IGEM:IMPERIAL/2009/Pup
Pup: Prokaryotic ubiquitin-like protein
Function
Small Ubiquitin-like molecule suggesting that prokaryotes too use ubiquitin-like tagging to target proteins for the proteasome (ie: destruction).
Organism
Link
http://www.nature.com/nrmicro/journal/v7/n7/full/nrmicro2148.html
Extracts
Bacterial 'ubiquitin'
One of the mysteries of the bacterial proteasome system was how proteins were targeted for degradation in the apparent absence of ubiquitin or UBLs. To begin to answer this question, natural substrates of the M. tuberculosis proteasome were first identified. Two biosynthetic enzymes, FabD (malonyl CoA-acyl carrier protein) and PanB (ketopantoate hydroxymethyltransferse), were identified as degradation substrates31. Both proteins accumulated in proteasome-defective M. tuberculosis; however, unlike proteins conjugated with ubiquitin, FabD and PanB did not seem to be modified, because they migrated through protein gels only at their predicted molecular weight. This observation, in addition to the lack of apparent protein modifiers in bacteria, led to the hypothesis that proteasomal degradation signals were inherent to the substrates.
Despite the identification of endogenous M. tuberculosis substrates, attempts to degrade FabD with recombinant proteasomes and Mpa in vitro were unsuccessful, suggesting that other cofactors were required for full proteasome function. To address the hypothesis that Mpa needed to interact with other degradation cofactors, a bacterial two-hybrid screen in Escherichia coli was performed with Mpa as bait. The screen resulted in the identification of Rv2111c, a protein of unknown function encoded with the proteasome core genes of M. tuberculosis13. Purified Rv2111c interacted non-covalently with Mpa but did not promote FabD degradation by proteasomes and Mpa in vitro.
Other bacteria with Pup
Pup, PafA and Mpa are found in numerous genera of pathogenic and non-pathogenic actinobacteria, and almost always in strains that encode proteasomes (exceptions include Bifidobacterium spp. and Corynebacterium spp.) (Fig. 3). All sequenced Corynebacterium strains have a Pup homologue based on BLAST (basic local alignment and search tool) analysis37, but it was previously noted that they do not encode proteasomes38. Inspection of the Corynebacterium glutamicum pup region reveals that mpa, pup and pafA are conserved and pup is likely to be transcriptionally and translationally coupled with pafA, an organization that is not observed in proteasome-bearing bacteria (Fig. 3). The different organization of this region compared with the same locus in M. tuberculosis suggests that a deletion of the proteasome core genes occurred that left the remaining proteasome accessory factor genes intact. Interestingly, the corynebacterial Mpa homologues lack a C-terminal extension with a penultimate tyrosine that is essential for function31, 39. Site-directed mutagenesis of the penultimate tyrosine of M. tuberculosis Mpa resulted in failed substrate degradation and reduced protection against NO39. Furthermore, a transposon mutation that deleted the last two amino acids of Mpa (tyrosine and leucine) attenuated M. tuberculosis virulence in mice as much as a null mutation in mpa31. The archaeal ATPase PAN also has a conserved penultimate tyrosine that is required to open proteasome cores for protein degradation25. These observations suggest that the Mpa C terminus probably interacts with the proteasome core to activate degradation. Because Corynebacterium spp. do not appear to have proteasomes or have proteases that have dramatically diverged from proteasomes, it is possible that Corynebacterium Mpa homologues have evolved different C termini. In any case, it remains to be determined if mpa, pafA and pup are expressed in this genus or other bacterial genera.
