Patent Application
20160304571
The invention relates to protein synthesis and modification. In particular the invention relates to lysine-linked protein modifications.
Lysine residues are key determinants for post-translational protein modification and their use key post-translational modifications such as ubiquitination, methylation, and acetylation are well known. Selectively modifying specific lysine residues within a protein remains a problem.
Ubiquitination is a reversible post-translational modification in which a specific lysine residue in an acceptor protein forms an isopeptide bond with the C-terminus of the ubiquitin donor. While the role of ubiquitination in regulating protein stability via proteasomal targeting is well established, it is emerging that ubiquitin is involved in almost every aspect of biology, including cell signaling, intracellular trafficking and the response to DNA damage1,2. Ubiquitin forms covalent chains through each of its Lysine residues (K6, K11, K27, K29, K33, K48, or K63), or through is N-terminus, and it is proposed that the distinct functions mediated by ubiquitin in diverse biological processes may be encoded in the distinct properties of the different ubiquitin chains2,3.
While proteomic studies reveal that all chains types are present in vivo4,5, most is known about K48 chains and K63 chains, which are important in proteasomal degradation and cell signaling, respectively1,6. In contrast very little is known about the other so-called ‘atypical’ linkages, though they account for more than half of the ubiquitin linkages found in model organisms, where proteomics data exists5. A central challenge in studying the roles of specific ubiquitin chains is to synthesize homogeneous chains bearing defined linkages.
The present invention seeks to overcome problem(s) associated with the prior art.
The present inventors have solved the problem of specifically targeting individual lysine residues for modification within a polypeptide chain. One aspect of this is to genetically encode the chemical protection for the target lysine(s). This enables site-specific protection of specific residue(s) within the polypeptide chain.
More importantly, the inventors have developed techniques for differentially protecting the lysines in the polypeptide. Specifically, the inventors teach use of a chemical protection which differs from the genetically encoded protection.
In this way, a polypeptide may be produced having a target lysine protected site-specifically by genetically encoding protection of that residue. This polypeptide with its site-specific protected lysine can then be chemically treated to protect each of the remaining (unprotected) lysine residues with a different chemical protection group. This dual approach has the advantage that the finished polypeptide has two different types of chemical protection on its lysines. This enables the original target lysine to be selectively deprotected by chemistry leaving the other protection groups on the other lysines intact. In this way the target lysine is site-specifically deprotected and can therefore be modified whilst the other lysines remain unaffected.
Finally all of the unmodified lysines can be optionally deprotected to leave nature-identical lysine residues if desired.
The present invention is based on this ingenious differential chemistry approach to the selective protection/deprotection of target lysine(s) in the polypeptide sequence.
Thus in one aspect the invention provides a method of modifying a specific lysine residue in a polypeptide comprising at least two lysine residues, said method comprising
(a) providing a polypeptide comprising a target lysine residue protected by a first protecting group, and at least one further lysine residue;
(b) treating the polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue;
(c) selectively deprotecting the target lysine residue; and
(d) modifying the deprotected lysine residue of (c).
Suitably producing the polypeptide comprises
(i) providing a nucleic acid encoding the polypeptide which nucleic acid comprises an orthogonal codon encoding the target lysine;
(ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said target lysine residue protected by a first protecting group into the polypeptide chain.
Suitably said orthogonal codon comprises TAG, said tRNA comprises MbtRNACUA and said tRNA synthetase comprises MbPylRS.
Suitably the target lysine residue protected by a first protecting group is chosen from the group consisting of: Nε-(t-butyloxycarbonyl)-L-lysine.
Suitably the target lysine residue protected by a first protecting group is Nε-(t-butyloxycarbonyl)-L-lysine.
Suitably the protecting group for said further lysine residues is chosen from the group consisting of: N-(benzyloxycarbonyloxy) succinimide (Cbz-Osu). When the protecting group for said further lysine residues is N-(benzyloxycarbonyloxy) succinimide (Cbz-Osu), suitably it is supplied in basic DMSO.
Suitably the protecting group for said further lysine residues is N-(benzyloxycarbonyloxy) succinimide (Cbz-Osu).
Suitably step (b) comprises treating the polypeptide with N-(benzyloxycarbonyloxy)succinimide (Cbz-OSu) in basic DMSO.
Suitably the amount of Cbz-OSu used is determined as [molar amount equivalent to the amount of polypeptide being treated] multiplied by [number of lysines to be protected in polypeptide plus one]. This advantageously provides a slight excess of the protecting groups for reaction with the lysines to be protected and therefore helps to propel the reaction towards completion (saturation/homogeneity).
Suitably step (c) comprises treating the polypeptide with trifluoroacetic (TFA) acid in water.
Suitably the modification of step (d) comprises
(i) activating thioester by conversion to N-hydroxysuccinimidyl ester in the presence of Ag(I);
(ii) adding a polypeptide to be joined to the target lysine; and
(iii) incubating to allow formation of a specific isopeptide bond.
Suitably the modification of step (d) is carried out on the s-amino group of the target lysine residue.
Suitably multiple modifications may be made to the target lysine in step (d).
Suitably the method as described above further comprises the step: (e) deprotecting said further lysine residue(s). Suitably step (e) comprises treating the polypeptide with a mixture of trifluoromethanesulfonic acid (TFMSA):trifluoroacetic acid (TFA):dimethylsulfide (DMS) in the ratio 1:3:6.
One advantage of deprotecting all remaining lysines as after modification is complete is to restore the polypeptide as close as possible to its natural form.
Suitably step (a) comprises producing the polypeptide by genetically incorporating the target lysine residue protected by a first protecting group into the polypeptide chain during its translation.
Suitably the polypeptide is ubiquitin.
Suitably the modification of step (d) is the covalent linkage of a further polypeptide chain to the target lysine. Suitably the further polypeptide chain is ubiquitin. In this embodiment the invention may be advantageously applied to ubiquitination of polypeptide(s).
In some embodiments the polypeptide is ubiquitin and the further polypeptide chain is ubiquitin. In these embodiments the invention is advantageously applied to the manufacture of ubiquitin chains. These chains may be made in any of the K-linked forms of ubiquitin (e.g. K6, K11, K27, K29, K33, K48, or K63 linked ubiquitin) simply by selecting the appropriate lysine residue to target in the first polypeptide.
When multiple modifications are made, steps (c)-(d) may be repeated to produce a chain of polypeptides joined by covalent linkages through lysine residues. Clearly this may involved repeated deprotection at the end of each round of modification before moving on to the next reaction in the sequence of modifications. Alternatively no protection/deprotection may be needed for the subsequent modifications if the reaction chemistry used for them is already specific to the target lysine (or modified target lysine) from earlier round(s) of modification.
The invention also provides polypeptide(s) produced as described above. Said polypeptide(s) may comprise a K-linked ubiquitin chain. Said K-linkage may be a K6, K11, K27, K29, K33, K48, or K63 linkage. Suitably said K-linkage is a K6 or K29 linkage.
In another aspect, the invention relates to use of TRABID as a K29 deubiquitinase.
In another aspect, the invention relates to a method of cleaving K29 linked ubiquitin comprising contacting same with TRABID.
In another aspect, the invention relates to a ubiquitin polypeptide comprising at least one protected lysine residue. Ubiquitin polypeptide comprising one or more of K6Boc, K29Boc.
In another aspect, the invention relates to a nucleic acid encoding ubiquitin wherein at least one lysine codon is replaced with an orthogonal codon.
In another aspect, the invention relates to use of K-linked ubiquitins of the invention for example for use in activating or promoting a response to DNA damage, and/or for use in preventing or treating cancer such as early-onset breast or ovarian cancer. Such uses may advantageously be applied in medicine.
Preferred methods of the invention may also be referred to in the following specification as GOPAL (Genetically-encoded Orthogonal Protection and Activated Ligation).
Suitably the targeted reaction in the method according to the invention is used to link proteins together.
In another aspect of the invention, the proteins obtainable from said method are provided. Suitably, the proteins are ubiquitins.
In another aspect of the invention, homogenously linked ubiquitin chains in which the ubiquitin polypeptides are linked by the lysine amino acid residue at position 6 and the C-terminal are provided. Said ubiquitin chains are for use in activating or promoting a response to DNA damage and therefore for use in preventing or treating cancer.
The invention will now be described in relation to the drawings in which:
It is vital to the invention that the protection groups on the target lysine and on other lysine(s) in the polypeptide are different. It is through the chemical differences of these protection groups that the differential deprotection chemistry permits specific or selective modification of the target residue by enabling its specific or selective deprotection and therefore modification.
Suitably the desired reaction in step (d) is carried out on the ε-amino group of the lysine residue.
In another preferred embodiment, the other lysine side chains to be protected in step (b) are also the ε-amino group. In such a case, the method above also applies to the terminal amino group of the polypeptide chain.
In the method according to the invention, said genetic incorporation preferably uses an orthogonal or expanded genetic code, in which one or more specific orthogonal codons have been allocated to encode the specific lysine residue with the lysine side group chain protected so that it can be genetically incorporated by using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair can in principle be any such pair capable of charging the tRNA with the protected lysine and capable of incorporating that protected lysine into the polypeptide chain in response to the orthogonal codon.
The orthogonal codon may be the orthogonal codon amber, ochre, opal or a quadruplet codon. The codon simply has to correspond to the orthogonal tRNA which will be used to carry the protected lysine molecule. Preferably the orthogonal codon is amber.
It should be noted that the specific examples shown herein have used the amber codon and the corresponding tRNA/tRNA synthetase. As noted above, these may be varied. Alternatively, in order to use other codons without going to the trouble of using or selecting alternative tRNA/tRNA synthetase pairs capable of working with the protected lysine, the anticodon region of the tRNA may simply be swapped for the desired anticodon region for the codon of choice. The anticodon region is not involved in the charging or incorporation functions of the tRNA nor recognition by the tRNA synthetase so such swaps are entirely within the ambit of the skilled operator.
Thus alternative orthogonal tRNA synthetase/tRNA pairs may be used if desired.
Preferably the orthogonal synthetase/tRNA pair are Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNACUA).
The Methanosarcina barkeri PylT gene encodes the MbtRNACUA tRNA.
The Methanosarcina barkeri PylS gene encodes the MbPylRS tRNA synthetase protein. When particular amino acid residues are referred to using numeric addresses, the numbering is taken using MbPylRS (Methanosarcina barkeri pyrrolysyl-tRNA synthetase) amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Methanosarcina barkeri PylS gene Accession number Q46E77):
Said sequence has been annotated here below as SEQ ID NO. 1.
If required, the person skilled in the art may adapt MbPylRS tRNA synthetase protein by mutating it so as to optimise for the lysine species with particular protected side chains to be used. The need for mutation depends on the lysine residue and protection/caging group used. An example where the MbPylRS tRNA synthetase does not need to be mutated is when the lysine residues with protected side chains used in step (a) are Nε-(t-butyloxycarbonyl)-L-lysine. An example where the MbPylRS tRNA synthetase may need to be mutated is when the lysine side group chain in step (a) is protected by a larger chemical group such as a photolabile caging group.
Such mutation may be carried out by introducing mutations at one or more of the following positions in the MbPylRS tRNA synthetase: M241, A267, Y271, L274 and C313. Preferably the mutations may comprise M241F, A267S, Y271C and L274M.
tRNA Synthetases
The tRNA synthetase of the invention may be varied. Although specific tRNA synthetase sequences may have been used in the examples, the invention is not intended to be confined only to those examples.
In principle any tRNA synthetase which provides the same tRNA charging (aminoacylation) function can be employed in the invention.
For example the tRNA synthetase may be from any suitable species such as from archea, for example from Methanosarcina barkeri MS; Methanosarcina barkeri str. Fusaro; Methanosarcina mazei Go1; Methanosarcina acetivorans C2A; Methanosarcina thermophila; or Methanococcoides burtonii. Alternatively the tRNA synthetase may be from bacteria, for example from Desulfitobacterium hafniense DCB-2; Desulfitobacterium hafniense Y51; Desulfitobacterium hafniense PCP1; Desulfotomaculum acetoxidans DSM 771.
Exemplary sequences from these organisms are the publically available sequences. The following examples are provided as exemplary sequences for pyrrolysine tRNA synthetases:
Methanosarcina barkeri MS
Methanosarcina barkeri str. Fusaro
Methanosarcina mazei Go1
Methanosarcina acetivorans C2A
Methanosarcina thermophila, VERSION DQ017250.1 GI:67773308
Methanococcoides burtonii DSM 6242, VERSION YP_566710.1 GI:91774018
Desulfitobacterium hafniense DCB-2
Desulfitobacterium hafniense Y51
Desulfitobacterium hafniense
Desulfotomaculum acetoxidans DSM 771
When the particular tRNA charging (aminoacylation) function has been provided by mutating the tRNA synthetase, then it may not be appropriate to simply use another wild-type tRNA sequence, for example one selected from the above. In this scenario, it will be important to preserve the same tRNA charging (aminoacylation) function. This is accomplished by transferring the mutation(s) in the exemplary tRNA synthetase into an alternate tRNA synthetase backbone, such as one selected from the above.
In this way it should be possible to transfer selected mutations to corresponding tRNA synthetase sequences such as corresponding pylS sequences from other organisms beyond exemplary M. barkeri and/or M. mazei sequences.
Target tRNA synthetase proteins/backbones, may be selected by alignment to known tRNA synthetases such as exemplary M. barkeri and/or M. mazei sequences.
This subject is now illustrated by reference to the pylS (pyrrolysine tRNA synthetase) sequences but the principles apply equally to the particular tRNA synthetase of interest.
For example,
Thus suitably when sequence identity is being considered, suitably it is considered across the tRNA synthetases as in
It may be useful to focus on the catalytic region.
Thus suitably when sequence identity is being considered, suitably it is considered across the catalytic region as in
‘Transferring’ or ‘transplanting’ mutations onto an alternate tRNA synthetase backbone can be accomplished by site directed mutagenesis of a nucleotide sequence encoding the tRNA synthetase backbone. This technique is well known in the art. Essentially the backbone pylS sequence is selected (for example using the active site alignment discussed above) and the selected mutations are transferred to (i.e. made in) the corresponding/homologous positions.
When particular amino acid residues are referred to using numeric addresses, unless otherwise apparent, the numbering is taken using MbPylRS (Methanosarcina barkeri pyrrolysyl-tRNA synthetase) amino acid sequence as the reference sequence (i.e. as encoded by the publicly available wild type Methanosarcina barkeri PylS gene Accession number Q46E77) (SEQ ID NO. 1):
This is to be used as is well understood in the art to locate the residue of interest. This is not always a strict counting exercise—attention must be paid to the context or alignment. For example, if the protein of interest is of a slightly different length, then location of the correct residue in that sequence corresponding to (for example) L266 may require the sequences to be aligned and the equivalent or corresponding residue picked, rather than simply taking the 266th residue of the sequence of interest. This is well within the ambit of the skilled reader.
Notation for mutations used herein is the standard in the art. For example L266M means that the amino acid corresponding to L at position 266 of the wild type sequence is replaced with M.
The transplantation of mutations between alternate tRNA backbones is now illustrated with reference to exemplary M. barkeri and M. mazei sequences, but the same principles apply equally to transplantation onto or from other backbones.
For example Mb AcKRS is an engineered synthetase for the incorporation of AcK
Parental protein/backbone: M. barkeri PylS
Mb PCKRS: engineered synthetase for the incorporation of PCK
Parental protein/backbone: M. barkeri PylS
Synthetases with the same substrate specificities can be obtained by transplanting these mutations into M. mazei PylS. The sequence homology of the two synthetases can be seen in
Mm AcKRS introducing mutations L301V, L305I, Y306F, L309A, C348F into M. mazei PylS,
and
Mm PCKRS introducing mutations M276F, A302S, Y306C, L309M into M. mazei PylS.
Full length sequences of these exemplary transplanted mutation synthetases are given below.
The same principle applies equally to other mutations and/or to other backbones.
Transplanted polypeptides produced in this manner should advantageously be tested to ensure that the desired function/substrate specificities have been preserved.
Polynucleotides encoding the polypeptide of interest for the method described above can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli.
Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term “operably linked” means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
Vectors of the invention may be transformed or transfected into a suitable host cell as described to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Vectors may be used, for example, to transfect or transform a host cell.
Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
The target lysine is protected by a protecting group (e.g. step (a) of the method). Said protecting group is different from the protecting group used to protect the further lysine(s) (e.g. step (b) of the method). The method of deprotecting used to selectively remove the protecting group from the target lysine in step (c) of the method must be performed so as NOT to deprotect the further lysine(s) at the same time. Chemical protecting agents for lysine side chains are varied and can be chosen by the person skilled in the art depending on the type of deprotection methods to be used. However said protecting agent is suitably chosen so as to allow the lysine residue to be incorporated genetically and thus allow it to be incorporated by an orthogonal synthetase/tRNA pair in a cell.
Suitably the protecting agent used in step (a) is as described herein. More suitably, the lysine amino acid with protecting agent to be used in step (a) is Nε-(t-butyloxycarbonyl)-L-lysine.
Another embodiment employs a lysine amino acid protected with a photo-labile caging group. This has the advantage of permitting photo-decaging (deprotection) which can be easier than chemical deprotection.
The further lysine residues and, optionally the N-terminal amino group, have their side chains protected to allow for the specific modification of the target lysine. This is advantageously accomplished using a reaction where the protecting group can reach, or at least approach, saturation (100%) of the further lysine residues present in the polypeptidic chain.
It is advantageous to use a polypeptide which can be easily denatured and renatured without losing its properties as a protein. In this regard it may be advantageous if the polypeptide according to the invention is a small protein. It is further advantageous for the polypeptide to have few or no post-translational modifications such as for example glycosylation.
Exemplary polypeptides to be modified include histones and/or small transcription factors. Suitably the polypeptide to be modified is not too large. By not too large is meant suitably not more than a few hundred amino acids long; suitably 400 amino acids or fewer, more suitably approximately 300 amino acids or fewer. Multidomain proteins are less attractive for modification because of the increased chances of affecting interactions with other domain(s) (e.g. other members of a multiprotein complex).
Suitably the protection groups used may be as described herein. More suitably the protecting agent is N-(benzyloxycarbonyloxy) succinimide (Cbz-Osu). Suitably this may be used in basic DMSO.
Alternative or additional protecting groups and their manipulation (such as the chemistry for their addition/removal are described below.
BocLys is described in “Genetic incorporation of Nε-(t-butyloxycarbonyl)-L-lysine (BocLys)” (YANAGISAWA, T., ISHII, R., FUKUNAGA, R., KOBAYASHI, T., SAKAMOTO, K. & YOKOYAMA, S. (2008) Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol, 15, 1187-97)
Off—trifluoroacetic acid (TFA)
Orthogonal protecting groups that can be mildly attached to protein amines where X=
On—N-(benzyloxycarbonyloxy)-succinimide
2. o-nitrobenzyloxycarbonyl
On—N-(o-nitrobenzyloxycarbonyloxy)-succinimide
Off—hv˜365 nm, aq. Na2S2O4 (1,4-elimination)
3. p-nitrobenzyloxycarbonyl
On—N-(o-nitrobenzyloxycarbonyloxy)-succinimide
Off—aq. Na2S2O4 (1,6-elimination)
Off—K2CO3/aqueous MeOH, pH>9
On—N-(Fluorenylmethoxycarbonyloxy)-succinimide
Can be removed by any primary amine therefore appropriate attention must be paid to conditions when using this protecting group.
Off—K2CO3/aqueous MeOH, pH>9
Orthogonal protecting groups that can be mildly attached to protein amines where X=
On—citraconic anhydride/aqueous buffer pH 8
2. t-butyloxycarbonyl (Boc)
On—N-(t-butyloxycarbonyloxy)-succinimide
Cbz, photocages and Ardec may also be used with genetically encoded TFAc protection.
Genetic incorporation of Nε-(o-nitropiperonyloxycarbonyl)-L-lysine (ONPOC)
Regarded as compatible with base cleavable and mild acid cleavable protecting groups. Ardec may also find application in the invention.
Genetic incorporation of Nε-(allyoxycarbonyl)-L-lysine (AllocLys)
Off—Ni(CO)4, (PdPh3P)4/Bu3SnH/AcOH
This has the advantage of being orthogonal with everything except catalytic deprotection and probably not HF/TFMSA.
In another embodiment the further lysine side chains may be protected with a photo-labile caged lysine. In this embodiment, when the target lysine is also protected by a photolabile group, the protection of the target lysine(s) should be by a photolabile group released at a frequency of radiation which will not release the second protecting group. The present method enables targeting of one or more specific lysines present within a polypeptide chain for a specific reaction through recombinant techniques and chemoselective protein chemistry.
Suitably the method is an in vitro method. Advantageously the polypeptides of the invention may be applied in vitro or in vivo.
An advantage of the invention is the provision of a method to study the effect of lysine modifications in polypeptides and proteins. One example of such a modification dimethylation of lysine(s). This finds application in studying the effect of dimethylation of lysines in proteins.
In another preferred example, the reaction in step (d) is covalently linking the lysine residue to another protein. As the specific lysine side chain presents an s-amino after deprotection in step (c), it is preferable that the reaction be a peptide bond formation. In such cases, the other protein may also present lysine side chains and thus it is preferable if the other protein be protected in the same manner in step (b) together with the other lysine residues of the polypeptidic chain. Suitably the whole modified polypeptide is deprotected after synthesis.
As is mentioned above, the invention is useful in ubiquitination of polypeptide(s) and/or the study of same. Thus it is preferable if the polypeptide and/or other polypeptide comprise ubiquitin. Ubiquitin is a small protein that is easily denatured and renatured, allowing for its ease of production by recombination and selective protein chemistry according to the method of the present invention.
Thus the present method is a powerful tool that allows ubiquitination in a specific manner of any protein. When the effects of such ubiquitination are known, such as for example by linking a protein to a polyubiquitin linked by K48 of the ubiquitin polypeptide, this can be helpful in studying their proteosomal degradation.
In another example, the method can be repeated to allow one to link several proteins together. In the case that both the polypeptidic chain and the protein are ubiquitin, it is possible to use the invention to produce ubiquitin chains, for example homogenously and/or heterogeneously linked.
Thus another aspect of the invention are the resulting polypeptidic chains that are obtainable from the methods according to the invention, suitably a polypeptidic chain linked specifically to a protein by an isopeptide bond. One such example are ubiquitinated proteins, whereby the reaction in step (d) is to link the ubiquitin to another protein by peptide bond formation.
Another such example are the homogenously linked ubiquitin chains obtainable by the method. As shown below, a homogenously linked ubiquitin chain has been obtained according to the method where the covalent link is an isopeptide bond between a lysine amino acid residue at position 6 or 29 and the C-terminus of another ubiquitin polypeptide. It is to be understood that the chain can be continued by further homogenous linkages, further obtainable by the method according to the invention.
Another aspect of the invention is the homogenously linked ubiquitin obtained according to the method of the invention where the covalent link is an isopeptide bond between a lysine amino acid residue at position 6 and the C-terminus of another ubiquitin polypeptide. The linkages can be continued for more than 2 links.
Said ubiquitin chain can be used as a medicament. It can be used in activating or promoting a response to DNA damage. The chains have been shown to be linked to the BRCA1/Bard1 E3 ligase complex and thus the ubiquitin chains can be used in preventing or treating cancer, preferably where the cancer is early-onset breast or ovarian cancer.
As such, the ubiquitin chains can be treated as an oncological medicament and can be used in pharmaceutical compositions and administered by means well known in the art in the filed of oncological pharmacy.
The following non-limiting examples are illustrative of the present invention:
The specific formation of an isopeptide bond between the C-terminus of one ubiquitin (donor) and the amine of a specific lysine residue in another ubiquitin (acceptor) requires these functional groups to be differentiated from the other carboxylic acids and amines in both ubiquitin molecules (
The present Example is the preparation of a ubiquitin molecule according to the method of the invention to be an acceptor ubiquitin (for a successive isopeptide bond later on).
This method is represented partly by the right hand side of
The protocols used were
Cloning of Acceptor Ubiquitin (UbTAG6-His6 and UbTAG29-His6)
The human UBC ubiquitin gene was PCR amplified using a forward 5′-CG CGC gccATG GAG ATC TTC GTG AAG ACC CTG ACT GG-3′ primer (SEQ ID NO. 19) and the reverse 5′-GCC GGA TCT CCG CTC GAG TTA GTG GTG ATG ATG GTG ATG CCC ACC TCT GAG ACG GAG GAC-3′ primer (SEQ ID NO. 20), that introduce NcoI and XhoI restriction sites as well as a C-terminal His6 tag followed by a stop codon. The PCR product was digested with NcoI and XhoI and ligated into a similarly treated pCDF-PylT plasmid (which encodes MbtRNACUA on an lpp promoter and rrnC terminator and has a spectinomycin resistance marker20,42). The forward primer forced a mutation of the second ubiquitin codon and as such, Quikchange mutagenesis was required to mutate the second ubiquitin residue back to Gln. A second round of Quikchange mutagenesis was then used to introduce a TAG codon at position K6 or K29. The final plasmids were named pCDF-pylT-UbTAG6-His6 and pCDF-pylT-UbTAG29-His6 respectively.
BL21(DE3) cells (Merck biosciences) containing pBKPylS (a kanamycin resistant plasmid encoding MbPylRS on an E. coli GlnRS promoter and terminator) and pCDF-pylT-UbTAG6-His6 were grown to overnight (37° C., 230 rpm, in LB-KS: LB media containing 50 μg mL−1 spectinomycin and 50 μg mL−1 kanamycin). The culture was diluted 1:50 in to 2 L of fresh LB-KS and incubated (37° C., 230 rpm). At OD600-0.6, a solution of 200 mM 1 (10 mL in 2 M aq. NaOH) was added to the cells whilst stirring vigorously and the culture was immediately neutralized with 5 M HCl (4 mL). After 30 min protein expression was induced by the addition of isopropyl-βD-thiogalactopyranoside to 0.5 mM. After incubation (37° C., 230 rpm, 3 h) cells were harvested by centrifugation and resuspended in 50 mL ice-cold lysis buffer (20 mM Na2HPO4 pH 7.4, 25 mM imidazole) and frozen at −80° C. until required. Cells were thawed on ice and lysozyme (0.5 mg mL−1) and DNAseA (50 μg mL−1) were added. After 30 min the cells were sonicated and clarified by centrifugation (39000×g, 30 min). The clarified lysate was loaded, by gravity flow, onto a column containing Ni-NTA resin (3 mL, Qiagen). The resin was washed with lysis buffer (90 mL) and the protein eluted with elution buffer (20 mM Na2HPO4 pH 7.4, 250 mM imidazole). Fractions containing UbBocK6-His6 were determined by SDS-PAGE and were pooled and concentrated to <9 mL with an Amicon Ultra-15 3 kDa MWCO centrifugal filter device (Millipore). The sample was then dialyzed against 10 mM Tris pH 7.6 for 3 hours. 1 mM DTT was added to the UbBocK6-His6 sample, followed by UCH-L3 at a final concentration of 15 μg mL−1 The sample was incubated at 37° C. for 1 hour to remove the C-terminal His6 tag. UbBocK6 was isolated by size-exclusion chromatography employing a HiLoad 26/60 Superdex 75 Prep Grade column (GE Life Sciences) at a flow rate of 2 mL min−1. Fractions containing UbBocK6 were pooled and concentrated to 2.5 mL. The sample was desalted into H2O using a PD-10 column (GE Life Sciences) and the elution was lyophilized yielding approximately 20 mg of UbBocK6. UbBocK29 was prepared the same way except that cells were transformed with pCDF-pylT-UbTAG29-His6. The yield of UbBocK29 was 8 mg.
Global Protection of UbBocK6 and UbBocK29 with Cbz-OSu
Lyophilized UbBocK6 (20 mg, 2.3 μmol) was dissolved in DMSO (1.7 mL) followed by the addition of DIEA (67 μL). Whilst stirring, Cbz-OSu (4.81 mg, 19.3 μmol) was added and the reaction. After stirring for 2 h at 25° C. the reaction was transferred into cold ether (17 mL) and briefly vortexed. The precipitate was collected by centrifugation and the ether layer was discarded. The pellet was washed with ice-cold ether (17 mL) and then air-dried. For UbBocK29 this and subsequent procedures were repeated pro rata in account of the reduced expression yield of UbBocK29-His6.
The dry globally protected peptide, UbBocK6(Cbz7-8), obtained from 10 mg of UbBocK6 was dissolved in cold 9:6 TFA:H2O (1.28 mL) and incubated at 4° C. for 1 h. The selectively deprotected peptide, was then precipitated and washed with ice-cold ether (2×13 mL). The aqueous and ether layers were removed and the peptide was left to air dry.
To site-specifically install a protected lysine at position 6 of ubiquitin we took advantage of the Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNACUA)18, which directs the efficient incorporation of Nε-(t-butyloxycarbonyl)-L-lysine (1) into recombinant proteins in response to the amber codon in E. coli19-21. We created a ubiquitin expression construct in which the ubiquitin gene contains a TAG codon in place of the lysine codon at position 6, and is flanked by a 3′ His6 tag coding sequence (UbTAG6-His6). We produced UbBocK6-His6 (ubiquitin-his6 containing 1 at position) by expressing UbTAG6-His6 in cells containing the MbPylRS/MbtRNACUApair and 1 (2 mM). UbBocK6-His6 production was strictly dependent on the addition of 1. UbBocK6-His6 was purified by Ni-NTA chromatography and the His6 tag was removed by treatment with ubiquitin C-terminal hydrolase-L3 (UCH-L3) to give UbBocK6 (
To protect the six Nε-Lys amino groups and the N-terminal amine in UbBocK6 with Cbz groups we reacted the protein with 7 equivalents of N-(benzyloxycarbonyloxy)succinimide (Cbz-OSu) in basic DMSO22. After protection for 2 hours (
To reveal a single free Nε-Lys amino group at K6, as desired for isopeptide bond formation, we removed the Boc protecting group present in 1 with trifluoroacetic acid (TFA) in water, leaving the Cbz protection intact (
As mentioned above, when the other protein (in this case donor ubiquitin) also presents lysine side chains and the reaction involves linking a polypeptidic chain to another protein, it is advantageous to protect the lysine side chains present on the protein. This method is represented partly by the left hand side of
The ubiquitin gene was PCR amplified from a plasmid containing Ub1-75 using the forward 5′-GGT GGT CAT ATG CAG ATC TTC GTC AAG ACG TTA ACC-3′ primer (SEQ ID NO. 21) and the reverse 5′-GGT GGT TGC TCT TCC GCA CCC GCC ACG CAG TCT TAA GAC CAG ATG-3′ primer (SEQ ID NO. 22) that introduced NdeI and SapI restriction sites respectively. The reverse primer also inserted a codon for Gly 76. The PCR product was double digested with NdeI and SapI restriction enzymes and ligated into similarly treated pTXB1 vector (NEB) to create pTXB1-Ub1-76.
ER2566 E. coli. cells (50 μL) (NEB) were transformed with pTXB1-Ub1-76 and recovered with S.O.B. medium (250 μL). The cells were incubated for 1 h at 37° C. and then LB medium (100 mL) containing ampicillin (100 μg mL−1) was inoculated with the recovered cells (200 μL) and the culture was incubated overnight whilst shaking (230 rpm) at 37° C. LB medium (2 L) containing ampicillin (100 μg mL−1) was inoculated with the overnight culture (60 mL) and incubated whilst shaking (230 rpm) at 37° C. At O.D.600˜0.4, the cells were transferred to a 25° C. incubator and after 30 min the cells were induced with IPTG (0.5 mM). After 5 h the cells were harvested and suspended in 60 ml lysis buffer (20 mM Na2HPO4 pH 7.2, 200 mM NaCl, 1 mM EDTA) and frozen. The thawed cells were lysed by sonication on ice and were clarified by centrifugation (39000×g, 30 min). An empty XK 26/20 column was filled with chitin beads (20 mL) (NEB) and equilibrated with lysis buffer. At 4° C. the clarified lysate was loaded (flow rate: 0.5 mL min−1) onto the column using an ÄKTA FPLC system. The column was then washed with lysis buffer (˜400 mL) and equilibrated with 60 mL of cleavage buffer (20 mM Na2HPO4 pH 6, 200 mM NaCl, 100 mM MESNa, 1 mM EDTA). The flow was then stopped and the column incubated for 66 h at 4° C., to allow cleavage of the ubiquitin thioester (UbSR). Cleaved UbSR was eluted with elution buffer (20 mM Na2HPO4 pH 6, 200 mM NaCl, 1 mM EDTA). The fractions containing UbSR were determined by SDS-PAGE and were then pooled and concentrated to ˜5 mL using an Amicon Ultra-15 centrifugal filter device (Millipore). The protein was then further purified by semi-preparative RP-HPLC employing a Phenomenex 250 mm×10 mm, C18, 300 Å, 10 μm column. A gradient of 10% buffer A to 75% buffer B was applied at a flow rate of 5 mL min−1 over 30 min (buffer A=0.1% TFA in H2O, buffer B=10% buffer A in MeCN). Fractions containing ubiquitin thioester were verified by ESI-MS and were lyophilized.
Global Protection of Ub-MES Thioester with Cbz-OSu
Lyophilized UbSR (10 mg, 1.15 μmol) was dissolved in DMSO (833 μL) followed by the addition of DIEA (17 μL). Whilst stirring, Cbz-OSu (2.75 mg, 11.5 μmol) was added to the reaction. The reaction was stirred for 2 h at 25° C. and then transferred into cold ether (8.5 mL) and briefly vortexed. The precipitate was collected by centrifugation and the ether layer was discarded. The pellet was washed with ice-cold ether (8.5 mL) and air-dried.
A donor ubiquitin molecule was prepared biosynthetically as a C-terminal thioester (UbSR), by thiolysis of an intein fusion11 with a purified yield of 6 mg per L of culture (
UbK6(Cbz7-8) (2.1 mg, 220 nmol) and UbSR(Cbz7-8) (3.3 mg, 336 nmol) were dissolved in DMSO (90 μL). DIEA (4 μL), H-OSu (0.39 mg, 3.36 μmol) and AgNO3 (57 μg, 336 nmol) were added. The reaction was incubated in the dark at 25° C. for 16 h. The crude mixture was precipitated with cold ether (1 mL) and washed with cold ether (1 mL) and air-dried. The proteins were dissolved in an ice-cold cocktail (5 mg/mL) consisting of 55% TFA, 35% DMS and 10% TFMSA. After stirring at 0° C. for 90 min the proteins were precipitated with 10 volumes of cold ether followed by 0.5% (vol.) pyridine. A heavy precipitate formed which was washed with cold ether, collected and dried. The dried precipitate was dissolved in buffer (100 mM Na2HPO4 pH 7.4, 8 M urea, 500 mM NaCl) at a protein concentration of approximately 0.5 mg/mL and dialyzed overnight against the same buffer (1 L) using a 3 kDa MWCO membrane (Spectrum Labs). The sample was then transferred to a fresh dialysis membrane and dialyzed overnight against folding buffer (20 mM Na2HPO4 pH 7.4, 100 mM NaCl). The protein was buffer exchanged into IEX buffer A (ammonium acetate pH 4.5) using an Amicon Ultra-15 3 kDa MWCO centrifugal filter device (Millipore). The sample was filtered (0.45 μM) and loaded onto a pre-equilibrated MonoS 5/50 GL column (GE Life Sciences) at a flow rate of 0.5 mL/min using an ÄKTA FPLC system. The flow was increased to 2 mL/min and a gradient running to 60% IEX buffer B (ammonium acetate pH 4.5, 1 M NaCl) over 10 minutes was applied. Fractions (0.5 mL) were collected and those containing K6-linked diubiquitin were determined by SDS-PAGE. The fractions were pooled and exchanged into IEX buffer A again using a centrifugal filter device. The sample was reapplied to the equilibrated MonoS column and at a flow rate of 2 mL/min a gradient running to 60% buffer B over 45 minutes was applied. Fractions were collected in 1 mL volumes and those containing pure K6-linked diubiquitin were pooled and concentrated to 1 mg/mL (200-300 μg, 5-8% yield). K6-linked diubiquitin was then dialyzed overnight against storage buffer (10 mM Tris-HCl pH 7.6) using a Dispo Biodialyzer 5 kDa MWCO (The Nest Group Inc.). The K6-linked diubiquitin samples were frozen at −20° C. for storage. Preparation of K29-linked diubiquitin was carried out as described for K6-linked diubiquitin except UbK29(Cbz7-8) was used in the isopeptide bond forming reaction.
Thioesters can be activated and converted in situ to N-hydroxysuccinimidyl esters in the presence of Ag(I), allowing selective acylation with amines23 24. We realized that this chemistry might be applied to the formation of a specific isopeptide bond between UbK6(Cbz7-8) and UbSR(Cbz8-9). We mixed the donor ubiquitin thioester (UbK6(Cbz7-8)) with UbSR(Cbz8-9) at a molar ratio of 1:1.5 in DMSO, in the presence of DIEA, silver nitrate (AgNO3, 3.75 mM) and N-hydroxysuccinimide (H-OSu, 37.5 mM) (
To remove the Cbz groups we used a cleavage cocktail consisting of 1:3:6 trifluoromethanesulfonic acid (TFMSA):trifluoroacetic acid (TFA):dimethylsulfide (DMS) at 0° C. for 1 hour25. The deprotected ubiquitin chain was precipitated, washed and resuspended in PBS buffer containing 8M Urea to form an unfolded ubiquitin chain. Since ubiquitin folds reversibly and enzymatically synthesized K48- and K68-linked ubiquitin chains are purified and refolded in vitro from denatured material26 it seemed reasonable that we could refold the atypical ubiquitin chains. We therefore dialyzed the protein into PBS buffer lacking urea to slowly re-nature the linked diubiquitin. Subsequent cation exchange allowed removal of residual monoubiquitin species, resulting in highly purified K6-linked diubiquitin (
To synthesize K29-linked ubiquitin we simply repeated the procedure described, except that we used UbTAG29-His6, in which the amber codon is at position 29, in place of UbTAG6-His6. For the preparation of UbBocK29 a yield of 8 mg from a 2 L culture was obtained.
To demonstrate that the purified K6- and K29-linked diubiquitin synthesized using the method according to the invention was linked via an isopeptide bond at the genetically directed site, and fully deprotected we used ESI-MS, and MS/MS sequencing.
Protein total mass was determined on an LCT time-of-flight mass spectrometer with electrospray ionization (Micromass). Samples were dissolved in 1:1 acetonitrile/H2O containing 1% formic acid. In the case of Cbz protected peptides, samples were dissolved in 4:3:3 acetic acid/acetonitrile/H2O. Samples were injected at 10 μl min−1 and calibration was performed in positive ion mode using horse heart myoglobin. 30 scans were averaged and molecular masses obtained by maximum entropy deconvolution with MassLynx version 4.1 (Micromass). Theoretical masses of wild-type proteins were calculated using Protparam (http://us.expasy.org/tools/protparam.html), and theoretical masses for unnatural amino acid containing proteins were adjusted manually.
For MS/MS analyses samples were digested with trypsin overnight at 37° C. Samples were then desalted and analyzed by LC-MS/MS with a LTQ Orbitrap Mass Spectrometer (Thermo Scientific). Target peptides were fragmented by collision-induced dissociation.
ESI-MS reveals a single mass-peak for the purified proteins, which corresponds to an isopeptide linked, quantitatively deprotected diubiquitin. (K6-linked observed mass=17113 Da, K29-linked observed mass=17111 Da, calculated mass=17112 Da for diubiquitin;
We were able to solve the structure of K6-linked diubiquitin by X-ray crystallography.
K6 diubiquitin crystallised with cubic morphology at a protein concentration of 1-2 mg mL−1 in hanging drops equilibrated against 19-20% PEG 3350, 0.2 M zinc acetate. Before freezing in a nitrogen cryo-stream, the crystals were soaked in mother liquor supplemented with 15% PEG 400. The largest crystals (30 μm) diffracted to 3 Å at the European Synchrotron Radiation Facility (ESRF) on beamline ID 14-2. Initial phases were obtained by molecular replacement using one ubiquitin moiety from the deposited coordinates of a K63 diubiquitin structure (pdb-id 2JF5,7). Structure refinement was carried out with PHENIX45 and model building was carried out within COOT45. In final rounds of refinement, TLS temperature factor refinement was performed. Geometric weight optimization in PHENIX resulted in a model with the lowest R/Rfree factors. Data collection and refinement statistics are shown in Table 1.
Crystals grown from 20% PEG 3350 and 200 mM ZnAc formed in a cubic space group (P4332) and diffracted to 3.0 Å resolution. The structure of K6-linked diubiquitin was solved by molecular replacement and subsequently refined (the statistics for refinement are shown in Table 1), revealing one K6-linked diubiquitin molecule in the asymmetric unit. Each ubiquitin adopts a native conformation confirming that the success of the refolding step in GOPAL. While ESI and MS/MS demonstrated the formation of the K6 isopeptide bond, the flexible isopeptide linkage is not fully resolved in the electron density maps, and Gly76 does not display discernible electron density. This has previously been observed in polyubiquitin structures due to the high flexible of the linkage.
The structure of K6-linked diubiquitin reveals that it adopts an asymmetric compact conformation distinct from previously described ubiquitin chain structures (
The K6 ubiquitin-ubiquitin interaction interface displays several previously undescribed features. The Ile44-patch is a common ubiquitin interaction interface, observed in the majority of interactions with ubiquitin binding domains and deubiquitinases8,27. In this interaction patch Val70 and Leu8 flank the central Ile44 residue, providing an extended hydrophobic interface (
We were able to build a model hexamer from the asymmetric K6 dimer by iteratively superimposing (in Coot28) the proximal ubiquitin moiety of one dimer onto the distal ubiquitin of a second dimer, to generate a new distal moiety (
The pRSET-UCHL3 plasmid was a generous gift from Keith D. Wilkinson, and was used prepare pure UCH-L3 as previously described43. An OTUB1 expression vector was kindly provided by Benedikt Kessler (Oxford), and the protein was purified according to44. USP21 was cloned from a plasmid kindly provided by Sylvie Urbe (Liverpool). Purification of remaining deubiquitinases is described in7. USP2, USP15, and BAP1 were purchased from ENZO LifeSciences or Boston Biochem. DUB assays were carried out as previously described7.
Deubiquitinases may be endowed with preference for particular chain linkages27. However, since most ubiquitin chain types have not been synthesized, deubiquitinase specificity profiling is incomplete. A mixture of K6 linked and K29 linked diubiquitin molecules, in which other lysine residues in the distal and proximal molecules were mutated to arginine, were examined for cleavage with hOtul, an OTU family deubiquitinase, and two JAMM deubiquitinase complexes29,30. These deubiquitinases were found not to cleave either chain type, but these experiments are problematic because it is unclear whether the mutated ubiquitin chains reflect the properties of the native chain. Deubiquitinases cover a large surface area in particular on the distal ubiquitin molecule27 and mutation of surface Lys residues to Arg may interfere with deubiquitinase binding.
We analyzed twelve deubiquitinases representing approximately 10% of known human deubiquitnases and covering four deubiquitinase families—ubiquitin C-terminal hydrolases (UCH), ubiquitin specific proteases (USP), Ovarian Tumor (OTU) deubiquitinases, and JAMM/MPN+ deubiquitinases—for their ability to cleave K6- and K29-linked diubiquitin in in vitro deubiquitinase assays7. In control experiments we used the same assay to cleave enzymatically assembled K63-linked diubiquitin.
UCH enzymes are highly efficient in cleaving small unstructured peptides from the C-terminus of ubiquitin (such as the His-tag,
In contrast to UCH enzymes, USP deubiquitinases are highly active in cleaving ubiquitin polymers, but often without obvious linkage specificity7. An exception is the tumor suppressor CYLD, whose USP domain prefers K63-linkages over K48-linkages32. When tested with K6-linked diubiquitin, four USP domains (USP2, USP5, USP15, USP21) disassembled this chain type as efficiently as K63-linked chains (
OTU domain deubiquitinases hydrolyze polyubiquitin chains, yet some members display remarkable selectivity between K48- and K63-linkages. A20 and OTUB1 are specific for K48-linked chains, and do not hydrolyze K63-linked or linear chains7,29, while TRABID is K63-linkage specific7,17. The OTU domains tested (A20, TRABID, Cezanne and OTUB1) did not cleave K6-linked diubiquitin, and A20, Cezanne and OTUB1 also did not cleave K29-linkages (
JAMM domain deubiquitinases have also been reported to be K63-linkage specific30,34, and the molecular basis for recognizing the K63-linkage was unveiled in the recent crystal structure of AMSH in complex with K63-linked diubiquitin35. AMSH binds to the extended K63-linked diubiquitin molecule and makes specific contacts with residues surrounding Lys63. We find that AMSH is inactive against K6- and K29-linked diubiquitin, while cleaving K63-linked diubiquitin under identical reaction conditions with high activity.
Ubiquitination is a reversible post-translational modification that regulates a myriad of eukaryotic functions. Our ability to study the effects of ubiquitination is often limited by the inaccessibility of homogeneously ubiquitinated proteins. In particular, elucidating the roles of the so-called ‘atypical’ ubiquitin chains (chains other than Lys48- or Lys63-linked ubiquitin), which account for a large fraction of ubiquitin polymers, is challenging because the enzymes for their biosynthesis are unknown. Here we combine genetic code expansion, intein chemistry and chemoselective ligations to synthesize ‘atypical’ ubiquitin chains. We solve the crystal structure of Lys6-linked diubiquitin, which is distinct from that of structurally characterized ubiquitin chains, providing a molecular basis for the different biological functions this linkage may regulate. Moreover, we profile a panel containing 10% of the known human deubiquitinases on Lys6- and Lys29-linked ubiquitin and discover that TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage.
Ubiquitination is a reversible post-translational modification in which a specific lysine residue in an acceptor protein forms an isopeptide bond with the C terminus of the ubiquitin donor. Although the role of ubiquitination in regulating protein stability via proteasomal targeting is well established, it is emerging that ubiquitin is involved in almost every aspect of biology, including cell signaling, intracellular trafficking and the response to DNA damage1,2. Ubiquitin forms covalent chains through each of its seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63) or its N terminus, and it is proposed that the distinct functions mediated by ubiquitin in diverse biological processes may be encoded in the distinct properties of the different ubiquitin chains2,3.
Although proteomic studies reveal that all chain types are present in vivo4,5, we know the most about Lys48- and Lys63-linked chains, which are important in proteasomal degradation and cell signaling, respectively1,6. In contrast, very little is known about the other so-called ‘atypical’ linkages, though they account for more than half of the ubiquitin linkages found in the model organism Saccharomyces cerevisiae5. A central challenge in studying the roles of specific ubiquitin chains is to synthesize homogeneous chains bearing defined linkages. Indeed, access to Lys48- and Lys63-linked ubiquitin, via identification of the cellular machinery (E1, specific E2 and E3 enzymes) that allows their specific biosynthesis in vitro, has allowed the characterization of their biological roles2. The structures of these chains have revealed distinct features, which may provide the molecular basis by which different chains are recognized by specific deubiquitinases or ubiquitin-binding domains7,8. Moreover, access to homogeneous chains has facilitated the generation of linkage-specific antibodies9,10, allowing the roles of specific chain types to be probed in vivo. Overall, the capacity to synthesize Lys48- and Lys63-linked ubiquitin chains has greatly accelerated our understanding of how these chain types are specifically recognized and regulated to mediate distinct biological processes, and we recently reported a biosynthesis of Lys11-linked chains, which should give further insights into this linkage11.
Unfortunately, the specific enzymes required to synthesize the atypical ubiquitin linkages are simply unknown, severely limiting our ability to study the structure and function of these chain types. In principle, chemical ligation approaches might be used to synthesize ubiquitin chains. Indeed, a lysine derivative containing a ligation auxiliary has been used to generate a native isopeptide bond between a histone and ubiquitin without mutation of adjacent residues12,13. However, this strategy involves multiple rounds of native chemical ligation and selective deprotection, yields small amounts of material and is best suited for ligation sites close to the termini of a target protein. Other ligation auxiliaries that yield an isopeptide linkage have been reported14,15, but their utility for linking entire proteins via a native isopeptide linkage has not been demonstrated. Moreover, these auxiliaries generally create glycine-to-alanine or glycine-to-cysteine mutations at the C terminus of the donor ubiquitin via nontraceless ligation reactions14,16. Although these mutations may not be important for some studies, they are known to abrogate the action of deubiquitinases17 and may alter the structure and dynamics of the linkage in unpredictable ways.
Here we report a new approach for the synthesis of homogeneously linked ubiquitin chains that allows the synthesis of specific chains in the absence of the cellular machinery for their synthesis. Our approach uses a powerful combination of genetic code expansion and chemoselective protein chemistry, which we term GOPAL (genetically encoded orthogonal protection and activated ligation). We demonstrate the generality of GOPAL by preparing Lys6- and Lys29-linked ubiquitin chains, which constitute 10% and 3% of ubiquitin linkages in yeast, respectively5. We solve the crystal structure of a Lys6-linked diubiquitin molecule synthesized by GOPAL and reveal that the molecule adopts a compact conformation distinct from the structures of Lys48- or Lys63-linked chains. Moreover, access to Lys6- and Lys29-linked diubiquitin allows us to profile a panel of 11 deubiquitinases (constituting approximately 10% of human deubiquitinases) for their ability to cleave these linkages. Using a quantitative deubiquitinase assay, we reveal that the ovarian tumor (OTU) family deubiquitinase TRABID, which has previously been shown to have high specificity for Lys63-linked ubiquitin over Lys48-linked ubiquitin18, has a 40-fold specificity for the Lys29 linkage over the Lys63 linkage.
The specific formation of an isopeptide bond between the C terminus of one ubiquitin (donor) and the amine of a specific lysine residue in another ubiquitin (acceptor) requires these functional groups to be differentiated from the other carboxylic acids and amines in both ubiquitin molecules (
Protecting all but one lysine residue in a protein requires the differentiation of chemically identical amino acid side chains. Indeed, although several reactions are known that are specific for one type of residue, it has been much more challenging to site-specifically modify proteins on one residue in the presence of many chemically identical residues. We realized that this problem might be solved by genetically encoding the site-specific incorporation of a protected version of lysine. Subsequent protection of all other amines in the protein with a chemically orthogonal protecting group and specific removal of the genetically encoded protecting group would yield a lysine with a free amine at the site where the protected lysine was genetically encoded (
To site-specifically install a protected lysine at position 6 of ubiquitin, we took advantage of the Methanosarcina barkeri MS pyrrolysine tRNA synthetase (MbPylRS) and its cognate amber suppressor tRNA (MbtRNACUA)19, which directs the efficient incorporation of N□-(t-butyloxycarbonyl)-
To protect the six N□-Lys amino groups and the N-terminal amine in UbBocLys6 with Cbz groups, we reacted the protein with seven equivalents of N-(benzyloxycarbonyloxy)succinimide (Cbz-OSu) in basic DMSO23. After 2 h of protection (
A donor ubiquitin molecule was prepared biosynthetically as a C-terminal thioester (UbSR), by thiolysis of an intein fusion12 with a purified yield of 6 mg per liter of culture (
Thioesters can be activated and converted in situ to N-hydroxysuccinimidyl esters in the presence of Ag(I), allowing selective acylation with amines24,25. We realized that this chemistry might be applied to the formation of a specific isopeptide bond between UbLys6(Cbz7-8) and UbSR(Cbz8-9). We mixed the donor ubiquitin thioester (UbLys6(Cbz7-8)) with UbSR(Cbz8-9) at a molar ratio of 1:1.5 in DMSO, in the presence of N,N-diisopropylethylamine (DIEA), silver nitrate (AgNO3, 3.75 mM) and N-hydroxysuccinimide (H-OSu, 37.5 mM) (Supplementary
To remove the Cbz groups, we used a cleavage cocktail consisting of 1:3:6 trifluoromethanesulfonic acid (TFMSA)/trifluoroacetic acid (TFA)/DMS at 0° C. for 1 h26. The deprotected ubiquitin chain was precipitated, washed and resuspended in PBS buffer containing 8 M urea to form an unfolded ubiquitin chain. Because ubiquitin folds reversibly, and enzymatically synthesized Lys48- and Lys68-linked ubiquitin chains are purified and refolded in vitro from denatured material27, it seemed reasonable that we could refold the atypical ubiquitin chains. We therefore dialyzed the protein into PBS buffer lacking urea to slowly renature the linked diubiquitin. Subsequent cation exchange allowed removal of residual monoubiquitin species, resulting in highly purified Lys6-linked diubiquitin (Supplementary
To synthesize Lys29-linked ubiquitin, we simply repeated the procedure described, except that we used UbTAG29-His6—in which the amber codon is at position 29—in place of UbTAG6-His6. For the preparation of UbBocLys29, a yield of 8 mg from a 2-liter culture was obtained. The subsequent steps in generating Lys29-linked diubiquitin proceeded with efficiency comparable to that of the steps described in detail for the preparation of Lys6-linked diubiquitin.
To demonstrate that the purified Lys6- and Lys29-linked diubiquitin synthesized using GOPAL was linked via an isopeptide bond at the genetically directed site, as well as fully deprotected, we used ESI-MS and MS/MS sequencing. ESI-MS revealed a single mass peak for the purified proteins, which corresponded to an isopeptide-linked, quantitatively deprotected diubiquitin (Lys6-linked diubiquitin, observed mass=17,113 Da; Lys29-linked diubiquitin, observed mass=17,111 Da; diubiquitin calculated mass=17,112 Da;
We were able to determine a structure for Lys6-linked diubiquitin by X-ray crystallography. Crystals grown from 20% PEG 3350 and 200 mM ZnAc formed in a cubic space group (P4332) and diffracted to 3.0-Å resolution. The structure of Lys6-linked diubiquitin was solved by molecular replacement and subsequently refined (for statistics see Supplementary Table 1), revealing one Lys6-linked diubiquitin molecule in the asymmetric unit. Each ubiquitin adopts a native conformation, confirming the success of the refolding step. Although ESI-MS and MS/MS demonstrated the formation of the Lys6 isopeptide bond, the flexible isopeptide linkage is not fully resolved in the electron density maps, and Gly76 does not show discernible electron density. This has previously been observed to occur in polyubiquitin structures because of the high flexibility of the linkage (Supplementary
The crystal structure of Lys6-linked diubiquitin reveals an asymmetric compact conformation distinct from previously described ubiquitin chain structures (
The Lys6 ubiquitin-ubiquitin interaction interface has several previously undescribed features. The hydrophobic patch surrounding Ile44 is a common ubiquitin interaction interface, observed in the majority of interactions with ubiquitin binding domains and deubiquitinases8,28. In this interaction patch, Val70 and Leu8 flank the central Ile44 residue, providing an extended hydrophobic interface (
The structure of Lys6-linked diubiquitin is distinct from previously observed diubiquitin structures (
Deubiquitinases may be endowed with preference for particular chain linkages28. However, as most ubiquitin chain types have not been synthesized, deubiquitinase specificity profiling is incomplete. In previous work, a mixture of Lys6- and Lys29-linked diubiquitin molecules, in which additional lysine residues in the distal and proximal molecules were mutated to arginine, were examined for cleavage with hOtul, an OTU family deubiquitinase, and two JAMM/MPN+ deubiquitinase complexes30,31. These deubiquitinases were found not to cleave either chain type, but these experiments are problematic because it is unclear whether the mutated ubiquitin chains reflect the properties of the native chain. Most deubiquitinases interact extensively with the distal ubiquitin molecule28, and mutation of surface lysine residues to arginines may interfere with deubiquitinase binding.
We analyzed 11 deubiquitinases, representing approximately 10% of known human deubiquitinases and covering four deubiquitinase families—ubiquitin C-terminal hydrolases (UCH), ubiquitin-specific proteases (USP), OTU deubiquitinases and JAMM/MPN+ deubiquitinases—for their ability to cleave Lys6- and Lys29-linked diubiquitin in in vitro deubiquitinase assays7. In control experiments, we used the same assay to cleave enzymatically assembled Lys63-linked diubiquitin. To discover enzymes that cleave Lys6- or Lys29-linked diubiquitin in preference to Lys63-, Lys48- or Lys11-linked or linear chains, we performed our assays under conditions in which the enzyme efficiently cleaves one of these previously analyzed chain types. Any enzyme that does not cleave Lys6- or Lys29-linked diubiquitin under these conditions is unlikely to have these linkages within its repertoire of substrates. However, if the Lys6- or Lys29-linked diubiquitin is an efficient substrate under these conditions, then the substrate specificity of the enzyme merits further investigation.
UCH enzymes are highly efficient in cleaving small, unstructured peptides from the C terminus of ubiquitin (such as the His tag, Supplementary
In contrast to UCH enzymes, USP deubiquitinases are highly active in cleaving ubiquitin polymers, though often without obvious linkage specificity. USP2 cleaved Lys6- and Lys63-linked diubiquitin more rapidly than Lys29-linked diubiquitin (
JAMM domain deubiquitinases have also been reported to be Lys63-linkage specific31,35, and the molecular basis for recognizing the Lys63 linkage was unveiled in the recent crystal structure of AMSH-LP in complex with Lys63-linked diubiquitin36. AMSH binds to the extended Lys63-linked diubiquitin molecule and makes specific contacts with residues surrounding Lys63. We find that AMSH is inactive against Lys6- and Lys29-linked diubiquitin, although it cleaves Lys63-linked diubiquitin under identical reaction conditions with high activity.
OTU domain deubiquitinases hydrolyze polyubiquitin chains, yet some members show remarkable selectivity between Lys48 and Lys63 linkages. A20 and OTUB1 are specific for Lys48-linked chains and do not hydrolyze Lys63-linked or linear chains7,30, whereas TRABID has a preference for Lys63 linkages7,18. Similarly, Cezanne has a preference for Lys11 linkages over Lys48 and Lys63 linkages11. The OTU domains tested (A20, TRABID, Cezanne and OTUB1) did not cleave Lys6-linked diubiquitin, and A20, Cezanne and OTUB1 also did not cleave Lys29 linkages (
To investigate the specificity of TRABID more quantitatively, we developed a quantitative deubiquitinase assay. Previous work has generally followed the cleavage of a fixed concentration of diubiquitin at varying enzyme concentrations37 or the cleavage of high concentrations of ubiquitin chains as a function of time38. These experiments provide a qualitative measure of specificity but are not compatible with extracting quantitative kinetic parameters. A method for extracting kinetic parameters for diubiquitin has been reported, but it requires 125I labeling of the ubiquitin chains39,40. To characterize the specificity of TRABID, after the cleavage of Lys63- and Lys29-linked diubiquitin we performed quantitative western blotting (
We have demonstrated that GOPAL, a powerful combination of genetic code expansion and chemoselective chemical reactions, can be used to synthesize proteins linked by specific isopeptide bonds. We have used this method to synthesize Lys6- and Lys29-linked diubiquitin, allowing structural characterization of Lys6-linked diubiquitin and profiling of deubiquitinases for Lys6- and Lys29-linked ubiquitin cleavage specificity for the first time.
Taken together, the deubiquitinase assays demonstrate that Lys6- and Lys29-linked diubiquitin are recognized and hydrolyzed efficiently by USP family deubiquitinases. The data extend the characterization of deubiquitinase specificity, demonstrating that deubiquitinases that are specific for Lys48 over the other previously synthesizable linkages (for example, OTUB1, A20) maintain this specificity with respect to Lys6- and Lys29-linked diubiquitin. However, we now reveal that TRABID is 40-fold more active toward Lys29-linked ubiquitin than Lys63-linked ubiquitin, whereas it is inactive against Lys6 linkages. This provides an impetus to study the role of Lys29 linkages in biological pathways such as Wnt signaling18. It will be interesting to analyze whether TRABID has Lys29-modified substrates. In addition to its OTU domain, TRABID also contains three N-terminal Npl4-type ZnF (NZF) domains that bind to Lys63-linked and linear ubiquitin chains7. It is possible that the NZF domains also bind to Lys29-linked chains or regulate the TRABID interaction with Lys63 linkages in vivo. Overall, the observations from the deubiquitinase profiling support the view that deubiquitinases are intrinsically highly specific. Lys6- and Lys29-linked diubiquitin will be important tools in identifying further deubiquitinases for these linkages.
Access to Lys6- and Lys29-linked ubiquitin chains may allow us to generate antibodies against these new linkages9,10 to further understand their cellular roles. Specific ubiquitin-binding domains (UBDs) that discriminate between different ubiquitin chains have been described8, and the ability to synthesize Lys6 and Lys29 linkages should accelerate the discovery of UBDs that may specifically recognize these linkages.
The crystal structure of Lys6-linked diubiquitin reveals a new compact, asymmetric conformation in which the proximal and distal ubiquitin moieties interact through distinct residues (
GOPAL may be applied to the synthesis of other isopeptide-linked proteins, including SUMOylated and Neddylated protein targets, though the method in its current form does require that the protein can be reversibly refolded. More broadly, the strategy for differentiating chemically identical residues in a protein by the site-specific encoding of a protected amino acid, chemical protection of other occurrences of the amino acid and deprotection of the encoded protected amino acid will allow the range of residue-selective and chemoselective reactions that have developed over many years of protein and peptide chemistry45 to be applied for site-selective protein modification. As the method uses genetic encoding to define the site of modification or ligation, it is applicable to modifications at any site in a protein.
Global Protection of UbBocLys6 and UbBocLys29 with Cbz-Osu.
Lyophilized UbBocLys6 (20 mg, 2.3 □mol) was dissolved in DMSO (1.7 ml), and DIEA (67 □l) was added. While the mixture was stirred, Cbz-OSu (4.81 mg, 19.3 □mol) was added to the reaction. After 2 h of stirring at 25° C., the reaction was transferred into cold ether (17 ml) and briefly vortexed. The precipitate was collected by centrifugation, and the ether layer was discarded. The pellet was washed with ice-cold ether (17 ml) and then air dried. For UbBocLys29, this and subsequent procedures were repeated pro rata to account for the reduced expression yield of UbBocLys29-His6.
The dry, globally protected peptide UbBocLys6(Cbz7-8), obtained from 10 mg of UbBocLys6, was dissolved in cold 9:6 TFA/H2O (1.28 ml) and incubated at 4° C. for 90 min. The selectively deprotected peptide was then precipitated and washed with ice-cold ether (2×13 ml). The aqueous and ether layers were removed, and the peptide was left to air dry.
Global Protection of Ub-MES Thioester with Cbz-Osu.
Lyophilized UbSR (10 mg, 1.15 □mol) was dissolved in DMSO (833 □l), and DIEA (17 □l) was added. While the mixture was stirred, Cbz-OSu (2.75 mg, 11.5 □mol) was added to the reaction. The reaction was stirred for 2 h at 25° C. and then transferred into cold ether (8.5 ml) and briefly vortexed. The precipitate was collected by centrifugation, and the ether layer was discarded. The pellet was washed with ice-cold ether (8.5 ml) and air dried.
UbLys6(Cbz7-8) (2.1 mg, 220 nmol) and UbSR(Cbz7-8) (3.3 mg, 336 nmol) were dissolved in DMSO (90 □l). DIEA (4 □l), H-OSu (0.39 mg, 3.36 □mol) and AgNO3 (57 □g, 336 nmol) were added. The reaction was incubated in the dark at 25° C. for 16 h. The crude mixture was precipitated with cold ether (1 ml), washed with cold ether (1 ml) and air dried. The proteins were dissolved in an ice-cold cocktail (5 mg ml−1) consisting of 55% TFA, 35% DMS and 10% TFMSA. After being stirred at 0° C. for 90 min, the proteins were precipitated with ten volumes of cold ether and 0.5% (v/v) pyridine. A heavy precipitate formed, which was washed with cold ether, collected and dried. The dried precipitate was dissolved in buffer (100 mM Na2HPO4, pH 7.4; 8 M urea; 500 mM NaCl) at a protein concentration of approximately 0.5 mg ml−1 and dialyzed overnight against the same buffer (1 liter) using a 3-kDa MWCO membrane (Spectrum Labs). The sample was then transferred to a fresh dialysis membrane and dialyzed overnight against folding buffer (20 mM Na2HPO4, pH 7.4; 100 mM NaCl). The protein was buffer exchanged into IEX buffer A (ammonium acetate, pH 4.5) using an Amicon Ultra-15 3-kDa MWCO centrifugal filter device (Millipore). The sample was filtered (0.45 □M) and loaded onto a pre-equilibrated MonoS 5/50 GL column (GE Life Sciences) at a flow rate of 0.5 ml min−1 using an ÄKTA FPLC system. The flow was increased to 2 ml min−1 and a gradient running to 60% IEX buffer B (ammonium acetate pH 4.5, 1 M NaCl) over 10 min was applied. Fractions (0.5 ml) were collected, and those containing Lys6-linked diubiquitin were determined by SDS-PAGE. The fractions were pooled and exchanged into IEX buffer A again using a centrifugal filter device. The sample was reapplied to the equilibrated MonoS column, and at a flow rate of 2 ml min−1, a gradient running to 60% buffer B over 45 min was applied. Fractions were collected in 1-ml volumes, and those containing pure Lys6-linked diubiquitin were pooled and concentrated to 1 mg ml−1 (200-300 □g, 5-8% yield). Lys6-linked diubiquitin was then dialyzed overnight against storage buffer (10 mM Tris-HCl, pH 7.6) using a Dispo Biodialyzer 5-kDa MWCO (The Nest Group Inc.). The Lys6-linked diubiquitin samples were frozen at −20° C. for storage. Preparation of Lys29-linked diubiquitin was carried out as described for Lys6-linked diubiquitin, except UbLys29(Cbz7-8) was used in the isopeptide bond-forming reaction.
Detailed methods for the cloning of acceptor ubiquitin (UbTAG6-His6 and UbTAG29-His6), the cloning of donor ubiquitin Ub1-76-thioester, the preparation of donor ubiquitin Ub1-76-thioester, deubiquitinase generation and Lys63 dimer generation, and preparation of acceptor ubiquitin (UbBocLys6 and UbBocLys29) can be found in the Supplementary Methods, as can methods for qualitative and quantitative deubiquitinase assays using silver staining and quantitative western blot and the analytical procedures for analyzing kinetic data. Methods for (UbLys6)2 crystallization, structure determination and refinement, and protein mass spectrometry can likewise be found in the Supplementary Methods.
The coordinates of Lys6-linked diubiquitin have been deposited with the Protein Data Bank, accession code 2xk5.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1005046.6 | Mar 2010 | GB | national |
| 1014556.3 | Aug 2010 | GB | national |
This application is a continuation of U.S. patent application Ser. No. 13/636,618, filed Sep. 21, 2012 (currently pending). U.S. patent application Ser. No. 13/636,618 is a national stage under 35 U.S.C. 371 of International Application PCT/GB2011/000420, filed Mar. 23, 2011 (currently published). International Application PCT/GB2011/000420 cites the priority of Great Britain patent application number 1005046.6, filed Mar. 24, 2010 (abandoned), and the priority of Great Britain patent application number 1014556.3, filed Aug. 27, 2010 (abandoned).
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13636618 | Sep 2012 | US |
| Child | 15199392 | US |
