Identification and Characterisation of a Novel Acylpeptide Hydrolase from Sulfolobus Solfataricus: Structural and Functional Insights

A novel acylpeptide hydrolase, named APEH-3Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a “true” aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3Ss and apehSs (the gene encoding the previously identified APEHSs from S. solfataricus), which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea

Small Peptide Inhibitors of Acetyl-Peptide Hydrolase Having an Uncommon Mechanism of Inhibition and a Stable Bent Conformation

Acyl peptide hydrolase (APEH) catalyzes the removal of acetyl aminoacids from the N-terminus of peptides and cytoplasmic proteins. Due to the role played in several diseases, and to the growing interest around N terminal acetylation, studies on APEH structure, function, and inhibition are attracting an ever increasing attention. We have therefore screened a random tetrapeptide library, N-capped with selected groups, and identified a trifluoroacetylated tetrapeptide (CF3-lmph) which inhibits the enzyme with a Ki of 24.0 ± 0.8 μM. The inhibitor is selective for APEH, shows an uncommon uncompetitive mechanism of inhibition, and in solution adopts a stable bent conformation. CF3-lmph efficiently crosses cell membranes, blocking the cytoplasmic activity of APEH; however, it triggers a mild proapoptotic effect as compared to other competitive and noncompetitive inhibitors. The unusual inhibition mechanism and the stable structure make the new compound a novel tool to investigate enzyme functions and a useful scaffold to develop more potent inhibitors

A Highly Selective Oligopeptide Binding Protein from the Archaeon Sulfolobus Solfataricus▿ †

SSO1273 of Sulfolobus solfataricus was identified as a cell surface-bound protein by a proteomics approach. Sequence inspection of the genome revealed that the open reading frame of sso1273 is associated in an operon-like structure with genes encoding all the remaining components of a canonical protein-dependent ATP-binding cassette (ABC) transporter. sso1273 gene expression and SSO1273 protein accumulation on the cell surface were demonstrated to be strongly induced by the addition of a peptide mixture (tryptone) to the culture medium. The native protein was obtained in multimeric form, mostly hexameric, under the purification conditions used, and it was characterized as an oligopeptide binding protein, named S. solfataricus OppA (OppASs). OppaASs possesses typical sequence patterns required for glycosylphosphatidylinositol lipid anchoring, resulting in an N-linked glycoprotein with carbohydrate moieties likely composed of high mannose and/or hybrid complex carbohydrates. OppASs specifically binds oligopeptides and shows a marked selectivity for the amino acid composition of substrates when assayed in complex peptide mixtures. Moreover, a truncated version of OppASs, produced in recombinant form and including the putative binding domain, showed a low but significant oligopeptide binding activity

Structural and Functional Insights into Aeropyrum pernix OppA, a Member of a Novel Archaeal OppA Subfamily▿ †

In this study we gain insight into the structural and functional characterization of the Aeropyrum pernix oligopeptide-binding protein (OppAAp) previously identified from the extracellular medium of an Aeropyrum pernix cell culture at late stationary phase. OppAAp showed an N-terminal Q32 in a pyroglutamate form and C-terminal processing at the level of a threonine-rich region probably involved in protein membrane anchoring. Moreover, the OppAAp protein released into the medium was identified as a “nicked” form composed of two tightly associated fragments detachable only under strong denaturing conditions. The cleavage site E569-G570 seems be located on an exposed surface loop that is highly conserved in several three-dimensional (3D) structures of dipeptide/oligopeptide-binding proteins from different sources. Structural and biochemical properties of the nicked protein were virtually indistinguishable from those of the intact form. Indeed, studies of the entire bacterially expressed OppAAp protein owning the same N and C termini of the nicked form supported these findings. Moreover, in the middle exponential growth phase, OppAAp was found as an intact cell membrane-associated protein. Interestingly, the native exoprotein OppAAp was copurified with a hexapeptide (EKFKIV) showing both lysines methylated and possibly originating from an A. pernix endogenous stress-induced lipoprotein. Therefore, the involvement of OppAAp in the recycling of endogenous proteins was suggested to be a potential physiological function. Finally, a new OppA from Sulfolobus solfataricus, SSO1288, was purified and preliminarily characterized, allowing the identification of a common structural/genetic organization shared by all “true” archaeal OppA proteins of the dipeptide/oligopeptide class

Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189± 205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH2SO2F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH2SO2F. Refinement of the crystal model of SsSOD confirmed that a phenylmetha- nesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH2SO2F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD

Adaptive response activated by dietary cis9, trans11 conjugated linoleic acid prevents distinct signs of gliadin-induced enteropathy in mice

Purpose The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor-2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). Methods Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg−1/bw) and indomethacin (15 mg L−1) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg−1/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. Results GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. Conclusions The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice