The authors gratefully acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC) for the work on cellular 2A-like sequences.Publisher PDFPeer reviewe

de Felipe, Pablo

Luke, Garry A.

Odon, Valerie

Roulston, Claire

Ryan, Martin D.

Tilsner, Jens

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The potential consequences for cell Signaling by a class of NOD-Like Receptor proteins (NLRs) bearing an N-terminal signal sequence

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The authors gratefully acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC) for the work on cellular 2A-like sequences.Peer reviewe

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The Potential Consequences for Cell Signaling by a Class of NOD-Like ReceptorProteins (NLRs) Bearing an N-terminal Signal SequenceMartin D Ryan1*, Claire Roulston1, Pablo de Felipe2, Valerie Odon3, Jens Tilsner4 and Garry A Luke11Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK2Spanish Medicines Agency (AEMPS), Parque Empresarial “Las Mercedes”, Campezo 1 –Edificio 8, 28022 Madrid, Spain3Department of Microbial Sciences, School of Biosciences and Medicine, University of Surrey, Guildford GU2 7XH, UK4James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK*Corresponding author: Martin D Ryan, Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews KY16 9ST,UK, Tel: 01334 463403; E-mail: martin.ryan@st-andrews.ac.ukReceived date: April 17, 2017; Accepted date: May 10, 2017; Published date: May 19, 2017Copyright: © 2017 Ryan MD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.CommentaryToll-like and NOD-like receptors (TLRs/NLRs) recognise pathogen-or damage-associated molecular patterns (PAMPs/DAMPs) and arekey initiators of cell signalling pathways by which the cell mounts anappropriate defence response. Whilst TLRs are type I transmembraneproteins, NLRs are cytosolic proteins. Determination of the genomesequence of the purple sea urchin (Strongylocentrotus purpuratus)revealed this echinoderm had a greatly expanded repertoire ofTLR/NLR innate immunity genes in comparison with vertebratespossessing an acquired immune system [1]. The open reading frame(ORF) of a number of NLR genes in S. purpuratus (and organismsfrom other phyla) commence with a leader sequence with sequencesimilarity to ‘2A/2A-like’ oligopeptide sequences found in many virusgenomes, which mediate an unusual translational ‘recoding’ event inwhich translation arrests at the C-terminus of 2A (mid-ORF; no stopcodon present), but then may recommence the synthesis of thedownstream protein as a separate, entirely discrete, translation product[2-5]. Bioinformatic analyses also suggested, however, that these S.purpuratus NLR 2A-like sequences might also function as co-translational signal sequences. We showed this type of 2A-like leadersequence possessed both activities [6], and in this commentary wespeculate upon the possibility that such a single gene could produceboth cytoplasmic and membrane-bound/secreted forms of such NLRs.‘2A’ and ‘2A-like’ oligopeptide sequences were first characterisedfrom a group of single-stranded positive (mRNA) sense RNA virusescalled picornaviruses. These short (~20aa) sequences were shown tomediate a co-translational ‘cleavage’ of a polyprotein at their own C-termini. This apparent ‘cleavage’ was, however, not a product ofproteolysis, but a newly-characterised form of translational ‘recoding’in which the synthesis of a specific peptide bond at the C-terminus of2A was ‘skipped’. Briefly, the model of this mechanism states that whenthe elongating ribosome translates the 2A sequence, the nascentpolypeptide sequence within the exit tunnel of the ribosome (2A)interacts with the tunnel to pause elongation and inhibit peptide bondformation. This is proposed to bring about the release of the nascentpeptide synthesised to that point, but then translation of the sequencesdownstream of 2A may recommence. In this manner the translationproducts are actually synthesised as discrete products rather than as asingle product which is then cleaved apart [2-5].Figure 1: NLR and Virus 2A sequences. The structure of the mRNAencoding the S. purpuratus NLR is shown encoding a single ORFcomprising (i) a 2A-like sequence at the N-terminus, then (ii) pyrin(PYR), (iii) nucleotide-binding (NACHT), (iv) putativetransmembrane (TMD) and (v) leucine-rich repeat (LRR; PAMP/DAMP recognition) domains (boxed areas). The virus RNA (vRNA)genome of the positive-stranded RNA Foot-and-Mouth DiseaseVirus (FMDV) encodes a single ORF (a ~2,300aa ‘polyprotein’;boxed areas) comprising a virus-encoded proteinase at the N-terminus (Lpro), the capsid proteins domain, 2A, then the domainscomprising the various genome replication proteins. The genomebears an oligopeptide cap structure (unlike cellular mRNAs) and ispolyadenylated. The 2A-like sequence of the S. purpuratus NLR isshown together with FMDV 2A. The C-terminal motif conservedamongst all 2As characterised to date is shown in bold, and the siteof translational recoding is shown by the vertical arrow.Many 2As possess only partial recoding activity: if, for example, aparticular 2A sequence recodes translation only 50% of the time, thenin 50% of the translation products the peptide bond is formed and theresulting translation profile is a mixture of the recoded (‘cleaved’)products (50%) plus the fusion protein (50%).Cellular 2A-like SequencesDatabase probing with a motif characteristic of 2A ([-D(V/I)ExNPGP-]) revealed the presence of ‘2A-like’ sequences in thegenomes of many other RNA viruses but, surprisingly, also multipleoccurrences within the genome of S. purpuratus. Here, 2A-likeJournal of Cell Signaling Ryan et al., J Cell Signal 2017, 2:2Commentary OMICS InternationalJ Cell Signal, an open access journal Volume 2 • Issue 2 • 148sequences were observed within two main types of cellular sequence:non-LTR retrotransposons [7,8] and certain NLRs [6]. In the lattercase our surprise was compounded in that these particular 2A-likesequences formed the N-termini of certain NLR proteins: until thatpoint all of the (virus) 2A/2A-like sequences we had studied wereinternal features used for the co-expression of protein domains up- anddown-stream of 2A (Figure 1). These cellular NLR-2As were ofapproximately the same length as virus 2As (~25-30aa), but compriseda higher proportion of hydrophobic residues and were predominantlyflanked by charged residues – suggestive of a signal sequence and,indeed, bioinformatic analyses confirmed these NLR-2As could besignal sequences. But how to show these NLR-2As could (i) mediatetranslational recoding and (ii) could function as N-terminal signalsequences? Given the lack of immortalised S. purpuratus cell-lines, thelimited availability of primary cell-lines, the lack of anti-NLR antibodyprobes etc., we chose to recapitulate the system using fluorescentreporter proteins (to study the effects upon protein localisation) and,since the function of N-terminal sequences is conserved acrosskingdoms, we used both immortalised, readily transfectable,mammalian (HeLa) cells and plant (Nicotiana benthamiana) leafepidermal cells. Our data showed that these NLR-2As could bothmediate (partial) translational recoding and act as N-terminal signalsequences [6]. When the NLR-2A recoded itself from the fluorescentreporter protein (mCherryFP), this protein localised to the cytoplasm.In the proportion of translation products where the NLR-2A did notrecode – remained as an N-terminal feature of mCherryFP - thenmCherryFP was secreted from the cell. Our data showed that inmammalian cells the NLR-2A was removed from the secretedmCherryFP, presumably by signal peptide peptidase.Innate Immune Signalling in S. purpuratusThe systems outlined above allowed us to demonstrate that theseNLR-2As were a new type of bi-functional translational recoding/signal sequence – but what are the implications for cell signalling inthe innate immune system of S. purpuratus? TLRs are type Itransmembrane proteins. Upon activation by PAMPs/DAMPs bindingto the leucine-rich repeat domain (LRR), TLRs recruit adapter proteinswithin the cytosol of the immune cell in order to initiate thepropagation of signal transduction pathways (Figure 2, Panel A). NLRsare, however, entirely cytosolic proteins detecting and signalling thepresence of PAMPs/DAMPs present within the cytoplasm (Figure 2,Panel B). Recognition of the ligand by the LRR leads to the NOD/NBD– nucleotide-binding (NACHT) domain mediating ATP-dependentself-oligomerisation and initiates cell signalling by the N-terminaleffector binding region comprising a protein-protein interactiondomain such as a caspase recruitment domain (CARD), pyrin (PYD),baculovirus inhibitor repeat (BIR) or transactivator domain (AD). Theend result of NLR signalling is the cell producing an inflammatoryresponse, undergoing autophagy or cell death.Database annotation of the S. purpuratus genome is based upondetection of similarities with other, previously characterised, proteindomains: for example, NCBI entry XP011670951.1 (predicted NACHT,LRR and PYD domains-containing protein 3-like [S. purpuratus])indicates a predicted Death Domain (aa’s 38-177), a P-loop NTPase(NACHT) domain (aa’s 222-414) prior to the C-terminal LRR domainbinding PAMPs/DAMPs. Interestingly, the transmembrane proteinstructure prediction algorithm MemBrain [9] predicts three maintransmembrane domains (TMDs) for this protein; the first centring onresidues 9/10 (propensity=0.97) – corresponding to the N-terminal2A-like signal sequence, the second centring on residues 441/442(propensity=0.92) – immediately after the NACHT domain, and thethird on residues 474/475 (propensity=0.78) – potentially prior to theLRR domain: could either/both of these putative TMDs between theNACHT and LRR domains act as internal signal-anchor sequences?Figure 2: TLR and NLR Innate immunity receptors. Toll-likereceptors bear an N-terminal signal sequence targeting the leucine-rich repeat domain (LRR; PAMP/DAMP recognition) of the proteinto either the extracellular space or the lumen of endosomes. Theinternal signal-sequence/stop-transfer domain arrests proteintranslocation with the signalling (PIR) domain of the protein beingcytoplasmic – the orientation of a type I transmembrane protein(Panel A). NOD-like receptors do not possess any sub-cellularsignalling sequences: the arrangement of protein domains shows theopposite polarity to NLRs, and comprises an additional centralNOD/NBD – nucleotide-binding (NACHT) domain (Panel B).The presence of a functional N-terminal signal sequence (functionalat least in mammalian and plant cells) together with putativetransmembrane/internal signal-anchor/stop-transfer domains betweenthe NACHT and LLR domains raises a number of questions withregards the sub-cellular localisation, potential membrane association(and, if this is the case, the protein orientation in the membrane) ofthese S. purpuratus NLR proteins. As shown in (Figure 3) (left panels),if the 2A-like signal sequence does not mediate a translational recodingevent (remaining fused to the NLR), then the protein could either besecreted from the cell (no effect of putative TMD/internal signal-anchor/stop-transfer sequences), or, become a type 1 transmembraneprotein (putative TMD/internal signal-anchor/stop-transfer sequencefunctional), although it is difficult to envisage a signalling function ofsuch a molecule either being completely secreted from the cell or withthis particular orientation within the membrane. As shown in (Figure3) (right panels) shows the possibilities if the 2A-like signal sequencedoes mediate a translational recoding event (2A-like signal sequencenot present at the N-terminus of the NLR): the NLR could becomecytoplasmic (no effect of putative TMD/internal signal-anchor/stop-transfer sequences), or, perhaps, become either a type I or a type IItransmembrane protein (internal signal-anchor/stop-transfer sequencefunctional). The orientation of the protein in the membrane isdetermined by the difference in the charges of the 15 residues flankingthe first internal signal-anchor/stop-transfer sequence, with the morepositive portion facing the cytosol [10].Citation: Ryan MD, Roulston C, Felipe PD, Odon V, Tilsner J, et al. (2017) The Potential Consequences for Cell Signaling by a Class of NOD-Like Receptor Proteins (NLRs) Bearing an N-terminal Signal Sequence. J Cell Signal 2: 148. Page 2 of 3J Cell Signal, an open access journal Volume 2 • Issue 2 • 148Figure 3: S. Purpuratus NLR Innate immunity receptors. Theorganisation of protein domains encoded by the mRNA is shown atthe top of the figure. Translation of the mRNA, however, canproduce 2, distinct, translation products. In the first case, the NLR2A-like signal sequence does not mediate translational recoding(remains fused to the NLR protein), the NLR could either besecreted from the cell, or, if the putative transmembrane domain(TMD) is recognised by the exocytic pathway, adopt a type Itransmembrane orientation with the LRR cytoplasmic and theNACHT oligomerisation and pyrin (PYR) domains oriented to theexterior of the cell (left panels). In this illustration we have assumedthat the 2A-like signal sequence is removed by signal peptidepeptidase (as is the case in mammalian cells). In the second case,where the 2A-like S-seq does mediate translational recoding, theNLR could be localised to the cytoplasm (putative TMD notrecognised by the exocytic pathway), or, if the putative TMD isrecognised by the exocytic pathway then adopts the orientation ofeither a type I or type II transmembrane protein – depending uponthe charge distribution surrounding the putative TMD (rightpanels).Again, it is difficult to envisage the function of such an NLRoriented as a type I transmembrane protein, but if it became a type IItrans membrane protein, then could it fulfil the same type of functionas a TLR? Interestingly, the 2A-like signal sequence could be acquiredby these S. purpuratus NLR genes by a process of ‘exon-shuffling’ sinceit is encoded by its own mini-exon preceding the bulk of the NLR gene:in the case of the NLR described above (XP011670951.1), encoding theentire signal plus six amino acids downstreamMDGFCLLYLLLILLMRSGDVETNPG↓PNTAVS; 2A-like signalsequence underlined, site of translational recoding indicated byvertical arrow).Our paper presented analyses of the function of these N-terminal2A-like signal sequences of NLR genes in S. purpuratus [6]. It shouldbe noted, however, that we have detected a similar 2A-NLR geneorganisation in other phyla; the cephalochordate Branchiostomafloridae, the sponge Amphimedon queenslandica, the mollusc Lottiagigantea and the arthropod Ixodes scapularis [11].These data, plus subsequent bioinformatic analyses presented here,raise a number of important questions as to the function of these‘NLR-like’ genes in these organisms. It may prove to be the case thatthey may have not only expanded the number of innate immunitygenes, but also expanded the functionality of some of these NLRproteins – potentially involving novel signalling pathways.AcknowledgementThe authors gratefully acknowledge the support of the UKBiotechnology and Biological Sciences Research Council (BBSRC) forthe work on cellular 2A-like sequences.References1. Hibino T, Loza-Coll M, Messier C, Majeske AJ, Cohen AH, et al. (2006)The immune gene repertoire encoded in the purple sea urchin genome.Dev Biol 300: 349-365.2. Ryan MD, Drew J (1994) Foot-and-mouth disease virus 2A oligopeptidemediated cleavage of an artificial polyprotein. EMBO J 134: 928-933.3. Donnelly MLL, Luke G, Mehrotra A, Li X, Hughes LE, et al. (2001)Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanismindicates not a proteolytic reaction, but a novel translational effect: aputative ribosomal ‘skip’. J Gen Virol 82: 1013-1025.4. de Felipe P, Hughes LE, Ryan MD, Brown JD (2003) Co-translational,intraribosomal cleavage of polypeptides by the foot-and-mouth diseasevirus 2A peptide. J Biol Chem 278: 11441-11448.5. Sharma P, Yan F, Doronina V, Escuin-Ordinas H, Ryan MD, et al. (2012)2A peptides provide distinct solutions to driving stop-carry ontranslational recoding. Nuc Acids Res 40: 1-9.6. Roulston C, Luke GA, de Felipe P, Ruan L, Cope J, et al., (2016) ‘2A-Like’signal sequences mediating translational recoding: A novel form of dualprotein targeting. Traffic 17: 923-939.7. Odon V, Luke GA, de Felipe P, Ruan L, Roulston C, et al. (2013) APE-typenon-LTR retrotransposons of multicellular organisms encode virus-like2A oligopeptide sequences which mediate translational recoding duringprotein synthesis. Mol Biol Evol 30: 1955-1965.8. Luke GA, Roulston C, Odon V, de Felipe P, Sukhodub A, et al. (2013) Lostin translation: The biogenesis of non-LTR retrotransposon proteins. MobGenet Elements 3: e27525.9. http://www.csbio.sjtu.edu.cn/cgi-bin/MEMBRAIN.cgi10. Hartmann E, Rapoport TA, Lodish HF (1989) Predicting the orientationof eukaryotic membrane-spanning proteins. Proc Natl Acad Sci USA 86:5786-5790.11. Roulston C (2015) Occurrence and function of cellular 2A sequences.PhD Thesis University of St Andrews. Citation: Ryan MD, Roulston C, Felipe PD, Odon V, Tilsner J, et al. (2017) The Potential Consequences for Cell Signaling by a Class of NOD-Like Receptor Proteins (NLRs) Bearing an N-terminal Signal Sequence. J Cell Signal 2: 148. Page 3 of 3J Cell Signal, an open access journal Volume 2 • Issue 2 • 148

The potential consequences for cell Signaling by a class of NOD-Like Receptor proteins (NLRs) bearing an N-terminal signal sequence

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