhibitors offer high selectivity and potency for DUBs, however, exhibit other limitations such as their cellular delivery. On the other hand, small molecule based inhibitors with high


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reactivity towards catalytic Cys falls short in terms of selectivity. Developing new inhibitors, which offer both selectivity and strong affinity, would be highly desirable in DUBs based drug development.

Figure 6: Quinone based small molecules as DUB inhibitors and their mechanism of action. 4. Summary and Future Outlook Nearly four decades have passed since the first report of involvement of Ub in proteasome mediated protein degradation.106 The journey of unraveling Ubsignalling in mediating several cellular events has witnessed several breakthroughs in the field. This has expanded our understanding of Ub signalling beyond proteasomal degradation. The diversity in Ub chain linkage types and lengths makes the Ub signal more complex than ever realized. Enzymatic approaches, which were the sole source for accessing polyUb chains and polyubiquitinated proteins during initial phase of the study, have assisted in understanding fundamental features of this signal. However, these approaches have severe limitations to address various aspects of the complexity involved in Ub signalling. These shortcomings inspired chemical biologists to develop various synthetic approaches to access defined Ub 37 Environment ACS Paragon Plus


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chains, ubiquitinated proteins and various probes and assays to study ubiquitination and its machinery107,

108

as well as deubiquitination28. These

strategies have already proven extremely powerful in many studies culminating in better understanding the complexity of Ub signalling. Starting from the synthesis of Ub monomer to various complex polyubiquitinated proteins, the current chemical toolbox already offers access to various Ub conjugates with precise control over their atomic structures. The non-native linked Ub conjugates have also proven useful in studying structural aspects of the DUBs.58 On the other hand, native isopeptide bond linked Ub conjugates have expanded our knowledge about the linkage type and minimal lengths of Ub chain required for the degradation of specific protein44,67,109 and also aided in the identification of interacting proteins.110 In addition, specificity of various DUBs towards different linkages has also been elucidated.24 Advancements in the development of Ub and di-Ub based probes have significantly aided identification of several DUBs and understanding their structural aspects to reveal the basis of their selectivity.28 Additionally, different fluorescent-based assays have also been developed to map the activity of DUBs. This has led to the development of several DUBs inhibitors.73 The cross talk between PTMs, where multiple PTMs are involved in particular biological function, whether in the same protein or different proteins, is the basis of regulation of many cellular events. For example and as described above Ub itself is regulated by different PTMs. In addition, ubiquitination in the context of various systems is affected by other PTMs such as in the case of chromatin. Studying such systems in vitro add even more hurdles for the


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molecular biology approaches. Recently, for example, we were able to give the first direct evidence that phosphorylation of Tyr57 in H2A inhibits the removal of Ub from H2B within the same nucleosome.111 The Muir group was able to generate libraries of nucleosome with different combination of PTMs employing the concept of DNA-barcoding.112 This enabled the group to investigate the effect of different PTMs on various histone mark readers and writers highlighting the flexibility given by chemistry to access these complex systems for various studies. Altogether, these advancements significantly contribute in decoding the Ub code and in understanding its dysregulation, which leads to various diseases. However, efforts in the field continue to surprise us about the role of the Ub signalling in crucial cellular processes and the different modes of their regulations, which suggest that the mysteries of Ub signalling is still far from uncovered. For instance, recently the role of Ub branched chains in cell cycle regulation and in NF-κB signalling has been discovered.113 These unique chains with different topologies might also be involved in mediating other cellular events. The preparation of these conjugates would help to understand their structural and functional features and other biochemical aspects related to how DUBs process these branched chains. Similarly much has to be learned about the atypical chains and the different processes that they regulate. Proteins bearing atypical chains would assist studies of how enzymes assemble and disassemble these chains and what the interacting protein partners inside the cell are.



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Another important class of modifications related to Ub research is the Ub like modifiers. Several Ub like modifiers have been discovered such as SUMO, NEDD, ISG15.114 These modifier proteins bear Ub like globular β-grasp fold, however, present remarkable differences in their cellular functions. Similar, but distinct Ub types of enzymatic cascade mediate these PTMs. The physiological roles of these Ub like modifiers are continuing to emerge. However, in many cases little is known about the roles of these modifications in biological processes. Studies of this diverse class of modifiers have been hampered due to the limited knowledge of their conjugating enzymes, which limit their preparation. Since the connectivity between the Ub like modifiers and the protein substrate is also via an isopeptide bond, the lessons learned from the synthesis of various Ub conjugates could be applied to the preparation of proteins decorated with Ub like modifiers to decipher their role in different cellular events and associated diseases. Indeed, several labs have already pursuing these directions and exciting results from these studies will surely emerge.115-118 A powerful application of the area of chemical synthesis of proteins in general and ubiquitin based conjugates in particular, is the possibility of delivering these synthetic bioconjugates into the cell. This would allow examining novel biological aspects related to these biomaterials and enable more meaningful insight and correlation between the biochemical and cellular results. Protein delivery to cells is an exciting field and several new technologies have emerged, which could aid in such a mission.119,

120

The ability to modify the

ubiquitin conjugates with essentially any desired modification, native or artificial, could open new opportunities to probe a particular pathway and its


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relevance to certain diseases. Indeed, in a recent example it has been shown that by using synthetic Ub based activity probes in live cells one could follow the enzymatic cascade of the ubiquitination event, which may assist in understanding the regulation of these pathways and the associated pathogenesis as well as monitoring the cellular efficacy of inhibitors that directly target the E1-E2-E3 cascade.107 In another example, the site specific modifications of cellular histones using a protein trans-splicing approach combining chemical protein synthesis and the delivery of the histone modified fragment have also been shown, which could bring great excitement to the epigenetic field.121 In all these aspects and more, chemistry will play a key role by providing unique and novel reagents where many could not be prepared otherwise. The synthesis of required Ub conjugates for studying specific aspects of Ub signalling has now become possible for many open questions in this area. While it appears that the major chemical challenges have been solved in this field, certain questions may require new reagents, which could enforce new synthetic challenges. Hence there is still a great need to explore and develop novel synthetic methods, which could assist in reducing the lengthy synthetic steps involved in these strategies. Such advances could also enable the preparation of a generic oligo-Ub construction kit, which would expand the uses of these reagents to the non-synthetic community. Success in these directions will significantly accelerate studies that otherwise would not be possible. This vibrant field and its related areas will keep many laboratories engaged for many years to come and chemistry will certainly shine in many future discoveries.



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Acknowledgements We thank the Israel Science Foundation for financial support (AB). A. Brik is a Neubauer Professor and a Taub Fellow-Supported by the Taub Foundations; SMM is thankful to the Schulich Foundation for a postdoctoral fellowship. We also wish to thank Dr. Indrajit Sahu for his comments on this perspective.

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Hershko, A.; Ciechanover, A. Annu. Rev. Biochem. 1998, 67, 425.

(3)

Pickart, C. M. Annu. Rev. Biochem. 2001, 70, 503.

(4) Goldknopf, I. L.; French, M. F.; Musso, R.; Busch, H. Proc. Natl. Acad. Sci. U S A 1977, 74, 5492. (5)

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