The Potential Consequences for Cell Signaling by a Class of NOD-Like Receptor Proteins (NLRs) Bearing an N-terminal Signal Sequence

Abstract

Commentary Toll-like and NOD-like receptors (TLRs/NLRs) recognise pathogen-or damage-associated molecular patterns (PAMPs/DAMPs) and are key initiators of cell signalling pathways by which the cell mounts an appropriate defence response. Whilst TLRs are type I transmembrane proteins, NLRs are cytosolic proteins. Determination of the genome sequence of the purple sea urchin (Strongylocentrotus purpuratus) revealed this echinoderm had a greatly expanded repertoire of TLR/NLR innate immunity genes in comparison with vertebrates possessing an acquired immune system [1]. The open reading frame (ORF) of a number of NLR genes in S. purpuratus (and organisms from other phyla) commence with a leader sequence with sequence similarity to '2A/2A-like' oligopeptide sequences found in many virus genomes, which mediate an unusual translational 'recoding' event in which translation arrests at the C-terminus of 2A (mid-ORF; no stop codon present), but then may recommence the synthesis of the downstream 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 leader sequence possessed both activities [6], and in this commentary we speculate upon the possibility that such a single gene could produce both cytoplasmic and membrane-bound/secreted forms of such NLRs. '2A' and '2A-like' oligopeptide sequences were first characterised from a group of single-stranded positive (mRNA) sense RNA viruses called picornaviruses. These short (~20aa) sequences were shown to mediate a co-translational 'cleavage' of a polyprotein at their own C-termini. This apparent 'cleavage' was, however, not a product of proteolysis, but a newly-characterised form of translational 'recoding' in which the synthesis of a specific peptide bond at the C-terminus of 2A was 'skipped'. Briefly, the model of this mechanism states that when the elongating ribosome translates the 2A sequence, the nascent polypeptide sequence within the exit tunnel of the ribosome (2A) interacts with the tunnel to pause elongation and inhibit peptide bond formation. This is proposed to bring about the release of the nascent peptide synthesised to that point, but then translation of the sequences downstream of 2A may recommence. In this manner the translation products are actually synthesised as discrete products rather than as a single product which is then cleaved apart [2-5]. Figure 1: NLR and Virus 2A sequences. The structure of the mRNA encoding the S. purpuratus NLR is shown encoding a single ORF comprising (i) a 2A-like sequence at the N-terminus, then (ii) pyrin (PYR), (iii) nucleotide-binding (NACHT), (iv) putative transmembrane (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 Disease Virus (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 domains comprising the various genome replication proteins. The genome bears an oligopeptide cap structure (unlike cellular mRNAs) and is polyadenylated. The 2A-like sequence of the S. purpuratus NLR is shown together with FMDV 2A. The C-terminal motif conserved amongst all 2As characterised to date is shown in bold, and the site of translational recoding is shown by the vertical arrow. Many 2As possess only partial recoding activity: if, for example, a particular 2A sequence recodes translation only 50% of the time, then in 50% of the translation products the peptide bond is formed and the resulting translation profile is a mixture of the recoded ('cleaved') products (50%) plus the fusion protein (50%).