Prokaryotic ubiquitin-like protein (Pup), proteasomes and pathogenesis

Abstract

Proteasomes are ATP-dependent, multisubunit proteases that are found in all eukaryotes and archaea and some bacteria. In eukaryotes, the small protein ubiquitin is covalently attached in a post-translational manner to proteins that are targeted for proteasomal degradation. Despite the presence of proteasomes in many prokaryotes, ubiquitin or other post-translational protein modifiers were presumed to be absent from these organisms. Recently a prokaryotic ubiquitin-like protein, Pup, was found to target proteins for proteolysis by the Mycobacterium tuberculosis proteasome. The discovery of this ubiquitin-like modifier opens up the possibility that other bacteria may also have small post-translational protein tagging systems, with the ability to affect cellular processes.

Fig. 1. Overview of the eukaryotic Ub-proteasome system

Ubiquitin (Ub) is encoded by four different loci in yeast as part of a larger polypeptide. Processing proteases expose C-terminal Gly-Gly that are activated by adenylation with an E1 enzyme. The E1 enzyme subsequently transfers Ub to an E2 enzyme, where a thioester bond is formed. The E2 then transfers Ub to any number of E3 ligases. The E3 ligase family can be sub-divided into HECT (Homologous to the E6-AP Carboxyl Terminus) and RING (Really Interesting Gene) domain ligases: RING ligases hold both the E2 and substrate, and facilitate the direct transfer of Ub from the E2 to the substrate; in contrast, HECT ligases form a thioester bond with Ub prior to transfer to a substrate lysine. E3 ligases dictate the type of Ub linkages that are formed. Proteins with Lys (K) 48 linked chains are usually targeted for degradation by the 26S proteasome. Other types of Ub linkages (mono- and poly-K63 and others) can result in degradation but generally serve other functions. See text for additional details.

Fig. 2. Proposed model of the Pup-proteasome pathway in Mtb

Unlike Ub, Pup is not processed proteolytically from a larger precursor protein. Pup appears to be de-amidated at the C-terminal Gln. From this point, it has been proposed that PafA phosphorylates the γ-carboxylate of the C-terminus of Pup, but this has not been established. The attachment of Pup to the substrate Lys can potentially be via either the α- or γ-carboxylate. It is not known if poly-pupylation occurs, nor is it known if Pup is removed by a de-pupylase (“DPUP”) prior to degradation, and recycled like Ub.

Fig. 3. Comparison of the pup regions of bacteria with and without proteasomes

(A) pup-containing bacteria. Mycobacterium tuberculosis: pup (red); proteasome core genes (β-subunit gene prcB; pink; α-subunit gene prcA; orange); proteasome accessory factor A (pafA; green); and Mycobacterium proteasomal ATPase (mpa; cyan). Homologues in other bacteria are shaded in the same color schemes. pafB and pafC do not appear to be involved in pupylation or degradation in Mtb^, . PafD (hatched green) is 40% identical and 60% similar to PafA (e-value 10⁻⁷³) but its role in proteasome function or pupylation has not been established. (B) Bacteria that have paf homologues but no apparent pup or proteasome genes. Data were collected from http://mbgd.genome.ad.jp/