The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe proteasome is a ubiquitous nano-machine for protein degradation in eukaryotes and archaea (). It is a ∼670 kDa barrel-shaped complex of four stacked rings (), each composed of seven identical (archaea) or distinct (eukaryotes) subunits. The outer rings consist of catalytically inactive α subunits, whereas the inner rings are composed of proteolytic β subunits. α and β subunits are similar in sequence and structure, and are thought to have emerged by the duplication of a proto-β subunit.The proteasome can act by itself as a 20S proteasome (), or its α subunits may interact with various regulators that affect its choice of substrates (, ). The proteasome's most prominent function, targeted protein degradation, requires interaction with hexameric unfoldases of the AAA+ (ATPase with diverse cellular functions) superfamily (), which can also act as chaperones on their own (). In recent years, experiments have also emphasized the significance of the proteasome's ATP-independent functions (), such as the degradation of oxidized proteins through interaction with PA28αβ () or the degradation of acetylated histones through interaction with PA200 ().The proteasome is absent from bacteria, barring some branches of Actinobacteria (, ) and Nitrospirae (). While one theory attributes the occurrence of the proteasome in actinobacteria to horizontal gene transfer (HGT) (), another argues that the original proteasome evolved in an ancestral actinobacterium, from where it was inherited by archaea and eukaryotes (). Both theories, however, assume (href="#bib12" rid="bib12" class=" bibr popnode">Bochtler et al., 1999) that the proteasome as such evolved from its simpler and widely distributed bacterial homolog HslV (heat shock locus V). This homolog, unlike the proteasome, is a homomeric assembly of just two stacked hexameric rings (href="#bib11" rid="bib11" class=" bibr popnode">Bochtler et al., 1997) and thus lacks the antechamber constituted by the α subunits. Despite this, HslV is similar to the proteasome in its ability to interact with an unfoldase of the AAA+ superfamily, HslU, which recognizes intrinsic features of misfolded proteins (href="#bib34" rid="bib34" class=" bibr popnode">Gur et al., 2011). HslU and proteasomal unfoldases, however, belong to different clades of AAA+ ATPases (href="#bib5" rid="bib5" class=" bibr popnode">Ammelburg et al., 2006) and use different interfaces for interaction with their respective proteolytic machinery (href="#bib68" rid="bib68" class=" bibr popnode">Sousa et al., 2000, href="#bib75" rid="bib75" class=" bibr popnode">Yu et al., 2010), suggesting that they were recruited independently. While several studies indicate that HslU is not always bound to HslV (href="#bib6" rid="bib6" class=" bibr popnode">Azim et al., 2005) and possesses chaperone-like activities on its own (href="#bib65" rid="bib65" class=" bibr popnode">Seong et al., 2000), HslV has not been shown to function in the cell on its own in an ATP-independent manner. Unlike the essential, constitutively expressed eukaryotic proteasome, the non-essential heat-shock-induced HslV complements a set of other unrelated self-compartmentalizing proteases, such as FtsH, Lon, and Clp, under stress conditions (href="#bib34" rid="bib34" class=" bibr popnode">Gur et al., 2011, href="#bib45" rid="bib45" class=" bibr popnode">Kanemori et al., 1997).In 2008, a novel bacterial β subunit homolog, termed Anbu (ancestral β subunit) was identified (href="#bib70" rid="bib70" class=" bibr popnode">Valas and Bourne, 2008). It was proposed that Anbu, not HslV, gave rise to the proteasome, and that this event took place in actinobacteria. This interpretation was, however, questioned as Anbu, unlike other self-compartmentalizing proteases, is not associated with an AAA+ ATPase on the genomic level, but frequently co-occurs in an operon with a transglutaminase, an ATP-grasp protein with putative peptide ligase function, and a unique α-helical protein, Alpha-E (href="#bib43" rid="bib43" class=" bibr popnode">Iyer et al., 2009, href="#bib70" rid="bib70" class=" bibr popnode">Valas and Bourne, 2008), hinting at a specific peptide-synthesis system in which Anbu would act as a peptidase (href="#bib43" rid="bib43" class=" bibr popnode">Iyer et al., 2009).In this paper, we study the molecular characteristics of Anbu and, based on bioinformatic analysis, its place in proteasomal evolution. We determined crystal structures of two Anbu proteins and could decipher the Anbu structure in solution via small-angle X-ray scattering (SAXS). We find that Anbu forms a dodecameric open-ring assembly that locally resembles the architecture of the self-compartmentalizing proteasome, but is not closed for steric reasons. Based on these findings, we draft a scenario in which the Anbu complex constitutes an evolutionary intermediate at the origin of the proteasome system.