An Approach for the Generation of Ubiquitin Chains of Various Topologies Based on
Bioorthogonal Chemistry
Dissertation
submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.)
Presented by
Xiaohui Zhao
at the
Faculty of Science Department of Chemistry
Date of the oral examination: 14.02.2017 First referee: Prof. Dr. Andreas Marx Second referee: Prof. Dr. Martin Scheffner Chairperson: Jun. Prof. Dr. Michael Kovermann
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-398677
Zusammenfassung ... I Abstract ... I
1 Introduction ... 1
1.1 Ubiquitylation ... 1
1.1.1 Writing - an enzymatic cascade ... 1
1.1.2 Erasing - a deubiquitylation pathway ... 3
1.1.3 Reading - ubiquitylation signaling ... 4
1.1.4 Regulation - a multilayer network ... 4
1.2 Diversity of ubiquitylation signaling ... 5
1.2.1 Mono- and multimono-ubiquitylation ... 5
1.2.2 Polyubiquitylation ... 6
1.3 Bioconjugate chemistry in Ub chain formation ... 9
1.3.1 Cysteine - based chemistry ... 9
1.3.2 Click reaction ... 11
1.3.3 Native chemical ligation ... 13
1.3.4 Other chemical methods ... 14
1.4 Ubiquitin - based probes ... 15
2 Aim of the thesis ... 18
3 Results and discussion ... 19
3.1 Ubiquitin dimers ... 19
3.1.1 Introduction ... 19
3.1.2 Generation of Ub dimers ... 21
3.1.3 Characterization of Ub dimers ... 25
3.1.4 E6AP auto-ubiquitylation assay ... 28
3.1.5 Conclusion ... 29
3.2 Ubiquitin trimer ... 30
3.2.1 Introduction ... 30
3.2.2 Generation of Ub trimer Ub3-PA48 ... 31
3.2.3 Conclusion ... 34
3.3 Homogeneous ubiquitin chains ... 35
3.3.1 Introduction ... 35
3.3.2 Generation of bifunctionalized Ub monomers ... 36
3.3.3 Reaction kinetics of Ub polymerization... 38
3.3.4 Analysis of characteristic polymerization behavior ... 41
3.3.5 Characterization of homogeneous Ub chains ... 43
3.3.4 Conclusion ... 44
3.4 Branched ubiquitin chains ... 46
3.4.1 Introduction ... 46
3.4.2 Generation of branched Ub chains ... 47
3.4.3 Characterization of branched Ub chains ... 50
3.4.4 Conclusion ... 53
3.5 Ubiquitin dendrimers ... 54
3.5.1 Introduction ... 54
3.5.2 Generation of the trifunctionalized Ub monomer ... 55
3.5.3 Generation and characterization of Ub dendrimers ... 56
3.5.4 Conclusion ... 57
3.6 Ubiquitin-based photo-crosslinking probes ... 58
3.6.1 Introduction ... 58
3.6.2 Generation of probes Ub2-BP48SH and Ub2-BP48Bio ... 59
3.6.3 Photo-crosslinking of probe Ub2-BP48SH and Iso T ... 62
3.6.4 Detection of Ub-accociated proteins in HEK cell lysate ... 63
3.6.5 Conclusion ... 65
4 Conclusion and outlook ... 66
5 Materials ... 68
5.1 Reagents ... 68
5.2 Kits ... 70
5.3 Equipment and software ... 70
5.4 Primer and oligonucleotides ... 71
5.5 Plasmids ... 72
5.6 Enzymes ... 73
5.7 Antibodies... 73
5.8 Medium ... 73
5.9 Buffers and solutions ... 74
6 Methods ... 77
6.1 Construction of plasmids ... 77
6.1.1 Molecular cloning ... 77
6.1.2 Competent cells and transformation ... 79
6.2 Synthesis of the trifunctional linker ... 81
6.3 Generation and characterization of Ub chains ... 82
6.3.1 Protein expression, purification and modification ... 82
6.3.2 Generation of Ub chains ... 85
6.3.3 Characterization of Ub chains ... 88
7 Appendixes ... 91
7.1 Sequences ... 91
7.2 NMR ... 94
7.3 Mass spectrometry ... 96
8 Abbreviations ... 104
9 References ... 106
10 Acknowledgements ... 111
I
Zusammenfassung
Eine der verbreitesten Posttranslationalenmodifikationen (PTMs) in eukaryotischen Zellen ist die Ubiquitinierung, die durch die Modifizierung von Substratproteinen mittels des 76 Aminosäuren großen Proteins Ubiquitin (Ub) charakterisiert ist. Ubiquitinierung spielt in einer Vielzahl von zellulären Prozessen, wie zum Beispiel Proteinabbau, Zellteilung, DNA Reparatur, Autophagie oder intrazellulärem Transport eine bedeutende Rolle. Deregulierung der Ubiquitinierung steht in Verbindung mit einer Vielzahl an Krankheiten wie zum Beispiel neurodegenerative Erkrankungen oder Krebs.
Ubiquitinierung von Substratproteinen erfolgt durch eine enzymatische Kaskade bestehend aus aktivierenden Enzymen (E1), konjugierenden Enzymen (E2) und Ligasen (E3), durch die Ub letztlich auf das Substratprotein übertragen wird. Die kovalente Bindung von Ub zum Substaratprotein erfolgt durch die Bildung einer Isopeptidbindung zwischen der Karboxylgruppe des C terminalen Glycin eines Ubiquitinmoleküls und einer ε-NH2 Gruppe eines Lysin des Substratproteins. Die Sequenz von Ub weist 7 Lysine auf (K6, K11, K27, K29, K33, K48, K63), die ebenfalls als Substrate für die Ubiquitinierung dienen können, was zur Bildung von Ubiquitinketten führt. Resultierend daraus kann ein Substrat nicht nur mit einem Ub-Molekül modifiziert werden (Monoubiquitinierung), sondern auch mit Ub-Ketten (Polyubiquitinierung). Es ist bekannt, dass das menschliche Genom aus zwei E1 Enzymen, über 30 E2 Enzymen und etwa 600 E3 Enzymen besteht. Die spezifische Kombination von E2 und E3 Enzymen bestimmt die Form der Polyubiquitinierung. Neben homogenen Ketten wurden auch heterogene Ketten, verzweigte Ketten und gemischte Ketten, bestehend aus Ub und ubiquitinähnlichen Proteinen beobachtet. Die Modifizierung von Substratproteien durch Ub kann durch Deubiquitinasen (DUBs) umgekehrt oder modifiziert werden, wodurch der reversible und dynamische Charakter der Ubiquitinierung ersichtlich wird. Die Vielfältigkeit der Ubiquitinierung zeigt sich außerdem auf einer weiteren Ebene, die durch die Modifizierung von Ub oder den Enzymen des Ubiquitin Systems charakterisiert ist. Durch Phosphorylierung von Ub, E3 Ligasen oder DUBs können deren Funktionen und Eigenschaften auf vielfältige Weise modifiziert werden. Somit zeigt sich die Komplexität und Vielfältigkeit des „Ubiquitin Codes“, durch den eine Vielzahl an zellulären Prozessen, ausgehend von einem einzigen Protein gesteuert werden kann.
Zum aktuellen Stand ist der Ubiquitin Code jedoch nur teilweise entschlüsselt. So sind zum Beispiel die zellulären Funktionen von K6-, K27-, K29-, und K33-verknüpften, homogenen Ketten noch wenig erforscht. Ähnlich verhält es sich mit verzweigten Ketten deren Untersuchung mit K11/K48 verknüpften Ketten erst am Anfang steht. Funktionelle Untersuchungen der genannten Kettentypen
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waren lange schwierig, aufgrund der mangelnden Verfügbarkeit der entsprechenden Ketten. Die hohe Dynamik der Ubiquitinierung, sprich das Zusammenspiel von Synthese und Abbau der Ketten, behinderten eine systematische Untersuchung. DUBs, die in eukaryotischen Zellen in relevanten Mengen exprimiert werden, tragen zu dieser Dynamik maßgeblich bei. Aus diesem Grund ist die Herstellung von nicht hydolysierbaren, bindungs- und längen-spezifischen poly-Ub Ketten in ausreichender Menge von sehr großer Bedeutung. Der Einbau von reaktiven Funktionalitäten in diese poly-Ub Ketten erweitert zusätzlich das Einsatzspektrum.
In dieser Arbeit wurde eine vielseitige Methode entwickelt, mit deren Hilfe poly-Ub Ketten mit verschiedenen Topologien und spezifischer Bindung generiert werden können. Dazu zählen zum Beispiel Ub-Dimere und -Oligomere, homogene poly-Ub-Ketten, verzweigte poly-Ub-Ketten sowie Ub -Dentrimere. Dieser robuste und einfache Ansatz basiert auf Modifizierung von Proteinen im zellulären System, bioorthogonaler Modifizierung der gereinigten Proteine und Kupfer katalysierter Azid-Alkin Cykloaddition (CuAAC). Durch „selective pressure incorporation“ (SPI) wurde der effiziente Einbau von Azidohomoalanin (Aha) in den C-Terminus von Ub gewährleistet. Zusätzlich konnte eine Thiol-Gruppe an einer ausgewählten Position durch Mutation eines Lysins zu einem Cystein in die Ub Sequenz eingebracht werden. Neben dem so eingebrachten Cystein wiest die Ub Sequenz kein Cystein auf. Im Folgenden wurde das modifizierte Ub-Molekül mit einem Alkin funktionalisiert, durch Michael Addition von Propargylacrylat (PA) und der Thiolgruppe des Ub. Anschließend konnten mit den Azid- und/oder Alkin- modifizierten Ub-Molekülen und CuAAC poly-Ub-Ketten mit definierter Topologie generiert werden. Es konnte gezeigt werden, dass die triazol-verknüpften Ketten eine ähnliche Konformation aufweisen wie physiologische poly-Ub-Ketten, sowie unempfindlich gegenüber DUB-katalysierter Hydrolyse waren. Des Weiteren wurde ein biotinyliertes, auf diUb basierendes Molekül entwickelt, das zu photo-crosslinking Experimenten verwendet werden kann.
Hiermit konnte die Interaktion mit DUBs (z.B. Isopeptidase T) und weiteren Proteinen des Ubiquitinsystems in HEK293T Zellextrakten gezeigt werden.
I
Abstract
Modification of the protein by ubiquitin (Ub), a highly conserved 76-amino acid polypeptide, is one of the most prevalent post-translational modifications (PTMs) in eukaryotic cells. Ubiquitylation participates in numerous cellular processes, including protein degradation, cell division, DNA repair, autophagy, and intracellular trafficking. Dysregulation of the ubiquitylation machinery has been found relating to various human diseases, like cancer and neurodegeneration.
Ubiquitylation is mediated by a three-step enzymatic cascade: it is catalyzed by activating enzyme E1, conjugating enzyme E2, and ligase E3, by which Ub is finally transferred onto the substrate protein.
Isopeptide bond is formed between the carboxylate of a C-terminal glycine of Ub and the ϵ-NH2- group of a lysine within the target protein. Ub contains seven lysine residues (i.e., K6, K11, K27, K29, K33, K48 and K63) and thus, is a substrate for ubiquitylation itself. As a result, the target protein can be modified with either a single Ub molecule (mono-ubiquitylation) or a polyUb chain (poly- ubiquitylation). It is known that the human genome encodes two E1s, more than 30 E2s and nearly 600 E3 enzymes. The combination of E2 and E3 enzymes determines the poly-ubiquitylation types (also termed ubiquitin code) on substrate proteins, including homogeneous and heterogeneous chains, branched chains, and chains mixed with Ub-like proteins. Noteworthy, ubiquitylation is a reversible protein modification pathway, so that the Ubs tagged on the substrates can be erased or edited by a superfamily of isopeptidases known as deubiquitinases (DUBs). As a multilayer network, ubiquitylation process is regulated by none-Ub PTMs in versatile ways, such as phosphorylation on Ub molecules, E3 ligases or DUBs. In a word, ubiquitylation is one of the most complex and language- rich regulators of biological processes.
However, so far, most of the ubiquitin codes are still less well understood. Comprehensive investigations of the ubiquitylation types, the structural and functional studies in vitro or in vivo, have been significantly hampered by the restricted availability of specific polyUb chains, as well as the dynamic interplay of ubiquitylation and de-ubiquitylation. The isopeptide-linked Ub chains can be rapidly disassembled by DUBs that are highly abundant in eukaryotic cells, resulting in for example the identification of their interacting partners rather difficult. Thereby, generation of non- hydrolysable linkage- and/or length-defined Ub chains in sufficient quantities is of primary necessity.
In this thesis, a universal approach has been developed for the generation of polyUb chains of various topologies, including Ub dimers and oligomers, homogeneous Ub chains, branched Ub chains, and Ub dendrimers. This facile and robust approach is based on protein engineering in a cellular
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system, protein post-expression bioorthogonal modification, and protein conjugation by copper(I)- catalyzed azide-alkyne cycloaddition (CuAAC). In brief, efficient incorporation of a single unnatural amino acid azidohomoalanine (Aha) into the C-terminus of Ub via selective pressure incorporation (SPI) has been exploited. Due to the absence of cysteine, Ub can be readily equiped with sulfhydryl groups at desired positions in place of lysine residues. Subsequently, the engineered Ub is able to be functionalized with alkynes via Michael addition by propargyl acrylate (PA) in tubes. Taking use of the azide- and/or alkyne-functionalized Ub monomers, CuAAC enables the chemical assembly of versatile polyUb of defined chain topology. It is noteworthy to mention that the triazole-linked Ub chains have been examined adopting native-like conformation and resisting the DUB-catalyzed hydrolysis.
Moreover, a biotinylated diUb-based photo-crosslinking probe has been raised, which highlights the potential in crosslinking DUB enzymes (e.g., isopeptidase T) and detecting Ub-asscociated proteins in HEK293T cell extracts.
1
1 Introduction
1.1
UbiquitylationThe diversity of human proteome is far beyond the prediction based on the coding capacity of 30,000 human genes. So far, more than 27,000 protein isoforms have been identified and this number is estimated to be more than one million.[1,2] The expansion of protein diversity happens after translation level is called post-translational modifications (PTMs).[1] The major types of protein modification are phosphorylation, methylation, acetylation, glycosylation, ubiquitylation, and ubiquitin-like modification (SUMOylation, Neddylation, etc.). Among them, ubiquitylation and ubiquitin-like modification are unique, as they modify the substrate with a polypeptide.[3]
1.1.1 Writing - an enzymatic cascade
Ubiquitin (Ub) is a highly conserved 76-amino acid globular protein found in all eukaryotic cells. Ub contains seven lysine residues (i.e., K6, K11, K27, K29, K33, K48 and K63), which orient to distinct directions and have different solvent-accessible surface area (Figure 1.1). K6 and K11 are located next to the β1/β2 loop, a dynamic region of Ub, which may undergo conformational changes in the context of a chain or upon associated protein binding.[4] K27, K29 and K33 are located on the same α-helix. K27 is buried, while K29 has an intermediate level of solvent exposure, and K33 is greatly exposed. K48 and K63 are in the loop region and calculated highly exposed.[5,6]
Figure 1.1 Structural features of ubiquitin (PDB 1ubq), indicating left: the surfaces with Ile44 (red), Ile36 (magenta), Phe4 (orange) patches, and TEK-box (yellow), middle: seven lysine residues, N-terminal methionine and C-terminal glycine, right: acetylation sites (magenta) and phosphorylation sites (yellow).
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Ubiquitylation is mediated by a three-step enzymatic cascade (Figure 1.2): the activating enzymes E1 activate Ub by forming a high-energy thioester bond between the catalytic cysteine residue of E1 and the C-terminus of Ub via an ATP-consuming reaction. Subsequently, the activated Ub is transferred to the catalytic cysteine residue of conjugating enzymes E2. By the combined action of E2 enzymes with E3 Ub ligases, Ub is transferred onto the substrate protein by either a direct or a non-direct manner.[7-11] E3 ligases are classified into three major families: the HECT (homologous to E6-AP C- terminus) domain E3s, the RING (really interesting new gene) domain E3s, and the RING-between- RING (RBR) E3s. HECT and RBR E3 ligases form an intermediate thioester with Ub before transfer to the substrate, whereas the RING E3s act as adaptors and aid the transfer from E2 directly to substrate.[12-14]
Figure 1.2 Scheme for protein ubiquitylation, deubiquitylation and ubiquitylation signaling.
Substrate proteins are modified with either one Ub (mono-ubiquitylation) or a polyUb chain (poly- ubiquitylation), in which Ub moieties are connected via the ϵ-NH2-group of a lysine residue or the N- terminal α-NH2 group in the proximal Ub and the carboxylate of the C-terminal glycine of a distal Ub.
The formed linkage is commonly referred as an isopeptide bond. The polyUb chains can be short as oligomers, containing only two or three Ub molecules or as long as up to ten Ub moieties.[15]
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Proteomic studies have confirmed that each of the seven lysine residues can be used in the polyUb chain formation.[16,17] The distinct linkages exist in vastly different abundances, in yeast with K48 (29%), K11 (28%), and K63 (16%) being the most abundent linkages, and K6 (11%), K27 (9%), K33 (4%), and K29 (3%) being more rare. [18] However, in higher eukaryotes, for example mammalian HEK293 cells, K11-linkage only accounts for approximately 2% of the total linkage content.[19]
So far, it is known that the human genome encodes two E1s, more than 30 E2s and nearly 600 E3 enzymes.[20] The combination of E2 conjugating enzymes and E3 ligases determines the ubiquitylation of substrate proteins at specific positions with certain number of Ub moieties and defined linkages.
These ubiquitylation types are classified into mono- or multi-monoubiquitylation, homogenous or heterogeneous polyUb chain modification, branched chain or mixed chain with Ub-like proteins (UBLs)[21] modification (Figure 1.3). The theoretical unlimited number of chain types makes ubiquitylation the most complex and language-rich regulation machinery of biological processes.[21-23]
Due to the complexity of the ubiquitylation machinery, thereby from another point of view, the possibility for a (disease-causing) mutation in the regulation of substrate proteins is relatively high.
Dysregulation of the ubiquitylation process has been found relevance to various human diseases, such as cancer, neurodegenerative diseases, metabolic syndromes, autoimmunity, inflammatory disorders, infection and muscle dystrophies.[24]
1.1.2 Erasing - a deubiquitylation pathway
Ubiquitylation is a reversible protein modification pathway, which enables the strict regulation of substrate proteins in a spatiotemporal manner. Ub codes can be erased and edited by a superfamily of isopeptidases named deubiquitinases (DUBs, also known as deubiquitinating enzymes).[25-27]
Human genome encodes approximately 100 DUBs which are classified into five families: ubiquitin C- terminal hydrolase (UCH), ubiquitin-specific protease (USP), ovarian tumor protease (OTU), Josephin/Machado-Joseph disease protease (MJD) and JAB1/MPN/MOV34 metalloenzyme (JAMM, also known as JAMM/MPN+).[28,29] The UCH, USP, OTU and MJD families are cysteine proteases, in which the thiol group located in the active site acts as a nucleophile that attacks the carbonyl group of the isopeptide bond. While for JAMM metalloproteases, an activated water molecule directly hydrolyzes the isopeptide bond with the help of a zinc atom coordinated by two histidines and one glutamate. DUBs, especially USPs, normally contain multiple domains that probably mediate protein- protein interactions. In common, they are abundant of various ubiquitin binding domains (UBDs) which are critical in targeting the polyUb chains.
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In cells, deubiquitylation functions in three major ways.[28] The first and a key issue of DUBs is to generate free Ub molecules from the precursor, a linear fusion consisting of multiple copies of Ub.
Second, DUBs remove the Ub modification on the substrate, either the proteolytic signal thus rescuing the protein from proteasomal or lysosomal degradation, or the non-degradative signal to abolish the downstream protein binding. Meanwhile, disassembly of the anchored polyUb chains maintains the homeostasis of the free Ub pool. Third, by trimming the polyUb chains, DUBs can edit the Ub codes thus regulating the cellular processes in a spatiotemporal manner.
1.1.3 Reading - ubiquitylation signaling
Ub-binding proteins (also known as Ub receptors) interpret and transmit the information conferred by protein ubiquitylation. [30] In the Ub-binding proteins, some small (20-150 amino acids) ubiquitin binding domains (UBDs) are found interacting directly with monoUb and/or polyUb chains.UBDs also exist widely in ubiquitylation or deubiquitylation enzymes with different structural features and distinct functions. The already known UBDs are structural diverse and classified into ubiquitin- interacting motif (UIM), ubiquitin-associated domain (UBA), zinc finger ubiquitin-binding motif (NZF), polyubiquitin-associated zinc finger (PAZ), GRAM-like ubiquitin-binding in Eap45 domains (GLUE), ubiquitin-conjugating enzyme variant (UEV) and other subfamilies. [31,32]
Ub is often recognized by UBDs at a hydrophobic surface (Figure 1.1) consisting of Leu8, Ile44, His68 and Val70, which is known as Ile 44 patch. This hydrophobic patch also contributes to the Ub-Ub interaction in the polyUb chains, such as in K48-linked chains. Another hydrophobic surface is centered on Ile36 and involves Leu71 and Leu73 at the Ub tail motif. The Ile36 patch can mediate interaction between Ub moieties in chains as well, but it is mainly responsible for the linkage- selective recognition by various UBDs.[30,33] A surface containing Gln2, Phe4, and Thr12 is named Phe4 patch. It interacts with the USP domain of DUBs that might function in linkage-specific hydrolyzation.[34] In addition, the TEK-box, a three dimensional motif that includes Lys6, Lys11, Thr12, Thr14 and Glu34, is required for the elongation of K11-linked chains by Ube2S and anaphase- promoting complex (APC/C).[15,35]
1.1.4 Regulation - a multilayer network
Regulating the ubiquitylation machinery by none-Ub PTMs is mainly in three categories.[36] Firstly, Ub itself can be acetylated or phosphorylated (Figure 1.1). Acetylation at K11 or K48 inhibits E2- mediated elongation of K11- or K48-linked polyUb chains.[37] Eight phosphorylation positions (Thr7,
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Thr12, Thr14, Ser20, Tyr59, Ser65, and Thr66) have been observed in mammalian cells. Protein kinase PINK1 catalyzes the Ser65 phosphorylation that impacts the chain formation and disassembly, chain linkage distribution, Ub-Ub interaction, and substrate targeting.[38,39]
Secondly, enzymes involving in the ubiquitylation process can undergo none-Ub PTMs.
Phosphorylation-induced conformation change can stimulate or inhibit the activity of E3 ligases [40,41]
and DUBs.[42] A well-known example is Parkin, which is promoted to active conformation through the combinational phosphorylation of its Ubl domain and Ub Ser65.[43,44]
Thirdly, substrate proteins are intensively none-Ub modified (acetylation, etc.), which engages crosstalk with ubiquitylation.[45] For instance, acetylation can compete with ubiquitylation at specific lysine residues that enhances the stability of substrate proteins, or promotes their proteasomal degradation by enforcing ubiquitylation on the other lysine sites.[46,47]
Furthermore, interference of ubiquitylation processes with small molecules has been intensively studied.[48] In the writing process, the activity of E1, E2, and E3 enzymes can be specifically blocked by the inhibitors. For example, inhibition of a E3 ligase Mdm2, which targets the tumor suppressor protein p53 for proteasomal degradation, shows promising results in the clinical trials for cancer treatment.[49] In addition, DUBs and proteasome are well-investigated targets of small molecule inhibitors.[50,51] Velcade (Bortezomib) is highlighted the first FDA-approved compound towards the proteasome.[52]
Last but not the least, a novel concept termed small-molecule-based proteolysis-targeting chimeras (PROTACs) has been raised in recent years.[53,54] The bifunctional molecule serves as a bridge between a ubiquitously expressed E3 ligase (e.g. CRL4) and a harmful protein, thus enabling the ubiquitylation of the target protein for proteasomal clearance.
1.2 Diversity of ubiquitylation signaling
1.2.1 Mono- and multimono-ubiquitylation
Mono-ubiquitylation is defined the attachment of a single Ub molecule on the specific lysine residue of substrate protein.[55,56] Usually, mono-ubiquitylation functions in a non-proteolytic way, such as DNA repair and endocytosis. In response to DNA damage, proliferating cell nuclear antigen (PCNA) is mono-ubiquitylated at the highly conserved K164 residue, thus recruiting damage-tolerant DNA polymerases for the translesion synthesis (TLS).[57,58,59] However, it is found that monoUb fusing to polypeptides (up to 150 residues) is enough for proteasomal degradation.[60] Other
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monoubiquitylated substrates, like paired box 3 protein (PAX3)[61] and cell adhesion receptor Syndecan 4 (SDC4) [62], can be recognized and degraded by proteasome.
Multimono-ubiquitylation refers to the modification of a substrate protein on several lysine residues with Ub molecules. For some proteins, such as cell-cycle regulator cyclin B1[63] or p105[64], a precursor of the NF-κB transcription factor p50, it is necessary to be modified by multiple Ubs for proteasomal degradation.
1.2.2 Polyubiquitylation
Polyubiquitylation is the most complex and diverse ubiquitylation mode. According to the chain length and linkage types, it is classified here into Ub oligomers, homogeneous or heterogeneous Ub chains, and branched Ub chains.[65-67]
Ubiquitin oligomers
Ubiquitin dimers are the minimal structural units of polyUb chains. In vitro assay indicates that Ub dimers can be uptaken by the ubiquitylation enzymes and serve as the buiding blocks in the assembly of polyUb chains.[68] Ub dimers also convey signals in protein degradation by proteasome. A very recent research finds that multiple diUb-modified proteins, such as securin, geminin and cyclinB, are more efficient in promoting proteasomal degradation than the tetraUb modified proteins, given the same number of conjugated Ub moieties.[69]
Crystallographic and NMR structural studies are mainly based on Ub oligomers.[70] So far, the crystal structures of Ub dimers linked at respective lysine residue have been reported. K6-,[71] K11-,[72] and K48-linked diUbs favor compact structure, in which Ub units bind each other through the hydrophobic interaction. In the K48-linked dimer, the Ub moieties interact via the Ile44 patches and two such diUb modules pack tightly to form a tetramer.[73] By NMR analysis, a minor population of K48 dimer in which the Ile36 patch of the distal Ub interacts with the Ile44 patch of the proximal Ub was identified.[74,75] Actually, in solution, the compact and open conformations are in fast exchange so that this equilibrium is critical for the recognition of the exposed hydrophobic patches.[75] In contrast, M1- and K63-linked diUb adopt open conformation with less contact between individual Ub units, which makes the linkage rotationally unstrained and highly flexible. The hydrophobic patches are completely exposed that enables binding of downstream signaling proteins.[76,77] The structural informations of K27-, K29- and K33-linked dimers or trimers have been reported very recently, which reveal that they adopt flexible conformations.[78-81]
7 Homogeneous and heterogeneous ubiquitin chains
Homogeneous Ub chains are defined that all Ub moieties link to each other by the isopeptide bond between the carboxylate of the C-terminal glycine of the distal Ub and the ϵ-NH2-group of an identical lysine of the proximal Ub. In addition, the N-terminal amino group may also be used in chain formation, which is termed as M1-linked or linear Ub chain. This chain type is thought as the precursor of the Ub pool. The linear polymers are firstly translated and then disassembled to the single Ub molecules.[28] In contrast, heterogeneous Ub chains contain multiple linkage types among the Ub units.
It is well-known that K48-linked chains target the substrate proteins for degradation through the ubiquitin-proteasome system (UPS).[18,82-85] In addition, K11-linked chain could be considered as the second proteolytic signal which facilitates proteasomal degradation of cell-cycle regulators during early mitosis.[86,87] Other chain types mediate protein degradation in UPS less frequently. K29-linkage is likely to convey the degradation signal, as the linkage type is enriched following proteasome inhibition. It drives protein turnover in the Ub-fusion-degradation (UFD) pathway.[88] Moreover, in vitro studies show that K63-linked chain can also be recognized by the proteasome and promote substrate degradation.[89]
For the non-proteolytic functions, it was found that K63-linked chains are intensively involved in DNA repair and other signal transduction.[90,91] M1-linked chain is assembled by LUBAC E3 ligase complex and plays an important role in NF-κB activation.[92] Except for the functions in cell-cycle regulation, K11-linkage has been identified in various cellular processes, including endoplasmic reticulum associated degradation (ERAD), membrane trafficking and TNFα signaling. But it acts mostly in conjunction with other linkage types, so called heterogeneous chains.[18,86,87]
Little is known about K6-, K27-, K29- and K33-linked chain types, which also have low abundance in cells.[18] The enzymes assembling these chain types are poorly explored. Mass spectrometry analysis finds no increasing abundance of K6-linkage after proteasome inhibition, suggesting that it involves in non-proteolytic pathways.[93] Recent research shows a direct link of K27-linked chain to DNA repair, that E3 ligase RNF168 mediates K27-linked ubiquitylation of H2As upon DNA damage.[94] K33-linked ubiquitylation of ζ-chain of TCR (T cell antigen receptor) results in reduced phosphorylation and inhibits association with the kinase Zap-70.[95]
Branched ubiquitin chains
Ubiquitin chains in which a single Ub moiety is modified at two or more lysine residues are described as forked or branched Ub chains. The proteomic studies have discovered various branching sites,
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mainly adjacent sites, such as K6/K11, K27/K29 and K29/K33.[16,89] But theoretically, all seven lysine residues are possible to be branched. In addition to the factor of chain-length, the topologies of branched chains could be numerous.
So far, the physiological roles of only limited types of branched chains have been unveiled. Initial research by Goldberg and co-workers found that branched chains bind weakly to proteasome and are disassembled slowly by proteasome-associated DUBs.[89,96] Branched chain modified substrate proteins are poorly degraded by 26S proteasome, comparing to the rapid proteasomal clearance of the same substrate modified by K48-linked homogenous chain. However, recent study by Meyer and Rape reported that K11/K48-branched chains conjugating to the cell-cycle regulators can enhance substrate recognition and degradation by proteasome, comparing to substrates marked with K11- linked homogeneous chain.[97] They assumed that this enhancement might be due to the higher Ub density of branched structure. Furthermore, Nathan and co-workers showed that branched K11- linked Ub chains with K48-linkage stimulate proteasomal degradation of cyclinB1, which is more efficient than proteins modified by homogeneous chains.[98]
Figure 1.3 Diversity of ubiquitylation signalling.
9
1.3 Bioconjugate chemistry in Ub chain formation
In order to decode the ubiquitylation signaling, generation of sufficient amount of linkage-defined Ub chains or Ub-protein conjugates is of great significance. A straightforward way is to take advantage of the ubiquitylation enzymatic cascade in vitro. That is the so-called enzymatic assembling of polyUb chains by certain pairs of E2 conjugating enzymes and E3 ligases. Due to the deeper understanding of E2 and E3 enzymes, as well as the combinatory use of linkage-specific DUBs, various homogeneous Ub chains and Ub chain-protein conjugates have been formed.[99,100] However, enzymatic assembling has some limitations: First, available enzymes for specific chain types are underdeveloped. So far, enzymatic assembly of K27-linked chain is still unfeasible. And generation of branched chains and mixed chains at specific site by enzymatic assembling is impossible. Second, mutant versions of Ub, normally arginine instead of individual or specific combinations of lysine residues, are widely used to assemble the linkage-defined chains in vitro or in vivo. But these mutations might have influence to the chain conformation or associated protein binding. Furthermore, E3 ligases frequently act promiscuously so that the isolation of desired chain type with defined chain length from the enzymatic reaction mixture needs a lot of efforts. Last but not least, enzymatic assembling is not able to generate polyUb chains with non-hydrolyzable linkage and additional modifications, for example dyes, biotin, reactive groups.
For these reasons, researchers have developed various chemical or semi-chemical synthesis methods to generate a broad range of polyUb chains, Ub-protein conjugates, Ub-based probes.[101-105]
1.3.1 Cysteine - based chemistry
Protein conjugation methods mainly rely on reactions at native nucleophilic amino acids, particularly cysteine. Cysteine can undergo disulfide exchange to form mixed disulfide bonds and alkylation with electrophiles like α-halocarbonyl (iodoacetamide) and Michael acceptors (maleimide or acrylate).[106,107] Cysteine has a low natural abundance within proteins. The 76-amino acid ubiquitin has no cysteine residue. Moreover, cysteine has unique nucleophilicity, in comparison with other reactive side chains, such as lysine, serine and histidine. Under physiological conditions, it potentially forms the nucleophilic thiolate ion with a general pKa value of 8.2,[106] which is of course highly dependent on the local environment in terms of the neighboring residues and media.[108] In the case of Ub, the calculated pKa value of a single cysteine mutation is lower than the pKa of corresponding lysine (except for K27C with an increased pKa).[6] Therefore, fine-tuning the pH value of the reaction buffer enables a certain degree of selectivity beyond multiple lysine residues.
10
As the pioneering work, Wilkinson and co-workers reported the synthesis of Ub dimers linked at sites 11, 29, 48, and 63. They engineered a distal Ub with a cysteine mutation in place of the C-terminal glycine and the proximal Ub bearing a cysteine at desired site instead of lysine. 1,3-dichloroacetone (DCA) was used to ligate the two thiol-bearing Ub monomers, thus producing a conjugate stable to chemical and enzymatic cleavage (Table 1.1b).[109]
Fifteen years later, Pratt and co-workers utilized a new generation of reagent, 1,3-dibromoacetone, to ligate a C-terminus thiol-functionalized Ub to a cysteine mutated α-synuclein (Table 1.1c).[110]
Przybylski and co-workers developed an approach to synthesize a K63-linked Ub dimer. Two copies of the N-terminal piece of Ub (residues 1-52) containing a cysteine residue reacted with an isopeptide- linked piece (residues 54-76) functionalized with chloroacetyl group to assemble the whole Ub dimer linked via thioether bond (Table 1.1d).[111]
Muir and co-workers explored a method based on disulfide exchange for the site-specific ubiquitylation of a histone. The Ub-intein fusion was treated with cysteamine that intein-mediated trans-thioesterification and subsequent sulfur-to-nitrogen acyl shift yielded C-terminus aminoethanethiol modified Ub. Histone H2B (K120C) activated by DTNP (2,2’-dithiobis(5- nitropyridine)) conjugated to the Ub via disulfide bond (Table 1.1e).[112]
Based on the same idea, Raines and co-workers conjugated an Ub variant with an additional cysteine at the C-terminus to the proximal Ub bearing a 2-nitro-5-thiobenzoate (NTB) activated cysteine instead of the specific lysine residue. All seven disulfide-linked Ub dimers were generated and the rate constant of dimer formation was measured. They found the rate of disulfide exchange reaction at different positions is in correspondence to the abundance of linkage types in cells (Table 1.1f).[6]
Strieter and co-workers developed a robust approach based on free-radical thiol–ene coupling (TEC) to synthesize various Ub oligomers and even longer Ub polymers.[113-115] Ub C-terminal hydrolase (UCH) YUH1 furnishes the allylamine (AA) to the C-terminus of the distal Ub variant (UbD77). And proximal Ub bears a cysteine mutation in place of the specific lysine. TEC was performed using a free- radical initiator, lithium acyl phosphinate (LAP), to construct the Ub dimer. The formed Nε-Gly-L- homothiaLys bond showed same character as the native isopeptide bond which enables the study of linkage-specific hydrolyzation by DUBs (Table 1.1g).[116]
11 Table 1.1 Cysteine - based chemistry in Ub chain formation.
Moiety 1 Moiety 2 Product
a Native b
Wilkinson[109]
c Pratt[110]
d
Przybylski[111]
e Muir[112]
f Raines[6]
g
Strieter[116]
1.3.2 Click reaction
The modified Huisgen cycloaddition, also referred to Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) or “click reaction”, represents one of the most important bioconjugation methods reported to date.[117,118] By using only catalytic amount of Cu(I), the reaction can yield a non-hydrolyzable and isosteric 1,4-disubstituted 1,2,3-triazole ring at rate constants of 10-200 M-1s-1.[119] Moreover, the reaction can be performed in aqueous media with a broad range of pH value (approximately 4 to 12) and at varieties of temperature (0-160 °C). In addition, the reaction has a good chemoselectivity, which doesn’t interact with all other functional groups in proteins. The concerned limitation of click reaction used in bioconjugation, like protein damage, could be carefully avoided with using minimal amount of copper catalyst in combination with a proper ligand under inert gas atmosphere.
12
The primary consideration of using this method is to introduce the azide and alkyne functionalities to the protein. Thanks to the developed protein engineering methods, including intein fusion expression following trans-thioesterification, amber codon suppression (ACS) and selective pressure incorporation, as well as solid-phase peptide synthesis (SPPS), peptides and proteins can be functionalized with azide or alkyne at any position.[120,121]
Selective pressure incorporation (SPI) is highlighted here that is used in the thesis to introduce an azide to Ub.[122] It takes advantage of the tolerance of the unnatural amino acids (UAAs) by the endogenous aminoacyl tRNA synthetase (aaRS). With electrical and structural similarities to the natural counterpart, UAAs can be charged onto the corresponding tRNA for protein translation. For example, azidohomoalanine (Aha) is a methionine analogue. The auxotroph cell line, E.coli B834 (DE3) that is not able to produce methionine needs to be cultivated in the minimal medium supplementing with a high amount of desired UAA. The bacteria have to utilize the UAA in the whole proteome, including the overexpression of the required proteins.
Our lab introduced an Aha to the C-terminus of a distal Ub, and a lysine derivative Plk bearing a terminal alkyne to the specific position of a proximal Ub. Click reaction was performed to generate all seven Ub dimers. The isolated dimers can serve as building blocks in the E6AP autoubiquitylation assay.[68] Furthermore, Aha and Plk were engineered into a single Ub that can be self-polymerized to longer Ub chains (Table 1.2a).[123-126]
Based on the same idea, Mootz and co-workers introduced the functionalities to the Ub in a reverse way. Intein-Ub fusion was treated by propargylamine to yield C-terminus alkyne functionalized Ub.
The substrate protein was modified with an UAA p-azidophenylalanine (AzF) through ACS. Click reaction resulted in the site-specific Ub-protein conjugate (Table 1.2b).[127] Furthermore, they explored a biorthogonal method to modify cysteine mutated substrate with iodoacetamide ethyl azide, thus functionalizing substrate protein with an azide functionality (Table 1.2c).[128,129]
13 Table 1.2 Click reaction in Ub chain formation.
Moiety 1 Moiety 2 Product
a Marx[68]
b
Mootz[127]
c
Mootz[128]
1.3.3 Native chemical ligation
Native chemical ligation (NCL) or expressed protein ligation (EPL) is one of the earliest developed method in generation of Ub-protein conjugates. The advantage over other chemical tools is that it can assemble Ub chains with native isopeptide linkage. In NCL, the thiol group of a proximal Ub attacks the C-terminal thioester of the distal Ub. This reversible transthioesterification is chemoselective and regioselective and leads to form a thioester intermediate which subsequently undergoes sulfur-to-nitrogen acyl shift to yield a stable isopeptide bond.[130,131]
Muir and co-workers are pioneers in applying this approach to synthesize the ubiquitylated peptide.
The photo-cleavable ligation auxiliary mediated the conjugation of Ub-thioester specifically to one of the three lysine residues in a target peptide (Table 1.3a).[132]
Chin and co-workers expanded amber codon incorporation to introduce δ-thiol-lysine at the desired site of proximal Ub in place of a lysine residue. Ub dimer bearing native isopeptide bond at the specific site was obtained through the NCL followed by desulfurization (Table 1.3b).[133]
Brik and co-workers applied SPPS combining NCL widely in the generation of isopeptide-linked Ub dimers, oligomers, as well as (poly)ubiquitylated α-synuclein, α-globin and H2B (Table 1.3c).[134-139]
14 Table 1.3 Native chemical ligation in Ub chain formation.
Moiety 1 Moiety 2 Product
a Muir[132]
b Chin[133]
c Brik[139]
1.3.4 Other chemical methods
Silver-mediated protein condensation is an alternative way in generation of isopeptide-linked Ub chains. Chin and co-workers explored a method named GOPAL (genetically encoded orthogonal protection and activated ligation) which combines ACS, intein fusion expression and Ag-mediated isopeptide formation. An Ub-thioester obtained via intein fusion thiolysis was globally protected by N-(benzyloxycarbonyloxy)succinimide (Cbz-OSu). Another Ub bearing a Boc-protected lysine was generated via ACS followed by orthogonal Cbz-protection of other six lysine residues. After removing the Boc, protein ligation was conducted, followed by global amine deprotection, thus yielding K6- and K29-linked Ub dimers (Table 1.4a).[71]
Fushman and co-workers further developed this method and applied orthogonal amine protection/deprotection reagents to generate isotope-labeled heterogeneous, mixed and branched Ub chains (Table 1.4b). [140,141]
Oxime ligation is a chemoselective condensation reaction between an aminoxy function and an aldehyde to form a linkage that is non-hydrolyzable by DUBs. This method was firstly utilized in the Ub field by Ovaa and co-workers. The Ub is functionalized at the C-terminus with an aldehyde generated in situ from an acetal via chemoenzymatic reaction. An aminoxy-modified peptide was synthesized by SPPS and ligated to Ub-aldehyde to yield the ubiquitylated peptide (Table 1.4c).[142]
Very recently, Virdee and co-workers described the genetically directed incorporation of Boc- protected or photocaged aminooxylysine to a specific position of Ub. After deprotection, the Ub- aminooxyl ligated to an Ub-aldehyde to form the Ub dimer. It is also applicable to functionalize a single Ub with both the aminooxyl function and the aldehyde, thereby generating K6-linked Ub chains via self-polymerization (Table 1.4d).[143]
15 Table 1.4 Other chemical methods in Ub chain formation.
Moiety 1 Moiety 2 Product
a Chin[71]
b
Fushmann[141]
c Ovaa[142]
d
Virdee[143]
1.4 Ubiquitin - based probes
Ubiquitin-based probes are those Ub molecules, either monomers or dimers, equipped with reactive chemical groups. Almost all the reported probes are defined as activity-based probes (ABPs), in which an electrophilic “warhead” positioned either at the C-terminus of a proximal Ub or within the linkage site between two Ub moieties. These Ub-based ABPs have been extensively utilized to identify the enzymes participating in the ubiquitylation and deubiquitylation processes, including numerous E1, E2, E3, and DUBs.[104,105,144]
The first-generation probes comprise the Ub monomers equipped with vinyl sulfone (Ub-VS)[145], vinyl methyl ester (Ub-VME)[146], bromoethyl (Ub-Br2)[146] and propargyl (Ub-PRG) [147]. This kind of probes contributed greatly to the identification of novel types of DUBs belonging to the cysteine proteases (Table 1.5).
16 Table 1.5 MonoUb-based probes for DUBs.
Probe Name
a
Ploegh[145]
Ub-VS
b
Ploegh[146]
Ub-VME
c
Ploegh[146]
Ub-Br2
d Ovaa[147]
Ub-PRG
The Ub-adenylate mimical probes, which locate Michael acceptors at the E1 activating enzyme binding site, have been raised to deepen the understanding of E1 reactivity (Table 1.6a,b).[148,149] To further explore the ubiquitylation machinery, an elegant Ub probe bearing a thioacrylate or a thioacrylamide was developed to capture the E2 conjugating enzymes and the corresponding E3 ligases (Table 1.6c).[150] Of note, a probe Ub-Dha is able to be used in the covalent capture of E1, E2 and E3 enzymes through the sequential trans-thioesterification of the ubiquitylation processes (Table 1.6d).[151]
Table 1.6 MonoUb-based probes for writing enzymes.
Probe a
Tan[148]
b
Statsyuk[149]
c
Virdee[150]
d Ovaa[151]
17
To understand the linkage-selective recognition and enzymatic reactivity of various DUBs, the second-generation probes based on Ub dimers have been improved (Table 1.7).[152-155] It is highlighted that Ovaa and co-workers synthesized the diUb-based probe comprising an alkyne functinality in its proximal Ub, which enabled the investigation of DUBs reactivity mediated by the S2 pocket binding (Table 1.7e).[156]
Table 1.7 DiUb-based probes for DUBs.
Probe a
Kessler[152]
b
Zhuang[153]
c Ovaa[154]
d Brik[155]
e Ovaa[156]
18
2 Aim of the thesis
The aim of this thesis is to develop a universal approach for the generation of ubiquitin chains of various topologies, including Ub dimers and oligomers, homogeneous Ub chains, branched Ub chains, and Ub dendrimers.
The synthesized chains should meet the following criteria: firstly, the unnatural linkage connecting the Ub units should mimic the isopeptide-linkage. The five-membered aromatic heterocycle, 1,2,3- triazole, has been demonstrated an analogue of electrical similarity to amide bond.[157]
Secondly, the synthesized chains should be resistant to DUB-catalyzed hydrolysis or in general to all proteases, while they would be applied in the whole cell extracts for studying the ubiquitylation signaling.
Thirdly, the synthesized chains should contain defined linkages. Tryptic digest combining LC-MS/MS analysis should be performed to verify that the Ub chains bear desired modifications sites. In addition, through Western blot analysis, antibodies raised for the native isopeptide-linked chains could probe the Ub chains connected by specific linkages.
Moreover, the synthesized chains should be obtained in quantities sufficient for biochemical studies.
The functionalized Ub monomers are able to be engineered in a cellular system followed by the protein post-expression modification in large batch sizes. Both the assembly of Ub chains via click reaction and the isolation of the Ub oligomers through high-resolution preparative gel filtration chromatography could be performed in a scale of milligram.
Last but not the least, the approach should be facile and robust which shows great potential in the ubiquitin research field. To meet this requirement, chemical linkers are either commercially available or easily synthesized. In addition, protein engineering and chemical modification are manupilated with standard molecular cloning and biochemical methods.
19
3 Results and discussion
3.1 Ubiquitin dimers
3.1.1 Introduction
Since Ub contains seven lysine residues (i.e., K6, K11, K27, K29, K33, K48 and K63), polyUb chains linked at these different positions diversify in structural characteristics and physiological functions.
As the minimal structural units of polyUb chains, Ub dimers have been widely used in exploring the linkage-dependent Ub-Ub interaction, linkage-specific binding of Ub-asscociated proteins, and linkage-selective enzymatic reactivity of DUBs.[66]
Generation of the linkage-defined Ub dimers is always a crucial topic in the Ub research field. In the past ten years, enzymatic assembling method and many chemical approaches thrived to enable the synthesis of all seven Ub dimers. But the isopeptide- or thioether-linked dimers can be readily disassembled by the abundant cytosolic DUBs. With requirement of the comprehensive studies in vivo and in whole cell extracts, non-hydrolysable Ub dimers are highly in demand. To this end, a toolkit for the generation of DUBs-resistant triazole-linked Ub dimers via click reaction has been exploited.[64] However, amber codon suppression (ACS) incorporation of unnatural amino acids to some specific positions of Ub is of low efficiency, which restricts this method in preparing Ub dimers of requied quantities.
To circumvent this limitation, a facile and robust approach has been described within this thesis, which is based on protein engineering in a cellular system, protein post-expression bioorthogonal modification, and protein conjugation by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).
Efficient incorporation of a single unnatural amino acid azidohomoalanine (Aha) into the C-terminus of the distal Ub via selective pressure incorporation (SPI) in the methionine auxotrophic E.coli B834 has been exploited. Due to the absence of cysteine, the proximal Ub can be readily equiped with a sulfhydryl group at desired position in place of the lysine residue. Subsequently, the cysteine-mutant Ub is able to be functionalized with an alkyne via Michael addition by propargyl acrylate (PA).
Enventually, the distal Ub bearing an azide and the proximal Ub bearing an alkyne can be conjugated via CuAAC to form a triazole-linked Ub dimer (Figure 3.1.1). The five-membered aromatic heterocycle, 1,2,3-triazole, has been demonstrated an good analogue of electrical similarity to the amide bond (Figure 3.1.2).[157]
20 Figure 3.1.1 Scheme for the generation of Ub dimers Ub2-PA11
and Ub2-PA48
.
Figure 3.1.2 Comparison of the triazole-linkage and the isopeptide-linkage.
Here, as a proof of concept, two Ub dimers linked at position 11 or 48 were synthesized and isolated by size exclusion chromatography (gel filtration) in milligram quantities. Western blot analysis using a K48-linkage specific antibody verified their folding into the native-like conformations. The triazole- linked Ub dimers have been demostrated the ablity to resist DUB-catalyzed hydrolysis by Isopeptidase T (Iso T/USP5) or HEK293T cell lysate. Furthermore, the synthesized dimers were able to be catalyzed by the enzymes (i.e., UBE1, UbcH5b and E6AP), thus serving as subunits in the polyUb chain assembly.
21
3.1.2 Generation of Ub dimers
Expression and purification of CxUb (x = 11 or 48)
Due to the absence of cysteine, Ub can be readily equiped with a sulfhydryl group at desired position in place of the lysine residue. To do so, the gene encoding human KxCUb (x = 11 or 48) was cloned into the pET3a vector. CxUb was expressed in E.coli BL21 (DE3) that was firstly transformed with the plasmid pET3a-KxCUb. Cells were harvested and lysed by sonication in 20 mM sodium acetate (pH 4.5) buffer. The cell lysate were cleared by centrifugation and heated to 65 oC for 20 min to precipitate most of the unwanted proteins. CxUb was further purified by cation exchange chromatography (CEC) on ÄKTA FPLC-system using 20 mM sodium acetate (pH 4.5) supplemented with 1 M sodium chloride as gradient elution buffer. The expression and purification of C48Ub was analyzed by 12.5% SDS-PAGE (Figure 3.1.3). The isolated proteins were combined and their buffer was changed to 20 mM Tris-HCl (pH 7.5) via dialysis. The concentration of pure CxUb was measured by BCA protein assay. The yield is above 10 mg/L cell culture.
Figure 3.1.3 Expression and purification of C48Ub. a) SDS-PAGE analysis of overexpression of C48Ub after IPTG induction at 37 oC for 5 h, input and elution fraction from cation exchange chromatography. b) Chromatogram (detection at 214 nm) of purification of C48Ub by CEC.
Modification of CxUb by propargyl acrylate (PA)
To functionalize the proximal Ub with a terminal alkyne, the purified CxUb reacted with commercially available propargyl acrylate (PA) at the cysteine residue via Michael addition. Briefly, CxUb was diluted to 100 µM in Tris-HCl buffer (pH 7.5), and then treated with 10 eq. TCEP at 37 oC for 30 min in order to reduce the disulfide bond. For the purpose of efficient and selective modification on cysteine, reaction conditions have been carefully optimized. It is known that the reaction buffer of basic pH value enhances the formation of thiolate, thus benefiting the cysteine reactivity with Michael acceptors. But high pH value would promote the unwanted reaction of PA with other
22
reactive amino acids, such as lysine and histidine. Finally, a weakly basic Tris-HCl buffer at pH 7.5 was utilized. For the same reason, the reaction was performed at mild temperature of 25 oC. Since PA is not water-soluble, it needs to be firstly mixed in a water-miscible organic solvent, and then supplemented to CxUb in Tris-HCl buffer. To this end, organic solvents DMF, DMSO, ethanol and acetonitrile of different volume ratio in Tris-HCl buffer were evaluated. Eventually, the PA- modification was conducted under the following condition: 20 µM TCEP-treated CxUb was incubated with 100 eq. PA in 20 mM Tris-HCl (pH 7.5) supplemented with 10% acetonitrile at 25 oC for 2 h.
To examine whether the cysteine has been completely modified, the reaction process was monitored by fluorescein-5-maleimide (F5M) labeling, which only tagged the unreacted sulfhydryl groups. In brief, a small portion of the reaction sample was removed and reacted with excess amount of F5M at 37 oC for 20 min. Without prior PA treatment, the cysteine-containing Ubs were readily labeled by F5M as evidenced by strong fluorescence signals in SDS-PAGE analysis (Figure 3.1.4). In contrast, PA- treated Ubs were not able to be tagged by F5M, which suggested the complete reaction of the cysteine.
It is a critical step to remove the excess PA from the reaction solution. Otherwise this rich amount of small molecules would have priority to react with the azide-functionalized distal Ub in the click reaction. It was found that stepwise dialysis against Tris-HCl buffer supplemented with 50% methanol and following with 20% methanol was an efficient, easy-handling and cost-effective way to achieve the goal. The protein precipitate in the dialysis tubing could be collected and dissolved in Tris-HCl buffer supplemented with 6 M guanidine hydrochloride, and then refolded by stepwise dialysis against decreased concentration of guanidine hydrochloride. The PA-modified CxUb was eventually dialyzed against Tris-HCl buffer, and then lyophilized in aliquot. When in use, the required amount of CxUb-PA was dissolved in water, and the concentration was measured by the BCA protein assay.
To verify the modification of CxUb with a single alkyne, electrospray-ionization mass spectrometry (ESI-MS) analysis was performed. The determined masses of C11Ub-PA (8649.5 Da) and C48Ub-PA (8649.4 Da) were in correspondence to the calculated mass of 8649.9 Da.
23
Figure 3.1.4 Modification of C11Ub and C48Ub by propargyl acrylate. a) Scheme for modification of CxUb and SDS-PAGE analysis of fluorescein-5-maleimide (F5M) labeling of PA-modified (+) or unmodified (-) CxUb. b) Structures of molecules used in the modification of CxUb.
Expression and purification of Aha75Ub
For the generation of azido-functionalized distal Ub, a methionine analogue azidohomoalanine (Aha) was introduced to the C-terminus of Ub via selective pressure incorporation (SPI). To this end, a fusion protein was finely constructed. In brief, Gly75 was mutated to methionine, and Gly76 was deleted to make the triazole-linkage of the similar length to the isopeptide-linkage. To make sure that only one Aha was incorporated, the ATG start codon (i.e. Met codon) was removed and Ub was fused to the C-terminus of glutathione s-transferase (GST). For the purpose of protein purification, a thrombin cleavage site (TCS) was inserted between GST and Ub.
The fusion protein GST-TCS-Aha75Ub was expressed in the Met auxotrophic E.coli B834 (DE3). New minimal medium (NMM) supplemented with limited amount of Met (60 µM) was used to cultivate the cells to a stationary growth state of an OD600 value around 1. Then the medium was changed to fresh NMM supplemented with 0.5 mM Aha and expression of protein was inducted by adding IPTG.
For protein purification, cells were lysed and GST-TCS-Aha75Ub was bound to glutathione agarose beads. The desired Aha75Ub was released from the beads by thrombin cleavage with a yield of above 5 mg/L culture. The purity of eluted protein was analyzed by SDS-PAGE (Figure 3.1.5).
To analyze whether Met was quantitatively replaced by Aha and verify the thrombin selective cleavage, ESI-MS analysis was conducted. The determined mass 9303.1 Da corresponds well to the calculated mass 9303.5 Da. It was found that the cleavage site of thrombin should be before _GSRRASVGS-, but sometimes it shifted to _RASVGS- or _ASVGS-. Masses of these two species, 9003.2 Da and 8847.0 Da were also identified by ESI-MS and double bands were resolved by SDS-
24
PAGE. It is envisioned that the minimal sequence extension at N-terminus has little effect on the globular structure and reactivity of Aha75Ub.
Figure 3.1.5 Expression and purification of Aha75Ub. a) Chemical structures of azidohomoalanine (Aha) and methionine (Met). b) Sequence of Aha75Ub. Arrows show the thrombin cleavage sites. The N-terminal extension is indicated in underlined text. c) Scheme for the fusion protein GST-TCS-Aha75Ub and the thrombin cleavage. d) SDS-PAGE analysis of expression and purification of Aha75Ub. The red star indicates the expression of fusion protein GST-TCS-Aha75Ub after IPTG induction. Red arrows show the eluted Aha75Ub bearing different length of extension.
Generation and purification of Ub dimers Ub2-PA11
and Ub2-PA48
With the alkyne-functionalized CxUb-PA and azide-functionalized Aha75Ub in hand, click reaction was finally performed to generate the Ub dimers in a milligram scale. The optimized reaction condition was described as follow: both Ub momomers in a final concentration of 100 µM, supplementation of 0.5 mM SDS, 5 mM THPTA, and 2.5 mM copper(I) complex Cu(MeCN)4BF4, under argon atmosphere, on ice for 1 h. For the isolation of synthesized Ub dimers, click reaction mixtures were applied directly onto a Hiload superdex column, separated by size exclusion chromatography (gel filtration). The eluted fractions were collected and analyzed by SDS-PAGE (Figure 3.1.6). Fraction 1 of the highest richness was the desired Ub dimers, while fraction 2 and 3 containing much less
25
proteins indicated the unreacted Ub monomers Aha75Ub and CxUb-PA. Subsequently, the isolated Ub dimers were combined and concentrated by Amicon Ultra Centrifugal Filter. The concentration was measured by the BCA assay.
Figure 3.1.6 Generation and purification of Ub dimers. a,b) SDS-PAGE analysis of the generation of Ub2-PA11 by click reaction (Input) and the elution fractions from gel filtration. Chromatogram shows the detection of the elution fractions at 214 nm. c,d) SDS-PAGE analysis of the generation of Ub2-PA48 by click reaction (Input) and the elution fractions from gel filtration. Chromatogram shows the detection of the elution fractions at 214 nm.
3.1.3 Characterization of Ub dimers
Western blot analysis
To verify whether the synthesized dimers folded into the native-like structures, Western blot analysis using a K48-linkage specific antibody was exploited. This monoclonal antibody was raised against the wild-type Ub chains linked at K48 via isopeptide bond.[158] Of note, Ub2-PA48
was selectively probed by the antibody (Figure 3.1.7a), which suggested that the triazole-linked dimers adopted a kind of conformation similar to the isopeptide-linked polyUb chains.
DUBs-stability assay
To examine the resistance of the synthesized Ub dimers to DUB-catalyzed hydrolysis, wild-type K48- linked Ub dimer Ub2-wt48
, triazole-linked Ub2-PA11
and Ub2-PA48
were incubated separately with 100 nM Isopeptidase T (IsoT/USP5). IsoT from the USP family is primarily responsible for the disassembly of unanchored polyUb chains in cells.[159] At the certain incubation time point (i.e., 0, 30, and 60 min) at 37 oC, aliquot samples were removed and the enzymatic reactions were stopped by adding 6x loading
26
buffer, heated at 95 oC for 5 min. The whole reaction mixtures were analyzed by SDS-PAGE shown in Figure 3.1.7b. The wild-type dimers were so fragile that they were hydrolyzed immediately after incubation with IsoT. After 30 min, all of them have been readily disassembled to monomers. In comparision, both triazole-linked dimers were intact even after one hour’s incubation. To further understand the enzymatic reactivity of IsoT, lower amout of enzymes (50 nM) was tested in the disassembly of Ub2-wt48 at the detailed reaction time (0, 5, 10, 30, and 60 min). Figure 3.1.7c displayed that IsoT was extremely active, so that after 5 min all the wild-type Ub dimers have been disassembled.
To demonstrate the potential application value of the synthesized Ub dimers in cells (lysate), Ub2-wt48
, Ub2-PA11
, and Ub2-PA48
were incubated respectively with human embryonic kidney (HEK293T) cell lysate.
At certain time point, samples were removed, mixed with 6x loading buffer and heated at 95 oC followed by SDS-PAGE analysis (Figure 3.1.7d). Comparing to the rapid disassembly of isopeptide- linked wild-type dimers, triazole-linked dimers were extraordinarily stable to the protease hydrolysis in the whole cell extract.
27
Figure 3.1.7 Characterization of Ub dimers by biochemical assays. a) Western blot analysis of Ub2-PA11 and Ub2-
PA48
using a K48-specific antibody. The same samples were resolved by SDS-PAGE followed by Coomassie staining. b) DUBs-stability assay of isopeptide-linked (Ub2-wt48) or triazole-linked Ub dimers (Ub2-PA11, Ub2-PA48) incubated with Isopeptidase T (100 nM) at the certain time (0, 30, or 60 min). c) Incubation of Ub2-wt48
with 100 nM or 50 nM Isopeptidase T at the certain time (0, 5, 10, 30, or 60 min). d) Deubiquitylation assay of Ub2-wt48
, Ub2-PA11
, or Ub2-PA48
incubated with HEK293T cell lysate. The whole reaction mixtures were analyzed by SDS- PAGE followed by Coomassie staining.