Repurposing a Prokaryotic Toxin-Antitoxin System ... - ACS Publications

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

Repurposing a prokaryotic Toxin-Antitoxin system for the selective killing

2

of oncogenically stressed human cells

3 4 5

Mark A. Preston1†, Belén Pimentel1†, Camino Bermejo-Rodríguez1, Isabelle Dionne1, Alice

6

Turnbull1, Guillermo de la Cueva-Méndez1,2*

7 8

1MRC

9

2Andalusian

10

Cancer Cell Unit. Hutchison/MRC Research Centre. Hills Road, Cambridge CB2 0XZ, UK Centre for Nanomedicine and Biotechnology (BIONAND). Parque Tecnológico de

Andalucia. C/Severo Ochoa, 35. 29590 Campanillas. Málaga. Spain.

11 12

†These

authors contributed equally to this work.

13 14

* To whom correspondence should be addressed. E-mail: [email protected]

15 16 17 18 19 20 21 22

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Abstract

2

Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until co-expressed

3

antitoxin partners are degraded in response to stress. Although not evolved to function in

4

eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is

5

neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a

6

synthetic system that becomes active in human cells suffering a specific oncogenic stress.

7

Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is

8

selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin

9

system functions autonomously in human cells, distinguishing those that suffer the oncogenic

10

insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results

11

provide a framework for developing personalized anticancer strategies avoiding off-target effects,

12

a challenge that has been hardly tractable by other means thus far.

13

Keywords:

14

Cancer cell killing, Gene therapy, Protein therapy, Smart therapeutic system, Toxin-antitoxin

15

system, Kid-Kis

16


Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Introduction

2

Prokaryotic organisms and many of their resident plasmids have evolved toxin-antitoxin (TA)

3

systems, many of which function as stress response elements. These encode intracellular toxins

4

that target vital cellular processes, but that pass unnoticed to bearing cells most of the time

5

because they are neutralized by co-expressed antitoxins. However, the latter are rapidly

6

degraded by cells in response to stress, and this unleashes the toxic activity of their cognate

7

partners. Many of these toxins are selective endoribonucleases, and their activation alters gene

8

expression profiles precisely and specifically, enabling affected cells (or plasmids) to respond

9

effectively to the inducing stress.1,2

10

For instance, the Kid-Kis TA protein pair functions as a rescue system in prokaryotic plasmid

11

R1.3-5 Bacterial cells carrying this plasmid produce toxin Kid and antitoxin Kis, and therefore they

12

proliferate normally. However, this only occurs as long as cells contain enough copies of R1 to

13

enable plasmid transmission to the two descendant cells arising after cell division. When this is

14

not the case (i.e., when R1 copies are insufficient), rapid proteolytic degradation of Kis frees Kid,

15

and the latter cleaves mRNAs at UUACU sites. Cleavage of host-encoded transcripts by Kid

16

inhibits cell division, which avoids production of plasmid-free cells. At the same time, cleavage of

17

plasmid-encoded mRNAs inhibits the expression of genes evolved to keep R1 replication rates

18

low. This results in a rapid increase of R1 copies in affected cells, eliminating the threat that they

19

produce plasmid-free descendants if cell division is restored. Once this is achieved, Kid is re-

20

neutralized by newly produced Kis, and cells are allowed to proliferate again.

21

The activity and selectivity of Kid are exquisitely adapted to the genetic environment that R1

22

encounters in its host cells (i.e. E. coli and other closely related enterobacteria). This ensures that

23

activation of the endoribonuclease in these organisms induces a response (i.e. reversible

24

cytostasis) that is well suited for plasmid survival, the ultimate goal of the rescue system. In

25

contrast, no selective pressure has ever operated to safeguard plasmid R1 existence in human

26

cells, and there the heterologous expression of Kid triggers apoptosis, an effect from which co-

27

expression of Kis protects.6 This raises the question of whether toxin Kid and antitoxin Kis could

28

be used to build synthetic devices capable of achieving the selective killing of predetermined

29

populations of human cells. These devices could find applications in the field of cancer therapy,

30

where the longed for goal of inducing apoptosis in diseased cells without harming healthy cells in



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

patients has proved very challenging to achieve so far.7-9 However, the successful

2

implementation of this approach requires that the TA pair is conferred the ability to operate

3

autonomously, and appropriately, in human cells. Hence, such devices must be able to

4

distinguish targeted cells from non-targeted cells, and to modulate Kid/Kis ratios differentially in

5

response to that distinction, so that cancer cells are killed by Kid whilst healthy cells remain

6

protected by Kis. Conveniently, the activation of oncogenes changes protein outputs in human

7

cells.10-12 Thus, it may be possible that regulatory elements driving these changes in response to

8

a given oncogenic insult could be used to turn cytoprotective Kid/Kis ratios into cytotoxic ones,

9

with the aim of killing selectively cancer cells that suffer that particular oncogenic stress (Figure

10

1a).

11

For instance, High-Risk Human Papilloma Viruses (HR-HPVs) are the etiological cause of

12

cervical cancer, which kills more than a quarter-million women worldwide every year, and also of

13

a substantial proportion of head and neck cancers.13,14 These viruses encode E6, an oncoprotein

14

that binds to specific proteins in infected cells and connects them to E3 ligases, such as E6AP,

15

which polyubiquitinate them and triggers their proteosomal degradation (Figure 1b).15,16 Inspired

16

by the way in which Kid becomes active in bacterial cells, here we asked whether linking Kis to a

17

domain recognized by E6 in one of its target proteins would produce an antitoxin variant still able

18

to neutralize its cognate toxin, but unstable in cells transformed by HR-HPV and, if so, whether

19

the resulting synthetic TA system would achieve selective killing of these type of cancer cells

20

(Figure 1c).

21

Results and Discussion

22

To investigate this, we linked the 5'-end of a FLAG-tagged kis (FKis) gene to the coding

23

sequence of a PDZ-containing domain bound by E6 in MAGI-1, a known target of the oncoprotein

24

in human cells (Figure 2a and Supplementary Figure 1).17,18 The resulting gene was cloned in a

25

bacterial expression vector and introduced in E. coli cells, and these were subsequently

26

transformed with a compatible plasmid from which expression of Kid (or of its RNase-dead

27

mutant Kid18) could be regulated tightly. We then analyzed the consequences for cell

28

proliferation of inducing Kid expression in bacteria producing PDZFKis, compared to samples

29

producing neutralizing control protein FKis, or no antitoxin at all. These experiments showed that


Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

PDZFKis was as effective as FKis in protecting E. coli cells from the growth arrest imposed to

2

them by Kid (Figure 2b).

3

Next we examined by yeast two-hybrid analysis whether PDZFKis interacts with Kid, as

4

suggested by our results above, and also with oncoprotein E6. Genes encoding PDZFKis (or

5

controls FKis and Kis) were cloned into a bait vector, and those encoding Kid18 or E6 from viral

6

serotypes 16 and 18 (16E6 and 18E6), which account for more than 70% cervical cancers

7

worldwide,19 were cloned into a prey vector. All possible bait-prey plasmid combinations were

8

introduced into an appropriate yeast strain, and the resulting samples were seeded on solid

9

media enabling the growth of cells only when they produced interacting bait-prey protein pairs.

10

This showed that PDZFKis interacts with Kid (i.e. Kid18), as well as with the two E6 oncoproteins

11

tested, and also that the latter occurred through the PDZ-containing region fused to the antitoxin,

12

as interaction was not observed in control Kis- or FKis-expressing cells (Figure 2c).

13

We then analyzed whether PDZFKis was polyubiquitinated in human cells expressing these

14

oncogenic E6 proteins. To do this, HEK293T cells were transfected with different combinations of

15

plasmids encoding PDZFKis, 16E6 or 18E6, and HA-tagged Ubiquitin (Figure 2d). PDZFKis was

16

immunoprecipitated from protein extracts produced from these samples 48 h post-transfection,

17

electrophoresed by SDS-PAGE, transferred to a nitrocellulose membrane and blotted with

18

antibodies against the HA tag linked to ubiquitin monomers expressed in these cells. This showed

19

that, compared to a weak background signal in control cells (lane 1; Figure 2d), a strong

20

polyubiquitination pattern was detected in PDZFKis from samples that co-expressed E6 proteins

21

(lanes 2 and 3; Figure 2d). This signal was particularly strong in the case of samples expressing

22

18E6 (lane 3; Figure 2d), supporting results elsewhere showing that MAGI-1 is a preferred

23

substrate for this protein, compared to its homologue 16E6.18

24

Next we compared the stability of PDZFKis in HEK293T cells producing either 16E6 or 18E6, or

25

none of these oncoproteins. Cells were transfected with mixtures of vectors expressing PDZFKis

26

(or its empty control), and either 16E6 or 18E6 (or their empty control). Western blot was carried

27

out on protein extracts made from these samples 48 h after transfection, using an anti-FLAG

28

antibody. This showed that the concentration of PDZFKis was lower in cells expressing E6

29

oncoproteins than in those that did not (Supporting Information, Figure 2a; compare lanes 3 and 6

30

with 2 and 5). To find out whether this was due to a lower stability of PDZFKis in human cells



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

containing E6, we performed experiments similar to those above, but now inhibiting protein

2

synthesis in our samples for different time lengths before analysis, using cycloheximide. This

3

confirmed that the concentration of PDZFKis decreased rapidly in cells expressing 16E6 or 18E6

4

(lanes 5-8; Figure 2e), whilst it remained fairly constant over time in oncogene-free control cells

5

(lanes 1-4; Figure 2e).

6

Finally, we investigated whether a synthetic TA pair composed of PDZFKis and Kid would enable

7

selective killing of human cells derived from cervical tumors induced by HR-HPV of serotypes 16

8

and 18. To analyze this we made mammalian bicistronic vectors expressing PDZFKis plus either

9

Kid (PDZFKK) or control Kid18 (PDZFKK18). These plasmids were used to nucleofect SiHa-

10

(HPV16), HeLa- (HPV18), and control C33A cells (no HPV), in conditions producing at least 85%

11

nucleofection. The number of viable and apoptotic (Annexin V-positive) cells in each sample was

12

determined every 24 h, and values in PDZFKK samples where normalized against those obtained

13

in their corresponding PDZFKK18 controls. These studies confirmed that the synthetic TA pair

14

induced a dramatic proliferative inhibition in HR-HPV-positive cells, which was not observed in

15

control cells (Figure 3a and Supporting Information, Figures 4 and 5). They also showed that this

16

effect was paralleled by an increase in the numbers of apoptotic cells in these samples which,

17

again, was not observed in control cells (Figures 3b and 3c, and Supporting Information Figures

18

6-8). Importantly, expression of PDZFKis was clearly observed in both C33A and HPV-positive

19

cells when they were nucleofected with PDZFKis-Kid18 (Supporting Information, Figure 2b). This

20

discarded the possibility that the absence of effects in control cells could be due to lack of

21

transgene expression from our plasmids in these cells.

22

Functional elements enabling translational and post-translational regulation of protein ouputs in

23

human cells provide an exceptional toolbox for the construction of smart therapeutic agents, as

24

they can be repurposed to interplay with central tasks in response to predetermined cellular

25

signals.19-25 Here, we have used the prokaryotic Kid-Kis TA pair to produce an engineered

26

protein-based system capable of detecting the presence of oncoprotein E6 in the molecularly

27

complex intracellular environment of human cells and, in response, induce its own activation.

28

When E6 is present, the system interfaces with cellular signalling pathways at three distinct (and

29

consecutive) levels: the first one leads to polyubiquitination of PDZFKis, the second leads to the

30

destruction of the latter protein, and the third, for which unleashed Kid is responsible, induces


Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

programmed cell death. Importantly, when E6 is absent none of the events above occur, and the

2

system passes unnoticed to cells. This approach, which may be adapted to other oncogenic

3

insults, should contribute to the pursuit of personalized anticancer agents (ie targeted to the

4

specific tumor profiles) that are both cytocidal and selective, thereby avoiding off-target effects. In

5

this context, we advocate the value of genetic circuits of the sort presented here for addressing

6

such a complex challenge, which has been hardly tractable by other means thus far.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Material and Methods

2

Oligonucleotides. Oligonucleotides used in this work are shown in Supporting Information, Table

3

1. Name NSEss NSEas SS-Kis

Main Features NdeI-SfiI-EcoRI sense NdeI-SfiI-EcoRI antisense SfiI-SpeI-Kis

SS-FKis

SfiI-SpeI-FLAGKis

Kis-B NSMAG293

Kis-BamHI NdeI-SfiI-hMAGI293

MAG733G4S

hMAGI1-733-4Gly-SpeI

NS-16E6

NdeI-SfiI-16E6

16E6-SP NS-18E6

16E6-SalI-PstI NdeI-SfiI-18E6

18E6-SP

18E6-SalI-PstI

SS-Kid

SfiI-SpeI-Kid

Kid-E NSBBNXHss

B-Kid Kid-X pA ss1

Kid-EcoRI NheI-SfiI-BamHI-BstXI-NcoIXhoI-HindIII sense NheI-SfiI-BamHI-BstXI-NcoIXhoI-HindIII antisense HindIII-SfiI-SpeI-BclI-BamHI sense HindIII-SfiI-SpeI-BclI-BamHI antisense BamHI-BstXI-NcoI-NheIXhoI-NotI-EcoRI sense BamHI-BstXI-NcoI-NheIXhoI-NotI-EcoRI antisense BstXI-Kid Kid-XhoI SphI-synthetic pA sense

pA as1

SphI-synthetic pA antisense

pA ss2 pA as2

synthetic pA-NotI sense synthetic pA-NotI antisense

H-PrCMV PrCMV-S EK-16E6 16E6-S EK-18E6 18E6-S

HindIII-PrCMV PrCMV-SfiI EcoRI-KpnI-16E6 16E6-SpeI EcoRI-KpnI-18E6 18E6-SpeI

NSBBNXHas HSSBBss HSSBBas BBNNXNEss BBNNXNE as

Sequence 5'-TATGGGGGCCAAAAAGGCCAGTG-3' 5'-AATTCACTGGCCTTTTTGGCCCCCA-3' 5'-AAAGGCCAAAAAGGCCACTAGTATGCATACCACCCGACTGAA GA-3' 5'-AAAGGCCAAAAAGGCCACTAGTATGGACTACAAAGACGATGA CGACAAGGGAGGAGGAATGCATACCACCCGACTGAAGA-3' 5'-AAAGGATCCTCAGATTTCCTCCTGACCAGT-3' 5'-GGAATTCCATATGGGGGCCAAAAAGGCCAGTATGCTTTCTGC AGAGGATAATTTAGGT-3' 5'-GGACTAGTTCCTCCTCCTCCCGACTTTGGGCTCTTCTTGGG A-3' 5'CTGCATATGGGGGCCAAAAAGGCCATGCACCAAAAGAGAACT GCAAT-3' 5'- GGCCGCTGCAGGTCGACTTACAGCTGGGTTTCTCTACGT-3' 5'-CTGCATATGGGGGCCAAAAAGGCCATGGCGCGCTTTGAGGA TCCA-3' 5'- GGCCGCTGCAGGTCGACTTATACTTGTGTTTCTCTGCGTCG T-3' 5'AAAGGCCAAAAAGGCCACTAGTATGGAAAGAGGGGAAATCTG GCT-3' 5'- AAAGAATTCTCAAGTCAGAATAGTGGACA-3' 5'-CTAGCGGGGCCAAAAAGGCCGGGGATCCGCATTGGTAGGAA TTACCACAACCATGGGGGCTCGAGA-3' 5'-AGCTTCTCGAGCCCCCATGGTTGTGGTAATTCCTACCAATGC GGATCCCCGGCCTTTTTGGCCCCG-3' 5'-AGCTTGGCCAAAAAGGCCACTAGTACCGGTTGATCAG-3' 5'-GATCCTGATCAACCGGTACTAGTGGCCTTTTTGGCCA-3' 5'-GATCCCCACAACCATGGGCTAGCCTCGAGGCATGGCGCGGC CGCG-3' 5'-AATTCGCGGCCGCGCATGCCTCGAGGCTAGCCCATGGTTGT GGG-3' 5'-GAATTCCCACAACCATGGAAAGAGGGGAAATCTGGCT-3' 5'-GAATTCCTCGAGTCAAGTCAAGTCAGAATAGTGGACAG-3' 5'CGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCG TTTGT-3' 5'-CCAACACCCGTGCGTTTTATTCTGTCTTTTTATTGCCGCATG3' 5'-TCATAAACGCGGGGTTCGGTCCCAGGGCTGGCGC-3' 5'-GGCCGCGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACA AACGAC-3' 5'-CCCAAGCTTGTTGACATTGATTATTGACTAGTTA-3' 5'-CGAATTCGGCCTTTTTGGCCCTAGAGATCTGACGGTTCACT-3' 5'-AAAGAATTCGGTACCATGCACCAAAAGAGAACTGCAAT-3' 5'-AAAACTAGTTTACAGCTGGGTTTCTCTACGT-3' 5'-AAAGAATTCGGTACCATGGCGCGCTTTGAGGATCCA-3' 5'-AAAACTAGTTTATACTTGTGTTTCTCTGCGTCGT-3'

4 5

Plasmids. To make our two-hybrid constructs we first introduced an SfiI restriction sequence in

6

the multicloning site of parental plasmids pGADT7 and pGBKT7 (Clontech). For this,

7

oligonucleotides NSEss and NSEas were annealed to each other, and the resulting DNA

8

fragment was subcloned between the NdeI and EcoRI sites of the plasmids above, which

9

generated pGADT72 and pGBKT72. We then produced DNA fragments SfiI-SpeI-Kis-BamHI and


Page 8 of 24

Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

SfiI-SpeI-FLAGKis-BamHI by PCR, using oligo pairs SS-Kis/Kis-B and SSFKis/Kis-B respectively,

2

and plasmid mR1wt as template.3 These DNAs were digested with SfiI and BamHI, and the

3

resulting products were subcloned between the same sites in pGADT72, generating plasmids

4

pGADT72-Kis and pGADT72-FKis. Next we used oligonucleotides NSMAG293 and MAG733G4S to

5

amplify a DNA fragment encompassing the coding sequence between aminoacids 293 to 733 of

6

the human MAGI-1, using cDNA from human W12 cells as template. The resulting PCR product

7

was flanked by NheI-SfiI sites and an ATG codon in its 5' end, and by a track of 4 glycine codons

8

and an SpeI site in its 3' end (NdeI-SfiI-ATG-MAGI1293-733-Gly4-SpeI). This PCR product was

9

digested with SfiI and SpeI and subcloned between these same sites in pGADT72-FKis,

10

producing pGADT72-PDZFKis. Sequencing revealed that the PDZ-contaning fragment fused to

11

FKis in this construct corresponded to an isoform of human protein MAGI-1 (e.g. isoform X16;

12

Accession: XP_006713473.1 GI: 578806754), which is 12 amino acids shorter than reference

13

protein (NCBI Accession: NP_001028229.1 GI: 74272284) in the region preceding the PDZ

14

domains (Supplementary Figure 1). Additionally, oligonucleotide pairs NS-16E6/16E6-SP and

15

NS-16E6/16E6-SP were used to amplify the E6 oncogenes from HR-HPV serotypes 16 and 18,

16

respectively, using HPV16 and HPV18 plasmids as template. The resulting PCR products were

17

digested with SfiI and PstI, and cloned into the same sites in pGBKT72 to generate pGBKT72-

18

16E6 and pGBKT72-18E6. Similarly, oligonucleotides SS-Kid and Kid-E were used to produce an

19

SfiI-SpeI-Kid18-EcoRI DNA fragment by PCR, using mR118 plasmid as template.3 This DNA was

20

digested with SfiI and EcoRI and subcloned between the same sites in pGBKT72 to generate

21

pGBKT72-Kid18.

22

For our experiments in E. coli we used p177Prara-Kid and p177Prara-Kid18, described previously.5

23

In these vectors, the expression of Kid or its inactive RNase mutant, Kid18, can be induced with

24

arabinose and repressed with glucose. We also made tetracycline-inducible vectors to express

25

Kis and PDZFKis in E. coli. For this, we annealed oligonucleotides NSBBNXHss and as with each

26

other, and the resulting DNA fragment was inserted between the NheI and HindIII sites in plasmid

27

pTet-HS3F,5 to produce tetracycline-inducible vector pTetHMCS2. Next, DNA fragments bearing

28

FKis and PDZFKis were excised with SfiI and BamHI from vectors pGADT72-FKis and

29

pGADT72-PDZFKis, and subcloned into those same sites in pTetHMCS2, which produced

30

pTetH-FKis and pTetH-PDZFKis.

31

For our experiments in human cells we used several vectors. Expression of PDZFKis, Kid and

32

Kid18 was carried out from plasmids bearing a constituve CMV promoter. To make these vectors,

33

annealed oligonucleotides pairs HSSBB ss/as and BBNNXNE ss/as were ligated to each other,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

and the resulting DNAs were subcloned, in a tripartite reaction, between the HindIII and EcoRI

2

sites of pUC18NotI,5 to produce plasmid p18MCS2. Then, an IRES-containing fragment was

3

excised from pIRES2-DsRed (Clontech) by restriction with BamHI and BstXI, and subcloned

4

between those same sites in p18MCS2, to produce p18MCS2-IRES. The latter, digested with SfiI

5

and BamHI, was used as the recipient vector for fragment SfiI-PDZFKis-BamHI from pGADT72-

6

PDZFKis, which produced plasmids and p18-PDZFKis. Then, oligos B-Kid and Kid-X were used

7

to amplify Kid and Kid18 from plasmids mR1wt and mR118,5 and the resulting products were

8

digested with BstXI and XhoI and subcloned between these same sites in p18-PDZFKis (i.e.

9

downstream of the IRES in this plasmid), which produced p18-PDZFKK and p18-PDZFKK18.

10

Then a polyadenylation sequence was produced by ligating annealed oligo pairs pA ss1/as1 and

11

pA ss2/as2 with each other, and inserting the resulting DNA between the SphI and NotI sites in

12

the plasmids above, to generate p18-PDZFKispA, p18-PDZFKKpA and p18-PDZFKK18pA.

13

Finally, the CMV promoter was amplified from plasmid pIRES2-DsRed using oligos H-PrCMV and

14

PrCMV-S. The resulting product was inserted between the HindIII and SfiI sites of the vectors

15

above to create final vectors pCMV-PDZFK, pCMV-PDZFKK and pCMV-PDZFKK18. We also

16

produced EcoRI-KpnI-16E6-SpeI and EcoRI-KpnI-18E6-SpeI DNA fragments by PCR, using

17

oligonucleotide pairs EK-16E6/E6-S and EK-16E6/E6-S, and plasmids pGBKT72-16E6 and

18

pGBKT72-18E6 as templates. These PCR products were digested with EcoRI and SpeI and

19

subcloned between EcoRI and XbaI of pIRES plasmid (Clontech), which generated pCMV-16E6

20

and pCMV-18E6. Besides the constructs above we also used pcDNA3-HA-Ub,26 and pEGFP-N1

21

(Clontech).

22

Toxin-neutralization assays. DH4B E. coli cells were transformed with plasmids pTetH,

23

pTetHFKis or pTetHPDZFKis and selected on LB plates supplemented with ampicillin (100

24

µg/ml). Each one of the resulting samples was then transformed with p177Prara-Kid or p177Prara-

25

Kid18, and selected on solid ZYP5052 minimal medium27 supplemented with ampicillin (100

26

µg/ml) and kanamycin (50 µg/ml), as well as glucose 0.2% (to repress Kid/Kid18 expression) and

27

anhydrotetracycline (A-Tet) 0.2 µg/ml (to induce Kis expression). Single fresh colonies of all

28

resulting samples were grown exponentially for a few doublings in the same medium as above,

29

and then washed and diluted to an OD600nm of 0.03 in basic ZY minimal medium. 5 ml of these

30

dilutions were spotted onto the same plates as above (which repressed completely the

31

expression of Kid) or in alternative plates where glucose was substituted by 0.5% glycerol plus

32

0.2% arabinose, to induce the expression of Kid. These plates were incubated at 30º C for 16 h


Page 10 of 24

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

before analysis. All assays were performed at least three times using different starting cell

2

colonies in each experiment.

3

Protein-protein interaction assays. All steps required to perform our two-hybrid analysis were

4

carried out as recommended by the system's manufacturer (Clontech). Briefly, AH109 cells were

5

transformed with plasmid pGADT72 or with one of its derivative plasmids pGADT72-Kis,

6

pGADT72-FKis, or pGADT72-PDZFKis, using the lithium-acetate method. Positive clones were

7

selected by growing cells onto solid SD medium lacking Leucine. Each one of the resulting

8

samples was transformed again either with plasmid pGBKT72 or with each one its derivatives

9

pGBKT72-Kid18, pGBKT72-16E6 or pGBKT72-18E6. This time positive clones were selected by

10

growing cells onto solid SD medium lacking both leucine and tryptophan (SD-LT). This produced

11

AH109 cells carrying all possible (sixteen) paired combinations of bait and prey plasmids required

12

for our analysis. Fresh single colonies of each one of these samples were inoculated in liquid SD-

13

LT medium, which were kept growing exponentially several generations before diluting them to an

14

OD600nm of 0.1. 15 ml aliquots of these dilutions were then spotted onto solid SD-LT medium

15

(not-selective for protein-protein interaction), as well as in SD medium lacking not only leucine

16

and tryptophan but also histidine and adenine (SD-LTHA), and incubated at 30º C for two days. In

17

SD-LTHA only cells expressing interacting bait and prey proteins can grow, and therefore this

18

selective medium revealed protein-protein interactions within our analyzed proteins pairs. Assays

19

were performed several times, using different colonies for each sample. Positive and negative

20

two-hybrid controls supplied by the system's manufacturer were also tested at the same time.

21

Ubiquitination assays. 107 HEK293T cells were transfected by the Calcium Phosphate

22

method28 with 5 µg of an equimolecular mixture of plasmids pCMV-PDZFK, plus either pCMV-

23

16E6 or pCMV-18E6, plus pcDNA3-HA-Ub. 48 h later, cells were collected with a scraper,

24

pelleted by centrifugation, resuspended in 185 µl of a pre-heated lysis buffer (20mM Tris-Cl pH

25

7.4, 1% SDS, 1mM DTT, 0.5 mM EDTA) and incubated at 98º C for 10 minutes. Samples were

26

then centrifuged at 16,000 x g for 5 minutes to eliminate cellular debris, and supernatants were

27

collected for analysis. 150 µl of the latter were diluted into 1.5 ml of NP40 lysis buffer (50 mM

28

Tris-Cl pH 7.4, 150 mM NaCl, 0.5% NP40, 50 mM NaF) to reduce the concentration of SDS in our

29

samples. Then, 2 µg of a mouse monoclonal anti-FLAG antibody (M2; SIGMA) were added to the

30

dilutions above, and the mixtures were incubated for 1h at 4º C on a rotating wheel. After this, 30

31

µl of an equimolecular mixture of Protein A- and Protein G-coated agarose beads (General

32

Electrics) were added to the samples above, before incubating them again for another hour at 4º



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

C on the rotating wheel. Beads were then collected by centrifugation at 1,800 x g for 30 seconds

2

at 4º C, washed once with 500 µl of NP40 lysis buffer, and resuspended in 50 µl of SDS loading

3

buffer before heating them at 98º C for 5 min. Beads in these samples were pelleted by a short

4

centrifugation and 15 µl of each supernantant resolved by SDS-PAGE, transferred to a

5

nitrocellulose membrane and analyzed by western blotting, using a mouse monoclonal anti-HA

6

antibody (HA-7; SIGMA) to highlight ubiquitinated proteins in our immunoprecipitated samples.

7

Protein stability assays. 106 HEK293T cells were transfected using Fugene 6 (Promega) with a

8

mixture of plasmids pCMV-PDZFK (0.2 µg) and either pCMV-16E6 or pCMV-18E6, or their empty

9

parental vector (0.5 µg). 48 h later cells were lysed by scraping in 150 µl TNESV (50 mM Tris-

10

HCl, pH 7.5, 1% Nonidet P-40, 2 mM EDTA, 100 mM NaCl, 10 mM sodium orthovanadate)

11

supplemented with complete proteinase inhibitors (Roche). Cell lysates were clarified by

12

centrifugation and protein concentration was determined by the BCA assay (Pierce). A total of 10

13

µg protein from each cell lysate was resolved by SDS-PAGE, transferred to a nitrocellulose

14

membrane and analyzed by western blot using mouse monoclonal anti-FLAG (M2; SIGMA) and

15

anti-β-actin (AC-15; Abcam) antibodies. In other experiments we transfected cells as before, but

16

in tetraplicates. 48 h later one sample in each group was processed as above, and 10 µg/ml of

17

protein inhibitor cycloheximide was added to the rest of samples, which were then processed

18

subsequently, at two hours intervals.

19

Cell proliferation and apoptosis assays. 2.5 x 105 HeLa, SiHa and C33A cells were

20

nucleofected with 0.6 µg of a 9:1 (mol/mol) mixture of pCMV-PDZFKK (or pCMV-PDZFKK18)

21

and control plasmid pmaxGFP. Conditions were optimized following manufacturer's instructions

22

to obtain 85% nucleofection efficiency and 90% cell viability when using the mixture containing

23

the plasmid expressing Kid18. Nucleofected cells were seeded in triplicate for each sample and

24

time point to be analyzed (24, 48, 72 and 92 hours), using 96-well plates. In the case of samples

25

of the 24 h time point group, 13,000 cells were seeded per well. The same numbers of cells were

26

seeded in the case of any HeLa and SiHa sample nucleofected with the plasmid mixture

27

containing pCMV-PDZFKK. In the rest of cases 6,500 cells were seeded instead. Relative cell

28

viability and cell death were analyzed simultaneously every 24 h using a Cellomics ArrayScan

29

High Content Analysis reader. For this, Hoescht 33342 (Invitrogen) was used to stain nuclei in our

30

samples, and an Annexin-V-Cy5 apoptosis detection kit (Bioscience Life Sciences) was used to

31

detect apoptotic cells, in each case proceeding as recommended by the manufacturer. Every 24

32

h, the total number of cells (Hoescht-positive) and the number of apoptotic cells (Annexin-V-


Page 12 of 24

Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

positive) per well were counted in the HeLa, SiHa and C33A samples nucleofected with pCMV-

2

PDZFKK, and obtained values were compared to those observed in the same cell type and time

3

point when nucleofected with control pCMV-PDZFKK18. We also calculated the nuclear

4

fragmentation index in our samples, normalizing it against that in non-nucleofected control cells.

5

This parameter is the coefficient of variation (i.e. the standard deviation divided by the mean

6

value) of the fluorescence intensity of Hoescht-stained nuclei within a cell population, which

7

increases with apoptosis. A minimum of 1500 cells were analyzed in each case. To determine the

8

statistical significance of the differences between relative values (Kid/Kid18) obtained in HeLa or

9

SiHa samples (HPV+) compared to C33A ones (HPV-) we first determined applied the Shapiro-

10

Wilk test, to determine the parametric or non-parametric distribution of our data. Once this was

11

established, the T-student or the Mann-Whitney test were applied to determine the statistical

12

significance with a 95% confidence interval. To demonstrate that expression of PDZFKis occurs

13

in both HPV-positive (SiHa) and control (C33A) cells containing pCMV-PDZFKK18, protein

14

extracts were produced from samples 24 hours after nucleofection with the bicistronic vector, and

15

they were then immunoblotted with anti-FLAG and anti-β-actin antibodies as described above for

16

the protein stability assays.

17

Acknowledgments

18

We thank C. Scarpini and M. Pett for sharing cDNA of W12 cells and HPV16- and HPV18-

19

derived plasmids, respectively, as well as K. Sato for sharing the pcDNA3-HA-UB plasmid. We

20

also thank all other members of the GCM laboratory at the MRC Cancer Cell Unit, as well as V.

21

de Lorenzo and L. Sanchez-Palazón, for valuable comments and discussion. This work was

22

supported by funds from the Medical Research Council UK (Programme Grant MC_U105365008

23

to G. de la Cueva-Méndez).

24

Supporting information

25

Contains: i) a table describing all oligonucleotides used in this work; ii) an alignment of amino

26

acids 293-733 in reference human protein MAGI-1 with amino acids 293-721 in MAGI-1 isoform

27

X16, which is the one fused to Kis in this work; iii) western blots showing decreased stability of

28

PDZFKis in HEK293T cells that express HR-HPV E6 oncoproteins, compared to those that do

29

not; iv) western blot demonstrating efficient expression of PDZFKis in control (C33A) and HPV-

30

positivre (SiHa) cells nucleofected with bicistronic expression vector pCMV-PDZFKK18; and v)

31

box and whisker plots from raw data obtained to produce tables shown in Figure 3 in the main

32

text. This information is available free of charge via the Internet at http://pubs.acs.org/.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Author contributions

2

G.C-M. conceived the project. M.A.P, B.P., C.B-R., and G.C-M. designed the experiments.

3

M.A.P, B.P., C.B-R., I.D., and A.T. performed the experiments and, together with G.C-M.

4

analyzed the data. G.C-M. wrote the manuscript.

5


Page 14 of 24

Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

References

2

1. Yamaguchi, Y., Park, J.H., and Inouye, M. (2011) Toxin-antitoxin systems in bacteria and

3

archaea. Annu Rev Genet. 45, 61-79.

4

2. Hayes, F., and Van Melderen, L. (2011) Toxins-antitoxins: diversity, evolution and function. Crit

5

Rev Biochem. Mol. Biol. 46, 386-408.

6

3. Pimentel, B., Madine, M.A., and de la Cueva-Méndez, G. (2005) Kid cleaves specific mRNAs

7

at UUACU sites to rescue the copy number of plasmid R1. EMBO J. 24, 3459-3469.

8

4. de la Cueva-Méndez, G., and Pimentel, B. (2007) Gene and cell survival: lessons from

9

prokaryotic plasmid R1. EMBO Rep. 8, 458-464.

10

5. Pimentel, B., Nair, R., Bermejo-Rodríguez, C., Preston, M.A., Agu, C.A., Wang, X., Bernal,

11

J.A., Sherratt, D.J., and de la Cueva-Méndez, G. (2014) Toxin Kid uncouples DNA replication and

12

cell division to enforce retention of plasmid R1 in Escherichia coli cells. Proc. Natl. Acad. Sci.

13

U.S.A. 111, 2734-2739.

14

6. de la Cueva-Méndez, G., Mills, A.D., Clay-Farrace, L., Díaz-Orejas, R., and Laskey, R.A.

15

(2003) Regulatable killing of eukaryotic cells by the prokaryotic proteins Kid and Kis. EMBO J. 22,

16

246-251.

17

7. Ismael, G.F., Rosa, D.D., Mano, M.S., and Awada, A. (2008) Novel cytotoxic drugs: old

18

challenges, new solutions. Cancer Treat. Rev. 34, 81-91.

19

8. Niraula, S., Seruga, B., Ocana, A., Shao, T., Goldstein, R., Tannock, I.F., and Amir, E. (2012)

20

The price we pay for progress: a meta-analysis of harms of newly approved anticancer drugs. J.

21

Clin. Oncol. 30, 3012-3019.

22

9. Dy, G.K., and Adjei, A.A. (2013) Understanding, recognizing, and managing toxicities of

23

targeted anticancer therapies. CA Cancer J. Clin. 63, 249-279.

24

10. Merkley, M.A., Hildebrandt, E., Podolsky, R.H., Arnouk, H., Ferris, D.G., Dynan, W.S., and

25

Stöppler, H. (2009) Large-scale analysis of protein expression changes in human keratinocytes

26

immortalized by human papilloma virus type 16 E6 and E7 oncogenes. Proteome Sci. 7, 29. doi:

27

10.1186/1477-5956-7-29.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

11. Pütz, S.M., Vogiatzi, F., Stiewe, T., and Sickmann, A. (2010) Malignant transformation in a

2

defined genetic background: proteome changes displayed by 2D-PAGE. Mol. Cancer 9, 254. doi:

3

10.1186/1476-4598-9-254.

4

12. Lin, C.Y., Lovén, J., Rahl, P.B., Paranal, R.M., Burge, C.B., Bradner, J.E., Lee, T.I., and

5

Young, R.A. (2012) Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151, 56-

6

67.

7

13. Marur, S., D'Souza, G., Westra, W.H., and Forastiere, A.A. (2010) HPV-associated head and

8

neck cancer: a virus-related cancer epidemic. Lancet Oncol. 11, 781-789.

9

14. Hellner, K., and Münger, K. (2011) Human papillomaviruses as therapeutic targets in human

10

cancer. J. Clin. Oncol. 29, 1785-1794.

11

15. Beaudenon, S., and Huibregtse, J.M. (2008) HPV E6, E6AP and cervical cancer. BMC

12

Biochem. 9, Suppl. 1:S4. doi: 10.1186/1471-2091-9-S1-S4.

13

16. Pim, D., Bergant, M., Boon, S.S., Ganti, K., Kranjec, C., Massimi, P., Subbaiah, V.K.,

14

Thomas, M., Tomaić, V., and Banks, L. (2012) Human papillomaviruses and the specificity of

15

PDZ domain targeting. FEBS J. 279, 3530-3537.

16

17. Thomas, M., Glaunsinger, B., Pim, D., Javier, R., and Banks, L. (2001) HPV E6 and MAGUK

17

protein interactions: determination of the molecular basis for specific protein recognition and

18

degradation. Oncogene 20, 5431-5439.

19

18. Kranjec, C., and Banks, L. (2011) A systematic analysis of human papillomavirus (HPV) E6

20

PDZ substrates identifies MAGI-1 as a major target of HPV type 16 (HPV-16) and HPV-18 whose

21

loss accompanies disruption of tight junctions. J. Virol. 85, 1757-1764.

22

19. de Sanjose, S., Quint, W.G., Alemany, L., Geraets, D.T., Klaustermeier, J.E., et al. (2010)

23

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-

24

sectional worldwide study. Lancet Oncol. 11, 1048-1056.

25

20. Ruder, W.C., Lu, T., and Collins, J.J. (2010) Synthetic biology moving into the clinic. Science

26

333, 1248-1252.


Page 16 of 24

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

21. Grünberg, R., and Serrano, L. (2010) Strategies for protein synthetic biology. Nucleic Acids

2

Res. 38, 2663-2675.

3

22. Culler, S.J., Hoff, K.G., and Smolke, C.D. (2010) Reprogramming cellular behavior with RNA

4

controllers responsive to endogenous proteins. Science 330, 1251-1255.

5

23. Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., and Benenson, Y. (2011) Multi-input RNAi-

6

based logic circuit for identification of specific cancer cells. Science 333, 1307-1311.

7

24. Church, G.M., Elowitz, M.B., Smolke, C.D., Voigt, C.A., and Weiss, R. (2014) Realizing the

8

potential of synthetic biology. Nat. Rev. Mol. Cell. Biol. 15, 289-294.

9

25. Lienert, F., Lohmueller, J.J., Garg, A., and Silver, P.A. (2014) Synthetic biology in mammalian

10

cells: next generation research tools and therapeutics. Nat. Rev. Mol. Cell. Biol. 15, 95-107.

11

26. Nishikawa, H., Ooka, S., Sato, K., Arima, K., Okamoto, J., Klevit, R.E., Fukuda, M., and Ohta,

12

T. (2004) Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains

13

catalyzed by BRCA1-BARD1 ubiquitin ligase. J. Biol. Chem. 279, 3916-3924.

14

27. Studier, F.W. (2005) Protein production by auto-induction in high density shaking cultures.

15

Protein Expr. Purif. 41, 207-234.

16

28. Pear, W.S., Nolan, G.P., Scott, M.L., and Baltimore, D. (1993) Production of high-titer helper-

17

free retroviruses by transient transfection. Proc. Natl. Acad. Sci. U.S.A. 90, 8392-8396.

18



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure legends

2

Figure 1. Rationale for the construction of a synthetic TA pair that kills oncogenically stressed

3

human cells. (a) Diagram depicting the composition and pursued behavior of the synthetic

4

system. Toxin Kid and/or antitoxin Kis are linked to an osPOM (i.e. oncogene-specific Protein

5

Output Modifier) element. This allows the system to distinguish cells suffering a particular

6

oncogenic insult (the input) from those that do not and, depending on this, to induce a change in

7

Kid/Kis ratios (the output), that triggers selective killing of oncogenically stressed cells (the

8

outcome). (b) Naturally ocurring osPOMs in humans. Cellular transformation by HR-HPV involves

9

the function of oncoprotein E6, which binds to and induces the polyubiquitination of specific target

10

proteins, and therefore their proteosomal degradation. Target protein domains responsible for the

11

above (shown in yellow) constitute natural E6-sPOMs. (c) The latter may be used to engineer a

12

synthetic TA system enabling selective killing of HPV-induced cancer cells. Fusion of an E6-

13

sPOM to Kis may produce an antitoxin variant still able to neutralize Kid, but bound by E6 and

14

therefore polyubiquitilated and highly unstable in HPV-transformed cells. Such system should be

15

unable to protect the latter cells, but not other cells, from Kid-induced apoptosis.

16

Figure 2. Functional characterization of PDZFKis. (a) Scheme of human MAGI-1 protein,

17

highlighting domains (boxed) required for HR-E6 recognition and subsequent proteosomal

18

degradation, and of the chimeric protein resulting from fusing FLAG-tagged (F) Kis to the latter.

19

(b) PDZFKis neutralizes Kid. E. coli cells expressing FKis, PDZFKis or none of these proteins,

20

were transformed with plasmids from which Kid (or Kid18) production is inhibited by glucose (Glu)

21

and induced by arabinose (Ara). Growth analysis of these samples in Glu- and Ara-plates shows

22

that PDZFKis neutralizes the toxin. (c) PDZFKis interacts with Kid and with HR-E6 proteins. Two-

23

hybrid AH109 yeast cells transformed with Kis-, FKis-, PDZFKis- or empty bait vectors (top

24

labels) and with either Kid18-, 16E6-, 18E6-, or empty prey vectors (right labels) were grown in

25

SD medium lacking only leucine and tryptophan (non-selective) or histidine and adenine too

26

(selective). Growth of the corresponding samples on selective medium reveals that PDZFKis

27

interacts with Kid and with the two HR-E6 proteins analyzed. (d) PDZFKis is polyubiquinated in

28

cells expressing HR-E6. HEK293T cells were transfected with pCMV-PDZFK, pCMV-16E6,

29

pCMV-18E6 and pcDNA3-HA-Ub as indicated. PDZFKis was immunoprecipitated from extracts

30

produced from these samples 48h post-transfection, using an anti-FLAG antibody. Precipitates

31

were resolved by SDS-PAGE and immunoblotted with a mouse monoclonal anti-HA antibody, to


Page 18 of 24

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

detect ubiquitilated PDZFKis (top panel). A Coomasie stained SDS-PAGE gel is also shown

2

(lower panel). Position of molecular weight markers, ubiquitinated proteins (HA-Ub), IgG and

3

PDZFKis are indicated. (e) PDZFKis is unstable in human cells expressing HR-E6 proteins.

4

HEK293T cells transfected with pCMV-PDZFK and either pCMV-16E6 (top) or pCMV-18E6

5

(bottom), or none of these plasmids (No E6) were treated with cycloheximide for the indicated

6

times, 48 h post-transfection. Extracts from these samples were resolved by SDS-PAGE and

7

immunoblotted with anti-FLAG and anti-β-actin antibodies, showing that HR-E6 destabilizes

8

PDZFKis in human cells.

9

Figure 3. The PDZFKis-Kid system selectively kills human cells transformed by HPV. (a)

10

PDZFKis-Kid inhibits the proliferation of HR-HPV-positive cell cultures. SiHa (HPV16), HeLa

11

(HPV18) and C33A (no HPV) cells nucleofected with PDZFKis-Kid (PDZFKK) or PDZFKis-Kid18

12

(PDZFKK18) bicistronic expression vectors were cultured, and their numbers counted every 24 h.

13

Values in PDZFKK samples were normalized to those in their PDZFKK18 controls, and resulting

14

ratios were represented, showing that exposure to PDZFKis and Kid inhibits the growth of SiHa

15

and HeLa (but not of C33A) cultures. (b) Increased Annexin-V binding to SiHa and HeLa cells

16

nucleofected with PDZFKK. Samples in (a) were incubated with Annexin-V-Cy5, and the number

17

of cells bound by this protein in PDZFKK- samples were normalized to those in their PDZFKK18

18

controls, showing that exposure to PDZFKis and Kid increased the number of apoptotic cells over

19

time in SiHa and HeLa (but not C33A) cultures. (c) Increased nuclear fragmentation in SiHa and

20

HeLa cells nucleofected with PDZFKK. Results in (b) were confirmed by comparing the

21

coefficient of variation of Hoechst staining, which increases with nuclear fragmentation during

22

apoptosis, in PDZFKK (black symbols) and PDZFKK18 (white symbols) samples, using non-

23

nucleofected control cells as the control reference. Error bars represent s.e.m. (n=3 for HeLa and

24

SiHa samples and n=6 for C33A samples). * is p

Repurposing a Prokaryotic Toxin-Antitoxin System ... - ACS Publications

Recommend Documents