The family of the Nine amino acid Transactivation Domain, 9aaTAD family, comprises currently over 40 members. The 9aaTAD domains are universally recognized by the transcriptional machinery from yeast to man. We had identified the 9aaTAD domains in the p53, Msn2, Pdr1 and B42 activators by our prediction algorithm. In this study, their competence to activate transcription as small peptides was proven. Not surprisingly, we elicited immense 9aaTAD divergence in hundreds of identified orthologs and numerous examples of the 9aaTAD species' convergence. We found unforeseen similarity of the mammalian p53 with yeast Gal4 9aaTAD domains. Furthermore, we identified artificial 9aaTAD domains generated accidentally by others. From an evolutionary perspective, the observed easiness to generate 9aaTAD transactivation domains indicates the natural advantage for spontaneous generation of transcription factors from DNA binding precursors. The transcription factors are versatile regulators of gene expression. Their DNA binding domains, DBD, specifically recognize regulatory elements and their transactivation domains, TAD, mediate activation of transcription. A number of tested TADs is functional in both yeast and mammals e.g. Gal4 and p53 transcription factors ,2.www.piskacek.org). The 9aaTADs are annotated on protein database UniProt (www.uniprot.org/9aaTAD).The Nine amino acid Transactivation Domain, 9aaTAD, is universally recognized by the transcriptional machinery in eukaryotes. Currently, the 9aaTAD family comprises of over 40 members including Gal4, Oaf1, Pip2, Pdr1, Pdr3, Leu3, Tea1, Pho4, Gln3, Gcn4, Msn2, Msn4, Rtg3, E2A, MLL, p53-TADI, p53-TADII, FOXO3, NF-kB, NFAT, CEBPA/E, ESX, ELF3, ETV1, KLF2/4, EBNA2, VP16, HSF1, HSF2, HsfA, Gli3, Sox18, PIF, Dreb2a, MTF1, OREB1, WRKY45, NS1, MKL1, VP16, EBNA2, KBP220, ECapLL, P201, AH, and B42 transcription factors. We and others have shown the 9aaTAD domains have competence to activate transcription as small peptides . We havePreviously, we predicted two distinct 9aaTAD domains for p53 protein with conserved proximal Leucines ,16. BothIn this study we aimed to determine the evolutional conservation of the 9aaTAD domains and to prove their competence to activate transcription.The construct pBTM116-HA (BHA) was generated by Klenow fill-in of oligonucleotides and subcloned in to pBTM116 (B) EcoRI. G1-G45 and H1-H45 were generated by PCR and subcloned in to pBTM116 EcoRI and BamHI sites. All constructs were sequenced by Eurofins Genomics. All construct information, primer sequences and further detailed information are available on request.β-galactosidase activity was determined in the yeast strain L40 crude extracts using the ONPG substrate . The aveEscherichia coli BL21. Cells were grown in LB medium at 37°C and induced with 1 mM IPTG for 2 h at 25°C. The cells were harvested by centrifugation, suspended in lysis buffer with complete protease inhibitors EDTA-free (Roche 04719948001). Protein extracts were cleared by centrifugation for 10 min at 12,000 g, diluted 10 times with GST buffer , applied on 20 uL GST beads slurry, incubated for 10 min with gently inverting and washed 4 times. Protein purity was estimated by SDS-PAGE. The yeast strain L40 crude extracts were produced by lysis (lysis buffer Roche 04719948001 with complete protease inhibitors EDTA-free) for 15 min at 25°C and clarified by centrifugation 14.000g for 10 min at 4°C.The GST-KIX expression constructs kindly provided by Isabelle Lemasson  were theFor both predicted p53 9aaTAD domains, we aimed to prove their competence to activate transcription.The human p53 9aaTAD constructs were generated with and without conserved 9aaTAD proximal regions including Leucines. We generated p53 9aaTAD constructs from several other species representing variability within the family . The facNext, we tested minimal p53 9aaTAD-I domain for interaction with the KIX domain. The LexA hybrid construct with HA-tag and minimal p53 9aaTAD-I domain was expressed in yeast L40 strain. The GST construct with human KIX domain was expressed separately in E.coli strain BL21. Under our experimental conditions, the purified GST-KIX protein was able to specifically pull down p53 9aaTAD-I from whole crude yeast extract .Finally, we also generated an artificial derivate of the human p53 9aaTAD-I, in which we followed variation found in the p53 TAD-II domain and substituted conserved Tryptophan in TAD-I domain for Phenylalanine conserved in TAD-II domain.All tested p53 9aaTAD domains have competence to activate transcription . The proThe transcription factors including Gal4, Oaf1 and Pdr1, members of the 9aaTAD family, are known to interact with the MED15 transcriptional mediator. Therefore we focused our attention on other MED15 interacting proteins such as Msn2 and Msn4 transcription factors , 22. TheInitially, we used the ExPASy SIB BLAST to identify the Msn2 and Msn4 orthologs. By using our online 9aaTAD prediction, we have identified the 9aaTAD domains in Msn2, Msn4 and their orthologs. First, we showed that the predicted 9aaTADs were located within the reported transactivation domains . Second,S.c.Msn2 and S.c.Msn4 but not in K.a.Msn2/4   had encoWe used the ExPASy SIB BLAST to identify the Pdr1 and Pdr3 orthologs and confirmed the 9aaTAD conservation in the family . Becausewww.piskacek.org) in Gal4 TAD replicas G80BP-A and G80BP-B originally shown in [In recent study , we reported artificial 9aaTAD domains identified by online 9aaTAD prediction , a part of DNA binding domain, DBD, which is not involved in transactivation. This Gal4 region can be fused with other peptides to form strong artificial 9aaTAD domains. We demonstrated the ability of the Gal4DBD to form strong artificial 9aaTAD domain by fusing it with the second half site of the Gal4 9aaTAD domain  ,36–40. TAlbeit the 9aaTAD domains have enormous variability and amorphous character, they are universally recognized by transcriptional machinery throughout eukaryotes . The 9aaIn this study, we showed that both p53 9aaTAD domains activate transcription as small peptides. Nevertheless, we showed that the p53 9aaTAD-I and 9aaTAD-II domains do not have one amino acid identical. We also demonstrated that their shared 9aaTAD motif enables residue swapping. In respect of the shared 9aaTAD motif in p53 protein, the modification of the first p53 9aaTAD-I domain towards the second 9aaTAD motif (in the p53 9aaTAD-II domain) did not result in a lost of the transactivation potential .Similar result has been reported for p53 mimetic ECapLL . We recoOur results have shown the 9aaTAD convergence in the rabbit p53 and Gal4 proteins, which have 44% overall identity and 78% similarity. Both 9aaTAD domains resembled another 9aaTAD domain, Sox18 . PreviouThe 9aaTAD proximal Leucines are well conserved in the p53 family, but less in other members of the 9aaTAD family, e.g. Msn2 activators. The Leucines are not conserved also in other 9aaTAD proximal regions demonstrating overall natural variability . From our results for the Msn2 and p53 activators, we demonstrated that the contribution of the 9aaTAD proximal regions is individually variable and not always essential for transactivation function. The 9aaTAD proximal regions might be part of the 9aaTAD helix e.g. E2A and MLL .The members of the 9aaTAD family share small size, highly variable pattern and hydrophobic and hydrophilic clusters. The 9aaTAD domains could be split in two subdomains and merged with other 9aaTAD subdomains without losing activity. The 9aaTAD domains could be largely and almost freely modified, and new 9aaTAD domain could be easily generated by accident or by intention.The 9aaTAD family is not exclusive transactivation domain in eukaryotes. There are numerous known transactivation domains unrelated to the 9aaTAD domain with different mode of binding to transcriptional mediators e.g. STAT2  or diffeDuring the evolution, the fast generation of numerous specific transcription factors has been crucial for tailored regulation of individual genes. The observed easiness of spontaneous generation of artificial 9aaTAD transactivation domains in the labs, "Activating regions: as many as you like" , indicatS1 FigThe protein level produced from the constructs 1-10p53, HaA and HaY in L40 strain were monitored by Westernblotting. The proteins comprise LexA a HA tags with a total size of about 21 kDa. The degradation product comprising almost LexA protein (LexA torso) has a total size of about 20kDa.(TIF)Click here for additional data file.S2 FigWe fused the part of the Gal4 DNA binding domain (92–100 aa), DBD, with the second half site of the Gal4 9aaTAD. The Gal4 DNA binding domain region (92–100 aa) substitute for the first half of the 9aaTAD in this and other artificial constructs. The amino acids in fusion region of both Gal4 DNA binding domain and the random peptide are essential for transactivation function. Notice: Gal4 region (1–84 aa) is sufficient for DNA binding. Blue asterisks referred to the results of this study , constru(TIF)Click here for additional data file.S3 FigThe full sequence of B42 peptide (1–79 aa) and the identified 9aaTAD within are shown. The B42 9aaTAD LexA hybrid construct was assayed in L40 strain for the transactivation activity.(TIF)Click here for additional data file.S4 FigK/Q, D/E, V, E, S/T, F, F, D, N/E). Noteworthy, the Gcn4 protein has an unusual Lys in the position 1 of the 9aaTAD-II, which is out of predictive recognition (sequence: KEWTSLFDN). The unusual amino acids in the 9aaTAD domains were found also in other members of the 9aaTAD family e.g. Cysteine and Glycine in rat and mouse p53 9aaTAD-I. We assigned many transactivation domains to the 9aaTAD family, which fit with size, share deliberated 9aaTAD pattern and the clusters of the hydrophobic/hydrophilic amino acids. The amorphous nature of the 9aaTAD domains does not offer any invariant or conserved residues, which let us to generate the absolute reliable pattern for all of them. Therefore i) our prediction is still uncertain, ii) generate many false positives, iii) pattern does not fit for all 9aaTAD variations of the orthologs, and iv) putative 9aaTADs need always to be experimental verified. Nevertheless, there are many examples, where the 9aaTAD prediction works well, e.g. MLL or p53 activators. MLL (Q03164) is 3969 amino acids long protein with only two predicted 9aaTADs, where one of them is confirmed transactivation domain. Over two hundred Gcn4 9aaTAD-I modifications were generated and their competence to activate transcription were assayed by Warfied et al., 2014. Despite of the authors' enormous effort to define the transactivation domain by this approach, they found merely Tryptophan-rich transactivation domains deprived of acidic residues . We tested the Tryptophan-rich activation domain m120 (AFWMWLFAT) derived from Gcn4 9aaTAD in the standard LexA hybrid assay. The Gcn4 mutant m120 has no activity , what indicated serious data inconsistency in the report by Warfied et al., 2014. Therefore we proceeded differently to characterise the Gcn4 TAD. Because of the Gcn4 TAD-I domain fulfils the deliberate criteria for 9aaTAD motif , we made subdomain swapping between putative Gcn4 9aaTAD and Gal4 9aaTAD and generated a hybrid construct S11 to prove predicted shared motif. The construct S11 has comparable transcriptional activity to the Gal4 9aaTAD in LexA hybrid assay, what proofs the concept for the 9aaTAD motif in Gcn4 TAD-I by the swapping experiment . Noteworthy, the hydrophobic patches in the core of Gcn4 9aaTAD-I (V_SFF) and Gcn4 9aaTAD-II (w_SLF) have high similarity with the 9aaTAD domain of the B.a.Gal4 (I_SLF).We identified the putative 9aaTADs in both reported Gcn4 transactivation domains , which amino acid variations are very close to mouse and bovine p53 9aaTADs Click here for additional data file.


S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen  Streptococcus are highly intertwined with humans and animals as commensal, opportunistic and pathogenic bacteria. Often streptococci show pronounced host tropism, but these bacteria can also cause zoonotic or anthroponotic infections in more uncommon hosts [Gram-positive bacteria of the genus on hosts .Streptococcus (S.) suis, we identified recently a novel IgG degrading enzyme designated IgdE [in silico modeling and mutational studies of the potential catalytic triad residues, IgdE was assigned to be a cysteine protease. This protease does not have homology to any protease previously described and is thereby the founding member of a novel cysteine protease family designated C113 within the CA clan in the MEROPS peptidase database (https://merops.sanger.ac.uk/) [S. suis is highly specific for porcine IgG and no other substrate has been identified. Immune evasion from Ig mediated immune defense seems to be of special importance for bacteria of the Streptococcus genus. Several Ig degrading enzymes of streptococci have been identified, such as the IgG specific proteases, IdeS of S. pyogenes [S. equi subsp. zooepidemicus and IdeE of S. equi subsp. equi [S. pneumoniae, S. oralis, S. sanguis and S.mitis have been described [S. suis, designated IdeSsuis [Starting with the observation of a Immunoglobulin (Ig) G degrading activity in culture supernatants of the important endemic pig pathogen ted IgdE . Throughpyogenes , IdeZ ofsp. equi . In addiescribed , 7 and rIdeSsuis . Most ofIgG is the major antibody in serum of most higher organisms , 10. HumPorcine IgG is divided into six subclasses . These sS. suis in other Streptococcus species as putative IgdE family proteases and compared them by phylogenetic analysis. The igdE genes of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for expression and purification of recombinant protein followed by screening for potential substrates of these putative proteases.In this study, we employed rigorous homology searches to identify several homologues of IgdE of S. agalactiae, also known as Group B Streptococcus, is commonly found as a commensal in cattle and humans [d humans , 23, butd humans  and genid humans .S. porcinus is a bacterium most commonly found in the respiratory tract of pigs [S. pseudoporcinus has recently been distinguished from S. porcinus as a separate species [ of pigs  and has  of pigs  and stil of pigs . S. pseu species  and has  species .S. equi subsp. zooepidemicus, a commensal and opportunistic pathogen of horses as well as other mammals, can cause severe zoonotic infections in humans, such as sepsis, meningitis and endocarditis [S. equi subsp. zooepidemicus can cause several different pathologies, including respiratory tract infections, uteritis and wound infections. The cause of the highly contagious upper respiratory tract disease strangles, S. equi subsp. equi, is believed to be a clonal descendent of an ancestral strain of S. equi subsp. zooepidemicus [carditis . In horsidemicus , 33.S. agalactiae was specific for human IgG1. IgdE of S. porcinus was specific for porcine IgG, while IgdE of S. equi subsp. zooepidemicus was specific for equine IgG7. IgdE of S. pseudoporcinus degraded both human IgG1 as well as porcine IgG thereby being the only identified IgdE family protease with multiple substrates. The substrate specificities, in regard of host species IgG, of these novel members of the IgdE protease family correlate well with the known host tropism of the respective Streptococcus species. The IgG subclass specificities of these proteases might implicate special importance of these specific IgG subtypes in immune defense against these Streptococcus species during certain stages of infection which the bacteria might counteract through expression of IgG subtype specific proteases.All putative IgdE family proteases tested in this study showed enzymatic activity and substrate specificity for IgG of specific hosts. IgdE of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus are designated IgdEagalactiae, IgdEporcinus, IgdEpseudoporcinus and IgdEequi in this study.The IgdE proteases of Streptococcus genomes were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/ on Aug-21-2015) and from PATRIC (ftp://ftp.patricbrc.org/patric2/ on Aug-25-2015). As a reference sequence for an IgdE protease the RefSeq sequence WP_014636499.1 of S. suis was used. The N-terminal signal peptide and the C-terminal region only present in sequences from S. suis were removed, leaving amino acids 38–520, hereafter called IgdE_domain.Coding sequences of all available S. suis. In order to distinguish these sequences, which are not members of the IgdE protease family, all sequences lacking the AxC motif were also removed.The IgdE_domain was used as query sequence in blastp searches  against the NCBI sequences as well as the PATRIC sequences. Sequences not containing the catalytic cysteine were removed from further consideration. Many of the sequences found are annotated as S-layer proteins or as containing an S-layer homology domain W. These are often present in two or more copies in the same genome, and have an SxC or GxC motif in the catalytic site instead of the AxC motif found in aa 300–302 of the original IgdE sequence of The obtained hits were in turn used as query sequences against the same databases, using the same parameters. From the list of matched sequences those that in the second round had a match overlapping with the region matched in the first round, when the IgdE_domain was used as query, were chosen. Sequences matching to the Transglutcore model with an E-value of at most 1e-6 or sequences not containing the catalytic AxC motif were excluded. The remaining sequences were trimmed at both ends to contain only the parts matching the IgdE_domain sequence. In cases where this resulted in identical sequences only one copy was kept.http://www.ebi.ac.uk/Tools/msa/clustalo/) [http://itol.embl.de/) [Clustal Omega version 1.2.1   was usedustalo/) . A Jonesustalo/) . To assembl.de/) .Escherichia coli strains were cultured in Lysogeny Broth (LB) or Lysogeny Agar (LA) under aerobic conditions at 30°C or 37°C. When appropriate, 50 μg/ml kanamycin or 25 μg/ml chloramphenicol was added.S. porcinus strain DSM20725 , S. pseudoporcinus strain LQ940-04T (ATCC), S. agalactiae strain CCUG420  and S. equi subsp. zooepidemicus strain 203  as templates using primer pairs designated in E. coli BL21 (DE3) pLysS for recombinant expression of the proteins.Genes of the IgdE homologues lacking the signal peptide were amplified from chromosomal DNA of E. coli BL21(DE3) pLysS isolates carrying pET_ZZ_1a igdEporcinus, igdEpseudoporcinus, igdEagalactiae or igdEequi were grown to OD600 0.5 at 30°C. Protein expression was induced with 0.5 mM IPTG for 5h at 30°C. Cells were lysed for crude soluble extracts by BugBuster HT Protein Extraction Reagent (Novagen) according to manufacture protocol or lysed by sonication in 20 mM sodium phosphate, 0.5M NaCl, 40 mM imidazole, pH 7.4 prior to further purification. The His-ZZ-tagged proteins were purified on HisTrap FF  using standard protocols. The tag was removed by enzymatic cleavage by Tev-protease for 20h at 4°C followed by a second round of purification on HisTrap FF . The flow through, containing untagged recombinant protein, was collected and buffer exchanged against PBS. Protein concentrations were determined by Nanodrop A280 measurements at appropriate dilutions. In case of IgdEequi no great overexpression was achieved and no purification attempt was conducted.E. coli expressing the igdE constructs were incubated with 0.5 mg/ml porcine, human, bovine, horse, goat, and mouse IgG , 0.25 mg/ml human IgG1 kappa, IgG2 kappa, IgG3 kappa, IgG4 kappa, IgA and IgM , 0.09 mg/ml purified recombinant horse IgG1, IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7 expressed in Chinese hamster ovary cells or FreeStyle 293-F cells  and 1% ™ Orange Protein Gel Stain (Invitrogen) or blotted to Hybond-P PVDF membrane  for Western Blot analyses. Membranes were blocked with 5% dry milk powder in 0.1% PBS-Tween, followed by incubation with horse-radish peroxidase conjugated primary antibodies or unconjugated primary antibody (according to Samples for SDS-PAGE were prepared with reducing sample buffer and heated to 95°C for 5 min. 12% SDS-PAGE was either stained with Coomassie blue (Sigma), Coomassie FluorIgG degradation reactions were separated by SDS-PAGE as previously explained and transferred to PVDF blotting membrane  by semi-dry blotting with blotting buffer consisting of 50 mM Sodium borate and 20% Methanol. The membrane was stained with Ponceau S 0.5% (Sigma) 1% acetic acid in MQ water and destained with MQ water. The membrane was quickly dried, where after the degradation product was neatly cut out. N-terminal Edman sequencing of the degradation product was performed by Proteome Factory . BLAST homology searches were used to identify the position of the obtained sequence in the IgG molecule.S. suis 05ZYH33, thereby excluding the N-terminal signal peptide and the C-terminal region only present in sequences from S. suis. Only sequences that, when used as query in a second round of homology searches, had an overlap with the region matched in the first round and carried the conserved catalytic cysteine residue were kept for further analysis  and analyzed by reducing SDS-PAGE. The findings showed that rIgdEagalactiae (pseudoporcinus (Specificity of the IgdE proteases from alactiae  and rIgdporcinus  were speagalactiae (pseudoporcinus (agalactiae and rIgdEpseudoporcinus were strictly IgG1 specific, and no degradation of human IgG2, IgG3 and IgG4 was observed. The equine IgG subtype specificity of rIgdEequi was tested by incubation of protease preparations with purified recombinant equine IgG1, IgG2, IgG3, IgG4, IgG5, IgG6 and IgG7 (equi and of all equine IgG subtypes only recombinant equine IgG7 was cleaved.Given the shown specificity of IgdE proteases for IgG of specific hosts, we were interested in IgG subtype specificity of these proteases. Recombinant IgdEalactiae  and rIgdporcinus  were theand IgG7 . Strikinagalactiae, IgdEporcinus, IgdEpseudoporcinus, and IgdEequi were subjected to N-terminal Edman sequencing. All obtained sequences corresponded to the hinge region of the respective IgG molecules (agalactiae and IgdEpseudoporcinus cleave both human IgG1, but cleavage sites are not identical, being located two residues apart from each other. IgdEagalactiae cleaves the human IgG1 heavy chain two residues N-terminal of the putative homodimer disulfide bond cysteine residues; while all other cleavage sites of IgdE family proteases are located directly adjacent to the putative homodimer disulfide bond cysteine residues in IgG hinge regions. The cleavage sites identified in porcine IgG generated through cleavage with both IgdEporcinus and IgdEpseudoporcinus were identical and found in porcine IgG2, IgG4 and IgG6 (equi corresponded to the hinge region of equine IgG7. The amino acid sequences analogous to those adjacent to the cleavage site in equine IgG7 are different in all the other horse IgG subtypes, providing a rationale for the observed subtype specificity.To determine the exact cleavage site of the IgdE proteases within the respective substrate IgG molecules, the 32 kDa cleavage products generated by IgdEolecules . IgdEagaand IgG6 . SimilarS. suis [Streptococcus. Locations of the genes of these putative proteases were conserved within, and to certain degree also between, different Streptococcus species suggesting that igdE genes are part of the core genome and not part of mobile elements (S. suis and two of S. agalactiae had, however, different neighboring genes than the other 22 sequences of S. suis and 16 of S. agalactiae, respectively. Putative IgdE family protease sequences were however only found in 55% of S. agalactiae, 46% of S. equi and 81% of S. suis genomes (igdE gene in some strains of these species or due to pore, incomplete or missanotated coding sequences of these genomes.Based on the identification of the founding member of a novel cysteine protease family, IgdE of S. suis , we idenelements . One seq genomes . This copseudoporcinus, showed only specificity towards IgG of one host species. The substrate specificity of these IgdE family proteases correlates well with the known host tropism of the respective Streptococcus species. However, IgdEagalactiae does not cleave bovine IgG, despite S. agalactiae being the cause of mastitis in cattle besides being an important human pathogen. The observed substrate preference for human IgG1 might reflect that most human invasive S. agalactiae isolates represent distinct subtypes from bovine isolates, as it has been suggested in a temporally and geographically matched isolate characterization study [pseudoporcinus showed double specificity for both human IgG1 and porcine IgG (S. pseudoporcinus is closely related to the pig pathogen S. porcinus, the specificity for porcine IgG is not that surprising. It is, however, astonishing that IgdEpseudoporcinus, being a human pathogen, has evolved the ability to cleave human IgG1, despite the close relationship to IgdEporcinus that does not possess this ability. Thus, it seems advantageous for S. pseudoporcinus to have a human IgG1 degrading protease, underlining the importance of IgG1 in immune responses towards bacterial pathogens. Mutational studies of IgdEpseudoporcinus and IgdEporcinus might be able to dissect the residues that mediate this substrate specificity.All investigated IgdE family proteases except IgdEon study . IgdEpsecine IgG . Since SStreptococcus species. Co-evolution of streptococcal opportunistic pathogens and their host could be reflected on a molecular level by co-evolution of IgdE family proteases and their substrate IgG heavy chain molecules. This is highly reminiscent of the co-evolution described between IgA and bacterial proteins targeting IgA, such as IgA-binding proteins and IgA-specific proteases, where reiterative episodes of natural selection are predicted to have shaped the interactions between the IgA and the bacterial proteins, reflecting an ‘arms race’ [We suggest that substrate specificity of IgdE family proteases contribute to the host tropism of some ms race’ , 42.S. agalactiae and S. pseudoporcinus towards human IgG1 and the IgdE family protease of S. equi subsp. zooepidemicus towards equine IgG7 is striking and surprising (S. agalactiae is a common cause of invasive neonatal infections in humans [S. equi in the horse is likely to significantly comprise IgG-mediated protection, given that IgG7 is one of the predominant subclasses in equine serum [agalactiae in immune evasion in the neonatal and adult host are currently ongoing in our group.The subtype specificity of the IgdE family proteases of rprising . The evon humans  and human humans . Therebyne serum . Due to ne serum , 46 it mne serum , 48 it mequi is the third IgG degrading protease of S. equi subsp. zooepidemicus described, beside IdeZ1 [equi is highly specific for equine IgG7, while both IdeZ1 and IdeZ2 have broader specificity, cleaving IgG of several host species. The abundance of genes encoding IgG degrading proteases in S. equi subsp. zooepidemicus implicates a special importance of an IgG cleaving phenotype of this species. These Ig-degrading proteases might be regulated by different gene regulation systems and thereby expressed during different stages of infection or colonization.IgdEde IdeZ1  and IdeZde IdeZ1 . IgdEequAll IgdE family proteases recognize IgG as substrates although the amino acid sequences at the cleavage sites in the respective hinge regions are quite diverse . Therefoagalactiae has the same cleavage site in human IgG1 as papain [agalactiae is highly specific for human IgG1 and has only one distinct cleavage site within the heavy chain. Interestingly, this cleavage site is not shared by IgdEpseudoporcinus that instead cleaves the heavy chain two residues closer to the C-terminus, just N-terminal of the putative homodimer disulfide bond cysteine residue. Differential cleavage sites implicate that targeting IgG1 has evolved independently in these two proteases, highlighting the importance for the bacteria to counteract IgG1. The cleavage sites of IgdEporcinus, IgdEpseudoporcinus and IgdEequi have a CPxCP motif just C-terminal of the cleavage site in common. This motif can, however, also be found in many IgG heavy chain molecules that are not substrates of any investigated IgdE family protease, supporting the idea that substrate specificity is determined by features others than cleavage site sequences.IgdEs papain . HoweverS. suis in pigs [S. equi in horses [Streptococcus species. This would especially be desirable in the cases of S. agalactiae and S. pseudoporcinus in humans and S. suis and S. porcinus in pigs. Antibodies elicited by such vaccines might both neutralize the proteolytic function of these potential immune evasion factors and potentially mediate antibody dependent cell cytotoxicity against the streptococcal pathogen.Secreted Ig degrading proteases have been shown to be protective antigens in experimental vaccine and infection studies with  in pigs  and S. en horses . The desi.e. similar to what has been proven for the IgG degrading enzyme of S. pyogenes IdeS [Lastly IgdE family proteases with pronounced species and subtype specificity might also be of biotechnological or therapeutical use, nes IdeS –61.


Gene suppression approaches have emerged over the last 20 years as a novel therapeutic approach for the treatment of neurodegenerative diseases. These include RNA interference and anti-sense oligonucleotides, both of which act at the post-transcriptional level, and genome-editing techniques, which aim to repair the responsible mutant gene. All serve to inhibit the expression of disease-causing proteins, leading to the potential prevention or even reversal of the disease phenotype. In this review we summarise the main developments in gene suppression strategies, using examples from Huntington’s disease and other inherited causes of neurodegeneration, and explore how these might illuminate a path to tackle other proteinopathy-associated dementias in the future. Gene suppression approaches refer to targeted molecular genetic therapies that serve to lower the expression of specific genes. The term “gene silencing” has also been used to describe these methods; however, this term should be considered a misnomer as complete gene inactivation does not occur, and might not be desirable. Such techniques have made enormous progress over the last 20 years, and show great promise for the treatment of inherited neurodegenerative diseases arising from a known genetic mutation.Neurodegenerative diseases that result from the toxic gain-of-function of a mutant protein or non-coding RNA that are without a significant loss-of-function are ideal candidates for gene suppression approaches. There are many strategies to lower the amount of toxic disease proteins that result from single gene mutations, including both post-transcriptional inhibition such as RNA interference (RNAi), anti-sense oligonucleotides (ASOs) and catalytic nucleic acids, and genome editing techniques such as zinc finger proteins (ZFPs) and CRISPR/Cas9. The great benefit of interventions at the gene suppression level, as opposed to interventions aimed at the toxic protein itself, is that the plethora of potentially negative downstream cellular pathogenic effects that may arise from the abnormal functioning of a single protein are all reduced as a consequence of treatment.In this review we will use examples from Huntington’s disease (HD), in respect of which a worldwide collaborative research effort has led to the swift progression of gene suppression approaches, from in-vitro and in-vivo development through to a clinical trial of a Huntingtin-lowering therapy that is currently in progress . HuntingPost-transcriptional gene suppression refers to approaches that trigger the cleavage, enhanced degradation or translational suppression of the target mRNA. They include RNAi, ASOs and catalytic nucleic acids (ribozymes and DNA enzymes). Ultimately all of these mechanisms serve to modulate translation efficiency, thus lowering the amount of protein expressed  see Fig.Fig. 1MeEndogenous RNAi is an evolutionarily conserved process within cells, whereby native, non-coding, double-stranded RNA sequences elicit post-transcriptional gene suppression, typically by causing the destruction of specific mRNA molecules, thereby regulating mRNA expression  see Fig. Two typWithin the nucleus, miRNAs are transcribed by RNA polymerase II (pol II) and pol III promoters to form stem–loop structures known as primary miRNAs (pri-miRNAs).pri-miRNAs are cleaved by the endoribonuclease enzyme Drosha to form a precursor miRNA (pre-miRNA) hairpin-like structure.pre-miRNAs are exported to the cytoplasm by Exportin-5.In the cytoplasm, the pre-miRNA is further processed by another endoribonuclease enzyme Dicer to create a mature miRNA complex.The antisense or “guide strand” of the (endogenous miRNA or exogenous siRNA) complex is loaded into the RNA-induced silencing complex (RISC), which is located in the cytoplasm, and the sense or “passenger” strand is degraded.The guide strand targets the RISC to its complementary mRNA. Perfect base pair matching in the case of siRNA leads to Argonaut-2 (Ago-2)-mediated cleavage of the target and complete translational inhibition. Imperfect complementarity in the case of miRNA leads to translational repression.This well-described mechanism is comprised of a number of steps :Within tsiRNAs differ from miRNAs in that miRNAs typically have incomplete base pairing to a target and inhibit the translation of many different mRNAs with similar sequences. siRNAs, in contrast, typically base-pair perfectly and induce mRNA cleavage only in a single, specific target.Intensive research has been carried out to generate effectors that manipulate the RNAi pathway to suppress the expression of a particular gene of interest. These have taken the form of siRNAs that include short hairpin RNAs (shRNAs), and artificial miRNAs, all of which aim to degrade mature, spliced mRNA in the cytosol.siRNAs are comprised of 20–25 base pairs, are processed by Dicer and ultimately lead to Ago-2-mediated cleavage of the target mRNA, to interfere with the expression of specific genes with complementary nucleotide sequences. They can be delivered into cells in liposome formulations , nanoparResearchers have therefore used RNA expressed from viral gene therapy vectors to achieve stable expression in brain regions of interest. The RNA from the viral genome is processed by Dicer to RNAi . shRNAs delivered into cells using viral vectors have been shown to cause extremely high levels of translational suppression , 17, altHTT mRNA levels and in the formation of HTT inclusions (which are a pathognomic feature of HD) [RNAi has been investigated widely in HD, and many potential scientific hurdles have been overcome. It is worth bearing in mind that the underlying principles of RNAi for the potential treatment of HD could also be applied to other causes of dementia. Initially, RNAi to lower the amount of Huntingtin protein (HTT) was shown in vitro to improve cell survival in cellular models of HD . In 2005e of HD) .HTT mRNA levels and inclusions, and reduced brain atrophy. Sustained effects including delayed onset of motor symptoms and improved survival were seen [Non-viral delivery systems have also been tested, using nanoparticles to deliver lipid formulated siRNAs via the intracerebral ventricular route in the R6/2 mouse model of HD. This lowered mutant ere seen . Deliverere seen .These studies all administered treatment in pre-symptomatic HD mice, but treatment in symptomatic HD mice has also successfully reduced HTT protein levels, reduced inclusions and improved histopathology . The impHTT mRNA, whilst leaving the mRNA of the wild-type allele unaffected, has proven to be very difficult in cells derived from HD patients . An alternative approach is to non-specifically lower both mutant and wild-type HTT to a degree which improves pathology whilst maintaining safety and tolerability in the face of wild-type HTT loss. A 40–60% reduction in both endogenous and mutant HTT extends the lifespan and prevents motor deficits, without causing toxicity in both fragment [The earlier siRNA studies in HD animal models did not affect the endogenous levels of wild-type HTT protein—only the disease-causing transgene was reduced. In addition, the models were also all expressing a small N-terminal fragment of the mutant protein  and notfragment  and fullfragment  mouse mofragment , 28.RNAi testing in HD non-human primates (adult rhesus monkeys) has also demonstrated the safety of partially reducing endogenous wild-type HTT following delivery of AAV vectors expressing artificial miRNAs and shRNAs. A 45% reduction in wild-type HTT did not induce any pathology or symptomatology  and sustTranslation of this finding to other genetic forms of neurodegenerative disease is not assured. The consequences of lowering levels of endogenous wild-type protein needs to be investigated carefully when non-allele-specific targeting is considered for conditions in which only the mutant protein is pathogenic. However, for neurogenetic disorders in which misfolding of the mutant protein corrupts native/wild-type protein, recruiting them to spread pathology, non-allele-specific targeting may be required for pharmacologic effect.ASOs are single-stranded DNA molecules consisting of 16–22 bases, which are complementary to the target pre-mRNA. ASOs have a more upstream site of action than RNAi effectors that act on mature, spliced mRNA species. Upon binding, ASOs recruit RNaseH1 (an endogenous enzyme that recognises RNA/DNA duplexes), which degrades the target pre-mRNA. Alternatively, ASOs may obstruct the interactors of the pre-mRNA leading to translational inhibition or modulation of splicing .Sulphur substitution for oxygen in the phosphate of the phosphate inter-nucleotide linkages or backbone of the ASO molecule, thus increasing resistance to nucleases and improving protein binding.Multiple potential alterations to the sugar moiety of the nucleotide which increase binding affinity to the target mRNA, increase resistance to nucleases and decrease toxicity to varying degrees. Importantly, most sugar modifications lead to failure to recruit the RNaseH1 enzyme. Therefore, when RNaseH1 activity is desired, nucleotides at the ends of the ASO can be modified to improve binding affinity and resistance to nucleases, while leaving a stretch of unmodified nucleotides in the middle that can engage RNaseH1.Over the last 20 years, modifications of ASOs have improved their suitability for therapeutic application in neurodegenerative diseases. These modifications include the following:Engineering ASOs with specific desired characteristics can be achieved by incorporating one or more of these chemical modifications into their design. A diverse range of ASOs is therefore possible, with different potencies, pharmacokinetics and pharmacodynamics, as well as different targets.ASOs are currently being tested in early-stage clinical trials in patients with HD and ALS , and havThis ASO has been tested in pre-clinical studies and is predicted to lower total HTT levels in a dose-dependent manner. It has been shown to reverse behavioural phenotypes in BACHD and YAC128 mouse models of HD  and to aRibozymes are naturally occurring RNA molecules comprising two flanking sequences that enable specific binding to target mRNA, and an effector catalytic core that cleaves the mRNA substrate. The mRNA fragments are then degraded by the cell .Hammerhead ribozymes (a specific class of ribozymes 30–40 nucleotides in length), delivered using an AAV vector, have been shown to result in 60% reduction of mHTT mRNA when delivered to HEK293 cells expressing mHTT exon 1 . Direct HTT mRNA constructs by 85% in a HEK293 cell model of HD [DNAzymes are synthetic single-stranded catalytic nucleic acids that also have substrate binding arms to target complementary mRNA and a cation-dependent catalytic core. They have been shown to knock-down mutant el of HD , but furGene suppression can also be achieved at the transcriptional level, using effectors that bind to specific DNA sequences. One such approach uses ZFPs which contain a zinc finger domain that can be manipulated synthetically to bind a DNA sequence of interest, fused to a functional protein domain. Zinc finger nucleases (ZFNs) cleave DNA at a particular target site, and zinc finger transcriptional repressors (ZFTRs) are transcription factor DNA-recognition motifs fused to a transcriptional repressor domain. Delivery of such agents would be through the use of viral vectors.HTT expression (without affecting wild-type HTT expression) and led to improvements in neuropathology and motor deficits [Potential advantages of this approach are the avoidance of on-target and off-target toxicities of RNA therapeutics and the elimination of all disease-causing mRNA splice variants, not just those that possess the target mRNA sequence . The prodeficits . The phaOther engineered nucleases include mega-nucleases, transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 systems . CRISPR/In addition to repressing gene transcription, the ability of these methods to target nuclease-induced scission and repair offers the exciting possibility of actually correcting genetic mutations (e.g. through the excision of the expanded CAG repeat in HD)—leading to true genome editing. There is thus the exciting potential to treat not just HD but any genetic cause of neurodegenerative dementia using these techniques.Because unmodified siRNAs do not readily cross the blood–brain barrier (BBB), or the plasma membrane of cells, researchers have used viral vectors to enable stable expression of shRNAs and artificial miRNAs within desirable brain regions. The most commonly used vectors are recombinant adeno-associated virus (AAV) and lentivirus (LV), both of which are non-pathogenic, trigger minimal immune response and cannot replicate in the host. AAVs remain as nuclear episomes and do not integrate into the host genome, but result in stable gene expression that is stronger than that of LVs. siRNA sequences constitutively delivered through miRNA expression systems expressed from a viral vector should minimise the requirement for repetitive administration. There are multiple capsid serotypes of AAV, allowing for specific cell-directed therapy , 48. UnfIn the case of HD the primary site of pathology is the striatum, thus RNAi treatments in animal models have generally been delivered by bilateral direct injection or infusion into the striatum, or the adjacent lateral ventricle (resulting in widespread delivery throughout the brain) . HoweverAAV serotype 9 (AAV9) crosses the BBB and transduces neurons and glia in the brain, as well as a variety of peripheral tissues, following a systemic, intravascular injection . A globaASOs also do not cross the BBB, but can be delivered into the cerebrospinal fluid (CSF), either through intrathecal or intra-ventricular injection, as they are soluble in artificial CSF. Once injected, ASOs are distributed to the brain parenchyma and taken up into both neuronal and glial cells. Unlike RNAi, ASOs are not regenerated within the cell and have a half-life that varies depending on the precise sequence and chemical structure. Repeated dosing of the drugs is therefore required to sustain treatment effect. ASOs display dose dependency and reversibility, and therefore the degree of protein lowering can be controlled to achieve safe yet effective levels.HTT mRNA throughout the brain and spinal cord. In NHP brains, ASOs delivered intrathecally were found in the spinal cord, in the cortex and to a lesser extent in the deeper brain structures [In the case of HD, ASOs delivered intra-ventricularly to rodents led to > 75% reduction of target ructures . Althougructures , the higructures .In the future, medical devices (e.g. implantable pumps) may be able to replace the need for repeated lumbar punctures to achieve regular intrathecal dosing of ASOs. This would reduce the burden of administration for both patients and medical professionals. Recently, the development of peptide-conjugated ASOs delivered through intravenous injection (IVI) has been shown to have broad peripheral and CNS distribution in a mouse model of severe SMA. A profound improvement in mean survival from 12 to 456 days was also seen [Off-target effects are unintended consequences following the administration of gene suppression therapies. They include interference with non-target mRNA transcripts and activation of the immune system in response to drug delivery. In the case of siRNAs and ASOs, there is potential to bind mRNA sequences that share sequence homology to the desired target, thus causing down-regulation of unrelated proteins . BecauseWhen a mammalian cell encounters a double-stranded RNA such as an siRNA, the cell may mistake it as a viral by-product and mount an immune response. siRNAs have been shown to up-regulate the expression of interferon (IFN)-stimulated genes through the activation of protein kinase R (PKR) . siRNAs Delivery of shRNA effectors has been shown to overload exportin-5, thus preventing the maturation of endogenous miRNA and effectively shutting down the cellular miRNA pathway , 63. SatHTT gene [In theory, certain gene suppression strategies may not be as effective in cases where there is evidence of abnormal RNA processing or toxicity of the RNA itself. In the case of HD, aberrant mRNA splicing has been shown to generate the production of a pathogenic HTT exon 1 fragment from the CAG-expanded HTT gene , which mHTT gene ; preventThe extent to which these alternative pathways of toxicity exist and contribute to the disease phenotype in other forms of dementia could impact on the success of various gene suppression approaches for treatment.Interpretation of drug effect in early-stage, proof-of-concept clinical trials can be facilitated by measuring of the ability of the therapy to engage with its target. Methods for assessing target engagement include imaging modalities and measurement of target RNA or protein in an easily accessible biofluid or tissue sample. To assess target engagement directly, brain-imaging techniques that allow for quantitation of target protein in vivo are usually required. Recently, novel radiotracers for PET imaging have been developed to detect brain tau deposition , which mQuantification of target RNA or protein in a biofluid or tissue sample is another useful method for evaluating target engagement. For neurodegenerative conditions, where the target tissue is in the brain, collection of target tissue samples is impractical; however, collection of CSF samples is straightforward, and changes in neuronal proteins measured in CSF can be expected to reflect changes in brain tissue. Assays to detect CSF β-amyloid and tau protein are already used in the clinical evaluation of AD, and are incorporated into most clinical trials of AD. Likewise, assays to detect alpha-synuclein are sometimes used to assess target engagement in PD trials . In the It is worth noting that levels of target proteins in the CSF are an indirect reflection of absolute protein levels at actual sites of pathology in the brain parenchyma. However, using research in animals, it is possible to develop models of the relationship between drug pharmacokinetics (PK) and pharmacodynamics (PD) that predict target engagement in the brain regions of interest. Such models relate the reduction of expression of a gene in target tissue with parameters that can be measured in humans, such as the protein product of that gene’s expression in CSF. Studies in non-human primates, where brain anatomy is similar to humans, can provide a guide for interpretation of target engagement markers in biofluids. In fact, animal data were shown to translate well to humans during clinical development of nusinersen, an ASO for the treatment of patients with spinal muscular atrophy (SMA). In this programme, autopsy material was available from a few patients who received nusinersen during clinical development, and levels of ASO and target engagement markers in tissue were found to be well aligned with predictions from the pre-clinical PK–PD models, supporting the premise that thorough characterisation of drug pharmacology in relevant animal species is important for planning and interpretation of data in humans .As with any disease-modifying treatment for neurodegenerative disease, timing of intervention is crucial. In the case of HD, predictive genetic testing is available to those whose family history puts them at risk, and mutation carriers could potentially start gene suppression treatment before any clinical symptoms appeared. The goal would then be to initiate treatment just before the onset of pathology and prevent the disease from ever being manifest. Objective biomarkers are required to pinpoint pathology onset and when to start treatment. Large, prospective, longitudinal natural history and deep phenotyping studies have been undertaken in HD  and suggASOs can be developed to target mRNA from both the mutant allele and the wild-type allele equally or to favour the mutant allele over the wild-type allele. The former, non-allele-specific, approach has the advantages of addressing the entire HD population and maintaining flexibility for ASO design across the entire gene, increasing the likelihood of identifying a specific, potent and well-tolerated ASO. The potential disadvantage for non-allele-specific ASOs lies in reduction of the wild-type allele. An allele-specific approach can be achieved by targeting either the CAG expansion or a sequence containing a specific single nucleotide polymorphism (SNP) found only on the mutant allele. Allele-specific approaches have the advantage of preferential reduction of the mutant protein. Potential disadvantages include blunted potency and tolerability due to severe limitations on sequence space, undesirable reduction of other proteins containing CAG repeats not linked to disease (with CAG-targeting ASOs) and addressing only a subset of the disease population (with SNP-targeting ASOs).HTT transgene [In the case of HD, embryonic knockout of Htt is lethal  and its ransgene . IntuitiASOs complementary to the expanded CAG have been produced which have up to 6-fold selectivity for mHTT over HTT –84; seleSNPs have been identified that reside only on the mutant HTT allele , 87, witHTT mRNA; this involves screening potential trial subjects for the presence of these SNPs before allocation to one or other of the compounds [As mentioned previously, ASOs could also be used in a SNP-targeting approach. An advantage of ASOs over siRNAs is that they can target SNPs present in intronic sequences, due to their capacity to bind pre-mRNA in the nucleus rather than only mature mRNA transcripts in the cytoplasm. ASOs targeting mutant-allele linked SNPs have been designed that lead to 50-fold selectivity of the mHTT allele over the normal allele –92. Threompounds .Rx) in ALS associated with a SOD1 mutation [The first human clinical trial of intrathecal delivery of ASOs for the treatment of a neurodegenerative condition was carried out in patients with amyotrophic lateral sclerosis (ALS) . Inheritmutation .As mentioned previously, another phase 1/2a clinical trial is also currently being undertaken by Ionis to test the safety and tolerability of a MOE-modified ASO targeting human HTT delivered by lumbar intrathecal bolus administration to early-stage HD patients. The trial includes neuroimaging assessments and exploratory endpoints to assess the effect on cognitive, motor and neuropsychiatric symptoms , is alsoSMN1, leading to a lack of survival motor neuron (SMN) protein and the death of motor neurons, which are particularly reliant on it. Nusinersen in fact alters the splicing of the mRNA of another gene, SMN2, that usually encodes a non-functional SMN protein, and in doing so increases the amount of functional SMN available in the cell. The phase 3 ENDEAR trial of this drug showed such promising efficacy in interim analysis that urgent work has already begun on an extended access programme to widen the availability of the drug to those not in a clinical trial [Patients with SMA type 1 (infantile onset) have been treated with lumbar intrathecal bolus injections of an MOE-modified ASO (Nusinersen), and have shown dramatic improvement . This isal trial . The drual trial  for all MAPT gene, which encodes tau protein, are causative for another neurodegenerative disease—frontotemporal lobar degeneration (FTLD). This study demonstrated improved survival and histopathology in a mouse model of tauopathy, and also showed tau protein reduction in CNS tissues and CSF in non-human primates following intrathecal bolus administration of ASOs targeting MAPT mRNA. This opens up the exciting potential for gene suppression treatments of tau as a therapeutic avenue for AD and other tauopathies such as progressive supranuclear palsy (PSP), and clinical trials of tau-lowering therapies are imminent.Recently DeVos et al.  have shoC9orf72 is also under investigation for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In the future, an ASO that suppresses alpha-synuclein gene (SNCA) expression to reduce alpha-synuclein protein might have utility in limiting the progression of PD [LRRK2)  has been shown to reduce the spread of alpha-synuclein pathology in the mouse brain [The possibility of treatment with ASOs that reduce pathogenic splice variants of on of PD . In addise brain . There mATXN2). Scoles et al. [ATXN2 mRNA  and tested their most promising candidate in two mouse models. Following intracerebroventricular injection, this ASO localised to Purkinje cells and reduced cerebellar ATXN2 expression and levels of cerebellar ATXN2. This was also associated with significantly improved motor function [Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant, adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-2 gene (s et al.  recentlyfunction .An ASO lowering ATXN2 has also been shown to increase the lifespan and improve motor symptoms in a mouse model of TDP-43 proteinopathy . Nearly Gene suppression technologies have made enormous progress in the last 20 years. Clinical trials of ASOs are already underway and trials of RNAi and genome editing using ZFNs are not far behind. The relative advantages of ASOs and RNAi are summarised in Table Challenges with drug delivery of gene suppression agents have been researched extensively and largely overcome. In the case of ASOs, lumbar intrathecal bolus delivery is proving to be a viable option, and in the case of RNAi, modification of viral vectors has allowed for tolerability and efficacy. Administration of viral vectors either through directed brain injection/infusion to target specific sites, or through peripheral vascular injection leading to global CNS effects, has been tolerated in animal models.The next few years will see further expansion in the clinical evaluation of gene suppression therapies for the treatment of dementia, both in terms of the number of neurodegenerative diseases targeted and the number of patients treated. Thus there is real hope that, in the face of aging populations and the ever-increasing incidence of dementia, we will in the near future be able to offer effective treatments to sufferers. Moreover, with advances in biomarker technology that can detect the increasingly understood evolution of pathology in asymptomatic individuals, the prospect of early disease interception to prevent the symptomatic expression of these neurodegenerative disorders is a tantalising possibility.


We show that anti-phase boundaries, created from current pulses (heat shocks), invert the polarization of selective domains resulting in reorganization of certain 71o domain boundaries into 109o boundaries. These boundaries subsequently interact and evolve with the partial dislocations, which migrate from domain to domain with the carrier-wind force . This work suggests that current pulses and carrier-wind force could be external stimuli for domain engineering in ferroelectrics with significant current leakage.Germanium telluride (GeTe) is both polar and metallic, an unusual combination of properties in any material system. The large concentration of free-carriers in GeTe precludes the coupling of external electric field with internal polarization, rendering it ineffective for conventional ferroelectric applications and polarization switching. Here we investigate alternate ways of coupling the polar domains in GeTe to external electrical stimuli through optical second harmonic generation polarimetry and  Polar metals such as GeTe could store information using electric domains but the high conductivity screens electric fields, preventing the use of usual domain control techniques. Here, the authors demonstrate that polar domains in GeTe can be manipulated using electrically generated heat shocks. R3m), characterized by the presence of bonding hierarchy (short and long Ge–Te bonds), which results in a net polarization along the [111] direction, and thus is a polar material3619–1021 cm−3) which degenerately dope it with holes, giving rise to p-type metallicity48xMnx1−Te (ref. 67121315Germanium telluride (GeTe), a IV–VI compound, exhibits several unique combinations of properties in the crystalline phase1234long the 1 directiin situ transmission electron microscope (TEM) characterization of the GeTe devices272829Here, we developed a variant of the optical second harmonic generation (SHG) polarimetry18192122232425x membrane based device platform compatible with both TEM and SHG polarimetry experiments−2)2O3 (insulating oxide), followed by 100 nm of Ag. The {111} termination ensured that the incident beam (both laser and electron) was always shone along the 〈111〉 direction , can give rise to eight different polar domains (four ferroelastic domains), which in general coexist in as-grown samplesα, β, γ and δ, each of them identifiable with the direction of the polarization vector. The Greek letters α, β, γ and δ refer to the ferroelastic domains and + or – refers to their polarization direction. We label domains as positive, if their polarization vectors are pointing out of the substrate, and as negative if they point into the substrate. α are a pair of inversion domains with the polarization vector ) parallel to the zone axis . β are the inversion pair of domains whose polarization directions Pβ  are perpendicular to the set of terminating {111} facets not parallel to the substrate. Domains γ and δ have polarization directions Pγ, Pδ in the other two 〈111〉 directions respectively, not perpendicular to any of the nanowire facet , β, γ, δ, and obtaining information from individual domains is challenging.The stable structure of GeTe can be understood as a rock salt structure rhombohedrally distorted along one of its body diagonals, [111], with a net polarization along that direction4re facet . Within lar axis . Hence io ({001} habit planes) or 109o (γ(+/−) and δ(−/+) domains as evidenced by the spot splitting along Domain walls in GeTe can be classified as 71ectively . Conversirection . It mustR3mPα(+) direction  was collected in the reflection mode at three particular polarizations, that is, 0o (x0), 60o (x60) and 120o (x120) with respect to the long-axis of the nanowire, corresponding to 〈110〉 directions in every domain . These intensities were fit to the expression:Developing SHG polarimetry on GeTe is challenging owing to the lack of standard samples (single-domain) and the non-linear material constants   relating SHG response to the incident field (Vi)d(=Vi)–dd . For thent field . The ratsequence . On 15 dectively .in situ TEM by recording structural changes in GeTe devices (I=0.75 mA) leaving behind AlxOy shell , where the domain boundary evolution was tracked while simultaneously applying a train of voltage pulses  of increasing amplitude until amorphization (γ(+/−) and δ(−/+). The {001} domain boundary contrast does not explicitly appear in the dark-field images, owing to them not being in the zone defined by 〈111〉 zone axis. However, the line defects along the nanowire, pre-induced by focus ion beam (FIB), act as tracers to the {001} domain wall dynamics (γ(+/−) and δ(+/−) domains separated by o boundaries between γ and δ domains reorganize into perpendicular To verify whether heat shocks through electrical pulses can couple with the polar domains, we performed hization . The SAEhization  showed sdynamics . While ndynamics , which sdynamics . SAED peundaries . Hence t6 V ref. . The par6 V ref.  could no6 V ref. . When th >2.7 V, . When thin situ TEM, we performed SHG polarimetry on nanowire devices at different positions in the virgin state and also after applying electrical pulses until amorphization  (Cθ) of the total intensity , and the rest of the domains contribute to coefficients of all the three terms. (ii) Coefficient of sin2(φ−θ) or Cθ produces a periodicity of π/2 to produce four lobes in polarimetry plots for φ varying from −180 to 180o, while coefficients of cos2(φ−θ) and sin2(φ−θ) (Aθ and Bθ) produce two lobes. The change in phase of the fits at θ=60o (2(φ−60o) and cos2(φ−60o) (two lobes) between virgin and programmed states suggests significant changes in domain fractions of either the variants of β or δ. θ=120o, both fit to a two lobed function, except for a stronger signal in virgin state. This indicates that neither the variants of β nor γ change significantly upon programming, and in conjunction with the polarimetry plots for θ=60o  and cos2(φ) (responsible for two lobes) in comparison with the coefficient of sin(2φ) (responsible for four lobes) post programming.The following aspects of equation (1) enable us to qualitatively predict the changes in domain fractions from the virgin to programmed state: (i) The domain whose polarization is perpendicular to SHG polarizer, contributes only to the coefficient of sin2at θ=60o , predomior θ=60o , a signithe θ=0o  polarimeo (δ(−/+) domain) near a pre-existing {001} domain wall separating the variants of γ(+/−) and δ(−/+) domains (domain boundaries can provide nucleation sites for APBs), δ(−) completely switches its polarization by 180o to nucleate δ(+). This polarization inversion, as confirmed from SHG polarimetry, results in the reorganization of the {001} boundary into γ and δ domains  and its ferroelastic rearrangement with γ(+) (evidenced by the slight change in the domain fraction of γ(+)  in GeTe29Heat shocks from a current pulse cluster Ge vacancies in a {111} plane, and beyond a certain cluster size, the Te planes above and below collapse forming an APB, a 2D extended defecto . Further domains , consist of γ(+) , formingo, 109o, 180o) with a well-studied rhombohedral ferroelectric BiFeO3 (BFO)o boundaries mobile or immobile, and are they charged or uncharged? The 180o boundaries contain a plane of Te-Te bonds (o boundaries (formed from APBs). So, while this study provides evidence that 180o boundaries are probably immobile, and an expectation that they are charged, these hypotheses need to be confirmed with further studies.The current study can lead to exploring several intriguing questions in leaky polar materials. For instance, given the crystallographic similarities of the various types of domain walls /δ (−) interface, say) to 110o (γ(+)/δ (+) interface) type. Thus it seems possible that upon nucleation of another APB in the γ domain, domain boundary flips again to 71o type (γ(−)/δ(+) interface), and this could go on forever. However, in our nanowire geometry once the APBs are nucleated on a domain (say δ), the partial dislocation migration−2, an energy comparable to GSF in Cu  of 175 mJ m−2  of a Te terminating plane by ∼200 meV, potentially making domain switching in thin films of GeTe useful for tunnel junction applications. Furthermore, the combination of polar metallic nature makes dilute magnetic materials such as Mn doped GeTe, where magnetic RKKY interactions arise from free-carriers, multiferroicFinally, any commercial applicability for polarization switching in these polar metals arises only if there are well-defined changes in measurable properties associated with switching. While FeRAM applications may seem far-fetched, specifically, in case of GeTe, it is recently reportedin situ TEM, we showed that the formation of an APB inverts polarization of selective domains, and domain walls are altered via interactions with partial dislocations, which can be externally controlled by current pulses. Furthermore, we clearly demonstrate that heat shocks or electrical pulses can couple to internal polarizations and address them in these materials. Leakage issues in ferroelectric materials preclude their efficient functionality, and strategies using chemical doping have been sought to reduce leakage45In conclusion, we demonstrated that extended defects created from heat shocks such as APBs and partial dislocations interact with polar domains and can be used to manipulate them in polar metals such as GeTe. In particular, by developing the SHG polarimetry technique on GeTe and utilizing it in conjunction with Tm=724 °C) was placed at the center of a tube furnace. Silicon (001) substrate evaporated with Au film (15 nm) and annealed at 720 °C for 10 min, was placed on the downstream side of the furnace (∼15 cm away from the middle). The furnace was ramped up to 720 °C, with 25 sccm of Ar (carrier gas) flow rate and a pressure of 120 torr. The reaction was carried out for an hour, after which the furnace was slowly cooled down to room temperature.GeTe nanowires were synthesized using metal catalyst mediated vapour–liquid–solid process. Bulk GeTe powder /Au (30 nm) electrodes were patterned on the membrane using photolithography. Nanowires were dry transferred onto the membrane, and two terminal devices were fabricated via Pt deposition using FIB technique. The devices were subsequently annealed at 250 °C for an hour, and ∼15 nm Al2O3 film =3–6 W m−1 K−1 (ref. −1 K−1 (ref. x membrane is etched down to 50 nm to enable electron as well as incident laser beam transparency (The K−1 ref. ) was conK−1 ref. ). Followsparency , inset.x membrane and focused with the spot size of∼2 μm. Its polarization was linearly controlled by a half-wave plate and chosen to be 0°, 60° and 120° with respect to NW's long axis. Under each excitation polarization , the SHG signals were imaged by a cooled charge-coupled device and detected by a spectrometer (Acton) with a 300 groove mm−1 500 nm blaze grating with a charge-coupled device detector (Princeton instruments) with a spectral resolution of 0.1 nm. The polarization properties of SHG signal was studied by placing a polarizer in front of the detectorA femtosecond pulsed Ti: sapphire laser (Chameleon), tuned from 680 to 1,080 nm with ∼140 fs pulse width and 80 MHz repletion rate, was utilized to excite SHG from individual GeTe nanowires via a home-built microscope equipped with a × 60, 0.7 NA objective (Nikon). It was incident through 50 nm thick SiNI–V sweeps were performed using Keithley 2602 . Keithley 2700 was used as the data acquisition system (DAQ), and resistances were measured using Keithley 2602 via low bias (−2 mV to 2 mV) I–V sweeps.Electrical pulses were applied using Keitheley 3401, and voltage-controlled d.c. http://www.crystalmaker.com/) by superimposing diffraction patterns of a bi-crystal of GeTe with the polarization vectors of each crystal (domain) at 71o and 109o to each other. The zone axis was set to another 〈111〉 direction, which is different from the polarization directions of each domain.Electron diffraction patterns were simulated using the crystal-maker software .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary ReferencesxOy shell behind, proving that neither static fields nor steady currents can couple with the ferroelectric domains in GeTe. Snapshots of this movie are shown in Fig. 2 of the manuscript.Bright field TEM mode movie recorded on a GeTe nanowire device  while applying a DC voltage sweep up to device failure at a current of 0.75 mA. We observe no structural changes until eventual device failure via melting and sublimation, leaving an AlStructural changes in GeTe nanowire device captured in dark field TEM mode while applying of train of voltage pulses (200 ns) with increasing amplitude all the way upto formation of an amorphous phase, and subsequent recrystallization. We see an initial reorientation of {001} domain walls into {1-10} domain walls from 2.6 V to 2.7 V. These {1-10} domain walls grow into a periodic pattern between 2.8 and 3 V. Following this reorganization (>3 V), we observe partial dislocation migration leading to amorphization at 3.16 V. Upon the application of 3.2 V pulse, the amorphous phase recrystallized, clearly revealing the partial dislocations which participated in the amorphization pathway. Snapshots of this movie are presented in Fig 3a-h of the manuscript, and corresponding structural changes are discussed in detail.Structural changes on the same GeTe nanowire device as shown in Movie S2 captured in dark field TEM mode while application of train of voltage pulses (200 ns) with increasing amplitude until the formation of an amorphous phase, starting from the recrystallized state (formed at the end of Movie S2). Partial dislocation migration and interaction with {1-10} domain boundaries starts at 1 V. Application of pulses with amplitude >2.7 V results in a massive migration of partial dislocations, which cause domain wall alteration and eventual blurring. For voltage pulse amplitude >3.2 V, we observe the intersection of various types of defects  resulting in the formation of defect-template. Addition of more partials to this template at 3.52 V results in formation of the amorphous phase via collapse of long-range order. Snapshots of this movie are presented in Fig 3i-l of the manuscript, and corresponding structural changes are discussed in detail.


The unique organization of these megasynthases, combined with the diversity of their catalytic domain building blocks, has fueled an interest in harnessing the biosynthetic potential of PKSs for the microbial production of both novel natural product analogs and industrially relevant small molecules. However, a limited theoretical understanding of the determinants of PKS fold and function poses a substantial barrier to the design of active variants, and identifying strategies to reliably construct functional PKS chimeras remains an active area of research. In this work, we formalize a paradigm for the design of PKS chimeras and introduce ClusterCAD as a computational platform to streamline and simplify the process of designing experiments to test strategies for engineering PKS variants. ClusterCAD provides chemical structures with stereochemistry for the intermediates generated by each PKS module, as well as sequence- and structure-based search tools that allow users to identify modules based either on amino acid sequence or on the chemical structure of the cognate polyketide intermediate. ClusterCAD can be accessed at  Polyketides and their derivatives are used broadly in medicine as antibiotics, antifungals and immunosuppressants, among other applications , motivatType I modular PKSs are composed of a series of catalytic domains whose identity and order determine the structure of the final polyketide product in a predictable manner, as shown in Figure The potential interchangeability of PKS catalytic domains make PKSs an appealing prospective toolkit for combinatorial biosynthesis. The structure of the two-carbon addition to the nascent acyl chain that is effected by a single PKS module is determined by the substrate specificity of the AT domain, the stereochemical outcome modulated by the KR domain, and the number of active reducing domains in the reductive cassette. Over 20 different malonyl-CoA analogs have been observed to be used as extender units in naturally occurring PKSs ,13,14. FIdentify a truncated PKS parent to use as a starting point for engineering by searching for a module known to produce a polyketide intermediate with maximal structural similarity to the compound of interest.Decompose the difference between the natural product of the truncated PKS starting point and the compound of interest into a series of catalytic domain exchanges.Select donor modules for the required catalytic domain exchanges on the basis of sequence similarity to the truncated PKS parent.However, in spite of recent structural and mechanistic insights gained from structural biology techniques, including X-ray crystallography –17 and cClusterCAD was developed with the aim of enabling the design of chimeric PKSs following this paradigm. As such, it includes a structural search tool to facilitate the identification of a truncated PKS parent to use as an engineering starting point given a small molecule target, and a sequence search tool to aid in the selection of donor modules for catalytic domain exchanges based on sequence similarity to the truncated PKS parent. However, we emphasize that ClusterCAD is intended to augment, rather than supersede, the biochemical intuition of PKS synthetic biologists.Sequence similarity between PKS modules, and structural similarity between their cognate polyketide intermediates, provide only a rudimentary method of predicting compatibility between PKS polypeptide sequences. It is thus also important to consider additional factors when designing a chimeric PKS. For example, modules that originate from well-characterized clusters, or that have been previously determined to be well-expressed in the host organism of choice, are particularly attractive choices for engineering.in silico simulation of biocatalysis using reaction SMARTS operators (http://www.daylight.com/dayhtml/doc/theory/), which were implemented using the RDKit cheminformatics software library (http://www.rdkit.org), yielding a predicted intermediate chemical structure for each PKS module. Known final chemical structures were taken from the MIBiG database if available, or were otherwise generated from the text description of the final structure in the MIBiG database using the ChemAxon JChem Base naming tool  (The gene clusters in ClusterCAD are sourced from the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database . To cons0, 2017) . Chemica0, 2017)  and an e0, 2017) ,34 was uStructure search tool: the structure search tool was designed to enable the identification of a truncated PKS parent to use as a starting point for PKS engineering, and takes as input either a small molecule chemical structure represented as a SMILES string, or a structure that is drawn in an interactive graphical user interface. This tool searches a database of the predicted polyketide intermediates produced by each of the PKS modules in ClusterCAD. Matches to the query structure are ranked using atom pair (AP) Tanimoto similarity scores (y scores , and rety scores  between Sequence search tool: the sequence search tool was designed to enable researchers to select candidate catalytic domains for domain exchange experiments, and supports a variety of queries that allow researchers to test hypotheses regarding which domain–domain interactions may be important in facilitating successful domain exchanges. The sequence search tool takes as input an amino acid sequence, and performs a protein–protein BLAST+ search against a database containing all of the subunits in ClusterCAD (usterCAD . The resThe primary objective of ClusterCAD is to provide computational assistance to the synthetic biologist seeking to engineer PKSs. ClusterCAD provides an intuitive interface for users to browse through the clusters in the database, as well as search tools developed to assist in the design of chimeric PKSs. An example entry of a PKS cluster as displayed in the ClusterCAD web interface, is shown in Figure ClusterCAD provides the first PKS database that specifically targets engineering applications. Existing PKS databases focus on identifying new metabolites through genome mining or on predicting metabolite structure from nucleotide or amino acid sequence . HoweverFrom an engineering perspective, ClusterCAD offers substantial improvements over existing databases. By taking advantage of resources such as the MIBiG database and the antiSMASH command-line tool for cluster identification and annotation respectively, we were able to focus on designing tools tailored to the needs of the synthetic biologist without attempting to optimize our software for genome mining or metabolite structure prediction. Furthermore, given the strong commitment from the synthetic biology and natural products communities to update MiBiG and antiSMASH with continuous improvements, populating ClusterCAD using an end-to-end pipeline that utilizes these tools means that ClusterCAD can easily be kept up-to-date as novel biosynthetic clusters are discovered and as our understanding of PKSs develops.While building on MiBiG and antiSMASH aids in automating the maintenance of ClusterCAD, it also means that inaccuracies in MiBiG or in the antiSMASH software may be propagated into ClusterCAD. In order to address this issue, we also employ extensive manual curation of each ClusterCAD entry in order to provide predicted polyketide intermediates that are as accurate as possible. ClusterCAD has a persistent corrections record that overwrites the output generated by antiSMASH. This enables ClusterCAD to incorporate new MiBiG entries and systematic improvements to antiSMASH, while also guaranteeing that clusters that have previously been manually corrected will remain correct. Because these records are persistent and override the automatically generated output, the manually curated clusters will only need to be further corrected if new empirical findings are generated. ClusterCAD thus combines computational annotations of AT substrate specificity and KR stereospecificity with domain expert application of heuristics obtained from the literature ,32,39,40ClusterCAD is further limited in that it does not support inclusion of chemistry effected by ‘on-line’ tailoring enzymes that act on ACP-tethered polyketide intermediates, but that are independent of the PKS . HoweverA substantial benefit of ClusterCAD is that it provides engineering tools that allow researchers to compare PKS modules based on amino acid sequence similarity and on cognate polyketide intermediate similarity. The substructure search in ClusterCAD is of particular utility when selecting candidate modules to serve as a starting point in a PKS engineering project. Previous experimental evidence supports the assertion that the structure of the intermediate can have a significant impact on the function of individual catalytic domains. KS domains, for example, have been reported to have ‘gatekeeping’ functionality, in which they are unable to condense intermediates that are chemically dissimilar for reasons ranging from overall steric bulkiness  to havinThe substructure search in ClusterCAD is one of the first that matches against polyketide intermediates associated with non-terminal PKS modules, making it more robust to false positives than similar search tools which only support substructure queries in the context of PKS final products . MatchinFurther, anecdotal evidence supports the assertion that seeking catalytic domains with greater sequence similarity improves domain exchange success rates . While tThe sequence search tool in ClusterCAD highlights the domains in each returned module that overlap with the query, and also allows the user to highlight results based on catalytic domain activity. Identifying junctions in PKS chimeras that reliably leave functionally important structural elements intact ,25 remaiet al. has demonstrated that it is possible to engineer a novel PKS capable of producing adipic acid (Identify a truncated PKS starting point that naturally produces a polyketide intermediate that is structurally similar to adipic acid. Hagen et al. selected the first module from the borrelidin PKS as a starting point based on previous biochemical studies establishing that BorMod1 tolerates succinyl-CoA as a starter, possibly because of structural similarity to the native priming unit, trans-1,2-cyclopentanedicarboxylic acid (lic acid . Adipic Identify a donor reductive loop with active ER, DH and KR from a module which naturally incorporates a malonyl-CoA extender unit. In order to produce adipic acid using BorMod1, incorporation of an extender unit lacking a α-substituent and reduction by a full complement of active reducing domains was required. Hagen et al. thus chose four reductive loop donors that naturally accepted malonyl-CoA, harbored a DH, ER and KR in the reductive cassette, and that came from a ‘standalone’ module, in which the corresponding open reading frame or subunit contained only a single module. Donor loops from ‘standalone’ modules were favored based on the intuition that domain exchanges using reductive cassettes derived from a ‘standalone’ module would retain more architectural similarity to BorMod1, itself a ‘standalone’ module, and thereby increase the likelihood of a functional chimera.et al., along with chimeras harboring reductive cassettes from the donor modules AurB, from the aureothin PKS and NanA2, from the nanchangmycin PKS. AurB, which is not contained in ClusterCAD, and SpnB were empirically determined to yield the most active chimeras. NanA2, which was not returned by ClusterCAD, but is contained in the database, yielded the least active chimera.Using the ClusterCAD sequence search tool, we sought to identify donor modules that not only met these criteria, but also exhibited maximal sequence similarity to BorMod1. We queried the ClusterCAD sequence search tool with the amino acid sequence of BorA2, the subunit which contains BorMod1, ranking the potential donor modules by bit score and filtering for ‘standalone’ modules containing a malonyl-CoA specific AT and a complete reductive cassette. IdmO, from the indanomycin PKS, followed by SpnB from the spinosyn PKS were the only two returned results that matched all three criteria. Chimeras utilizing these two donor modules were experimentally tested by Hagen Identify a DH domain that exhibits higher levels of activity on 3-hydroxy adipic acid. The chimeras constructed by Hagen et al. yielded a relatively large proportion of the incompletely reduced product 3-hydroxyadipic acid, potentially due to the DH from the donor reductive cassettes having a poor substrate compatibility with the terminal carboxyl moiety, an unusual feature in PKS biosynthetic intermediates. To overcome this substrate incompatibility, Hagen et al. further substituted a heterologous DH into the engineered BorMod1 variant. The DH from BorMod2 was selected based on the observation that it is known to demonstrate catalytic activity on a substrate containing a terminal carboxy moiety. The intuition employed by Hagen et al. was consistent with the sequence-similarity based output of the ClusterCAD search tool, which returned BorMod2 as the module containing an active DH that had the highest bit score with BorMod1.While engineered PKSs have long been an attractive strategy to produce pharmaceutically relevant natural products and their analogs, more recent work has also sought to harness the chemical diversity accessible by PKS to provide an environmentally friendly alternative to chemical synthesis for commodity chemicals. Previous work by Hagen pic acid , a widelhttps://github.com/JBEI/clusterCAD. This open source release provides Python object representations of catalytic domains, modules and clusters, that enable researchers to construct and simulate the activity of chimeric PKSs in silico. Furthermore, we sought to establish a method to describe PKSs in a human and computer readable manner using the JSON data-interchange format, as shown in Figure As the first PKS database that specifically targets synthetic biologists interested in PKS engineering, we also sought more generally to provide tools for computer-aided PKS design, and have released ClusterCAD under a BSD style license at To the best of our knowledge, ClusterCAD is the first web-based toolkit developed specifically to aid in designing chimeric PKSs. By providing chemical structures with stereochemistry for the polyketide intermediate produced by each module, as well as relative solvent accessibility and secondary structure predictions for each subunit, we provide rich information about each cluster to inform chimeric PKS design. Furthermore, our intuitive user interface, combined with the ability to query for modules based on either sequence- or structure-similarity provide powerful tools for PKS engineering that bring us closer to the promise of harnessing PKSs for combinatorial biosynthesis.