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Pyrococcus horikoshii TET2 Peptidase Assembling Process and Associated Functional Regulation*

  • Author Footnotes
    1 Supported by a Ph.D. scholarship from the French Ministry for Research and Technology.
    Alexandre Appolaire
    Footnotes
    1 Supported by a Ph.D. scholarship from the French Ministry for Research and Technology.
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Eva Rosenbaum
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Author Footnotes
    2 Supported by a French National Research Agency postdoctoral fellowship.
    M. Asunción Durá
    Footnotes
    2 Supported by a French National Research Agency postdoctoral fellowship.
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Matteo Colombo
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Vincent Marty
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Marjolaine Noirclerc Savoye
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Anne Godfroy
    Affiliations
    the Ifremer, UMR6197, Laboratoire de Microbiologie des Environnements Extrêmes, 29280 Plouzané, France
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  • Guy Schoehn
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Eric Girard
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Frank Gabel
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Bruno Franzetti
    Correspondence
    To whom correspondence should be addressed: Institut de Biologie Structurale, 41 Rue J. Horowitz, F-38027 Grenoble Cedex 1, France. Tel.: 0033-4-38-78-95-69; Fax: 0033-4-38-78-95-69;
    Affiliations
    From the Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble and
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  • Author Footnotes
    * This work was supported in part by the Agence Nationale de la Recherche Grants “MacroTET”-BLAN-07-3 204002 and “Archelyse” ANR-12-BSV8-0019-01 and the GRD Ecchis.
    This article contains supplemental Fig. 1.
    1 Supported by a Ph.D. scholarship from the French Ministry for Research and Technology.
    2 Supported by a French National Research Agency postdoctoral fellowship.
Open AccessPublished:May 21, 2013DOI:https://doi.org/10.1074/jbc.M113.450189
      Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.
      Background: TET aminopeptidases are 12-subunit complexes present in the three domains of life and are involved in important biological functions.
      Results: The TET assembling process has been characterized. The oligomerization triggers TET activity toward large polypeptidic substrates.
      Conclusion: The assembling of TET is a controlled process and regulates its activity in vivo.
      Significance: This work provides a new example of peptidase regulation driven by self-oligomerization.

      Introduction

      Efficient and controlled intracellular polypeptide breakdown is a primordial requirement that controls many cellular processes (
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      ). Aminopeptidases constitute a group of enzymes of critical importance to intracellular regulatory networks. In addition to their key role in energy and amino acid metabolism, they contribute in a crucial manner to the protein degradation pathways by trimming the peptides released by ATP-dependent proteases such as the proteasome (
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      ). These enzymes equally assume important roles in specific biological processes involving peptide signaling, such as the production of the major histocompatibility complex ligands (
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      Post-proteasomal and proteasome-independent generation of MHC class I ligands.
      ). Therefore, altered intracellular aminopeptidase activities have been associated with a variety of pathologies, including aging, cataracts, cystic fibrosis, angiogenesis, and cancers (
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      ).
      Numerous intracellular energy-independent peptidases co-exist in the cytosol, but only a few of them share the capacity for self-assembly as large homo-multimeric complexes. These include bleomycin hydrolase, leucine aminopeptidase, DppA, tripeptidyl peptidase II, Tricorn protease, protease1, pab87, and the TET
      The abbreviations used are: TET, tetrahedral aminopeptidase; SAXS, small angle x-ray scattering; AUC, analytical ultracentrifugation; PDB, Protein Data Bank; pNA, p-nitroanilide; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid.
      aminopeptidase (
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      ). All these systems confine the peptidase activity to inner cavities, accessible exclusively to unfolded polypeptides. Although most of them adopt a barrel-shaped architecture in which the active sites are lined up alongside a single central channel, the TET aminopeptidases form unique dodecameric edifices with a typical tetrahedral shape (
      • Franzetti B.
      • Schoehn G.
      • Hernandez J.F.
      • Jaquinod M.
      • Ruigrok R.W.
      • Zaccai G.
      Tetrahedral aminopeptidase: a novel large protease complex from archaea.
      ). The TET particle interior is accessible via the openings situated on each facet of the tetrahedron. The internal organization of TET peptidases revealed a highly self-compartmentalized system comprising a crossing network of four access channels extended by vast catalytic chambers in which three active sites are arranged in a circular fashion (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
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      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
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      • Maury O.
      • Vellieux F.M.
      • Franzetti B.
      • Girard E.
      Using lanthanoid complexes to phase large macromolecular assemblies.
      ). This spatial arrangement is structurally different from all other self-compartmentalized protease complexes of known three-dimensional structure.
      TET cytosolic enzymes belong to the M42 or M18 metallopeptidase families in the clan MH according to the MEROPS classification system (
      • Rawlings N.D.
      • Barrett A.J.
      • Bateman A.
      MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.
      ). The typical TET dodecahedral quaternary structure was initially described in archaea (
      • Franzetti B.
      • Schoehn G.
      • Hernandez J.F.
      • Jaquinod M.
      • Ruigrok R.W.
      • Zaccai G.
      Tetrahedral aminopeptidase: a novel large protease complex from archaea.
      ,
      • Russo S.
      • Baumann U.
      Crystal structure of a dodecameric tetrahedral-shaped aminopeptidase.
      ,
      • Borissenko L.
      • Groll M.
      Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.
      ). It was also found in bacteria (
      • Kim D.
      • San B.H.
      • Moh S.H.
      • Park H.
      • Kim D.Y.
      • Lee S.
      • Kim K.K.
      Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.
      ), and recently, the crystallographic structure of bovine and human tetrahedral aspartyl-aminopeptidases have revealed that the TET complexes are also present in eukaryotic cells (
      • Chen Y.
      • Farquhar E.R.
      • Chance M.R.
      • Palczewski K.
      • Kiser P.D.
      Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase.
      ,
      • Chaikuad A.
      • Pilka E.S.
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      • von Delft F.
      • Kavanagh K.L.
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      • Oppermann U.
      • Yue W.W.
      Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity, and M18 peptidase family.
      ). The quaternary structure of archaeal, bacterial, and eukaryal TET assemblies is highly conserved. Their tertiary structures show that they all exhibit a two-domain architecture consisting of a catalytic and a dimerization domain (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Kim D.
      • San B.H.
      • Moh S.H.
      • Park H.
      • Kim D.Y.
      • Lee S.
      • Kim K.K.
      Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.
      ,
      • Chen Y.
      • Farquhar E.R.
      • Chance M.R.
      • Palczewski K.
      • Kiser P.D.
      Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase.
      ).
      The high evolutionary conservation of TET peptidases in the three kingdoms of life suggests that they perform important biological functions. They were found to process polypeptides up to 27 amino acids in length in the absence of ATP (
      • Franzetti B.
      • Schoehn G.
      • Hernandez J.F.
      • Jaquinod M.
      • Ruigrok R.W.
      • Zaccai G.
      Tetrahedral aminopeptidase: a novel large protease complex from archaea.
      ,
      • Durá M.A.
      • Receveur-Brechot V.
      • Andrieu J.P.
      • Ebel C.
      • Schoehn G.
      • Roussel A.
      • Franzetti B.
      Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.
      ). Three different versions of TET complexes co-exist in the hyperthermophilic archaeon Pyrococcus horikoshii as follows: PhTET1, PhTET2, and PhTET3. These complexes are built up in an identical fashion and have comparable dimensions. PhTET2 can be defined as a leucyl-aminopeptidase that displays a preference for neutral and aliphatic substrates; PhTET3 is a lysyl-aminopeptidase that hydrolyzes preferentially basic residues, and PhTET1 is a glutamyl-aminopeptidase that shows high specificity toward acidic residues (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Receveur-Brechot V.
      • Andrieu J.P.
      • Ebel C.
      • Schoehn G.
      • Roussel A.
      • Franzetti B.
      Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ). The comparison of the surface electrostatic potential features of the proteolytic chambers and of the structures of the active site pockets suggest a mechanism of substrate (N-terminal amino acid) discrimination based on the PhTET internal surface electrostatic potential features (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ).
      The particular substrate specificities of the three TET variants in P. horikoshii suggest that they form a complementary set of enzymes (
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ). Because of their cooperative action, the archaeal TET peptidases can be designated as a “peptidasome” involved in the destruction of a vast variety of polypeptides. It has been suggested that, in peptide-fermenting organisms, the TET system plays an important role in the energy metabolism (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ) or in the intracellular protein degradation by hydrolyzing the peptides produced by the proteasome endopeptidase activity (
      • Borissenko L.
      • Groll M.
      Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.
      ). In addition, the TET peptidases could play more specific physiological roles as they can cleave physiologically relevant peptides. This hypothesis is supported by recent work on the eukaryotic tetrahedral aspartyl aminopeptidase that has been proposed to be a key player in the central nervous system, in particular by regulating the ocular and renin system (
      • Chen Y.
      • Farquhar E.R.
      • Chance M.R.
      • Palczewski K.
      • Kiser P.D.
      Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase.
      ).
      The TET enzymes are co-catalytic metallopeptidases typically binding, by means of five amino acid ligands, two atoms of zinc or cobalt per monomer. The catalytic mechanism also implies a glutamate and an aspartate residue. Cobalt ions have a clear stimulatory effect on the amidolytic activity of PhTETs, and the co-catalytic metals have been found to be important to maintain the PhTET oligomerization state (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Receveur-Brechot V.
      • Andrieu J.P.
      • Ebel C.
      • Schoehn G.
      • Roussel A.
      • Franzetti B.
      Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ,
      • Rosenbaum E.
      • Ferruit M.
      • Durá M.A.
      • Franzetti B.
      Studies on the parameters controlling the stability of the TET peptidase superstructure from Pyrococcus horikoshii revealed a crucial role of pH and catalytic metals in the oligomerization process.
      ). Unlike other self-compartmentalized peptidases, TETs are not processive enzymes that imply the detachment of the peptide moiety from the active site once the N-terminal residue has been cleaved (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Receveur-Brechot V.
      • Andrieu J.P.
      • Ebel C.
      • Schoehn G.
      • Roussel A.
      • Franzetti B.
      Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ,
      • Ando S.
      • Ishikawa K.
      • Ishida H.
      • Kawarabayasi Y.
      • Kikuchi H.
      • Kosugi Y.
      Thermostable aminopeptidase from Pyrococcus horikoshii.
      ). The mechanism of TET hydrolysis is extremely similar to the one of secreted monomeric aminopeptidase such as Vibrio aminopeptidase Ap1; it is the charge properties and the dimensions of the catalytic pocket of each monomer that trigger the specificity of the enzyme toward the N-terminal amino acid from the peptide chain (
      • Chevrier B.
      • Schalk C.
      • D'Orchymont H.
      • Rondeau J.M.
      • Moras D.
      • Tarnus C.
      Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family.
      ). Thus, in the case of TET peptidases, the biological significance for oligomerization and active site self-compartmentalization is not clear. To address this question, a site-directed mutagenesis strategy was used to slow down the natural oligomerization process of the PhTET2 complex. The structural properties of the purified PhTET2 dimer and of various oligomeric form intermediates were characterized by combining small angle x-ray scattering (SAXS), native gel electrophoresis, analytical ultracentrifugation (AUC), and electron microscopy. This allowed the dissection of the TET assembling pathway. The relationship between the aminopeptidase activity and its multimeric structure was also assessed by functional assays. Finally, density gradient fractionations and immunodetection experiments performed with Halobacterium and Pyrococcus cell extracts suggested the existence of a regulatory mechanism controlling the TET oligomerization state in vivo.

      DISCUSSION

      A key question regarding self-compartmentalized peptidase complexes are the mechanisms by which such well organized edifices assemble. In this study, a site-directed mutagenesis strategy has been used to slow down the self-assembling process of TET, a large dodecameric aminopeptidase present in the three domains of life. The disruption of stabilizing interactions located in the interface area between the subunits at the apices of the tetrahedron had a significant effect on the kinetics of the TET assembling process. This way, a stable dimeric species could be purified that was fully able to self-assemble into active dodecamers. SAXS studies showed that the dodecameric particle obtained from the mutant dimer displayed the same stability and quaternary structure as the wild-type TET complex, even under extreme temperatures.
      Native gel electrophoresis, AUC, and electron microscopy analyses proved that the mutant PhTET2 oligomerization does not proceed by an incremental addition of dimers but involves well defined intermediate species that are generated with time from the PhTET2 dimer precursor. Although tetramers and octamers were detected at the beginning of the oligomerization process, they are the products of an alternative pathway induced by the mutations. Based on SAXS analysis of the free PhTET2 dimer, electron microscopy study, and structural modeling from existing crystallographic structures of the PhTETs (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Borissenko L.
      • Groll M.
      Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.
      ), we propose that the natural pathway involves two hexameric intermediates, a tricorn and an open (Z-form) chain complex. Indeed, the tricorn represents a stable oligomeric form in solution. However, the Z-form is able to self-associate, leading to the extended interaction zone involving the catalytic domains of three subunits present in the highly stable biological dodecamers. Consequently, we propose that the open and closed hexamers are in equilibrium and represent the intermediates in the PhTET2 dodecamer assembling process (Fig. 8). The oligomerization interface that is present in the PhTET2 hexamers and dodecamers is well conserved in the three PhTETs (data not shown). This suggests that the oligomerization process and the associated functional activation that we described for PhTET2 may also be valid for PhTET1 and PhTET3.
      Figure thumbnail gr8
      FIGURE 8Oligomerization model of the dodecameric TET particles based on the results obtained by the combination of site-directed mutagenesis, SAXS, AUC, electron microscopy, and structural analysis described here. The dimer constitutes the building block and self-assembles into a closed hexamer, which involves the association of three dimers (red, magenta and blue). Two open “mirror” conformations of this complex are in equilibrium and finally associate to form the super-stable dodecameric particle.
      Self-compartmentalization is a hallmark of cytosolic peptidases that degrade peptides in an unspecific manner (
      • Lupas A.
      • Flanagan J.M.
      • Tamura T.
      • Baumeister W.
      Self-compartmentalizing proteases.
      ). In the proteasome, tricorn protease, and bleomycin hydrolase, the sequestration of the catalytic sites from the bulk environment into chambers represents a peptide filtering system that is necessary to prevent unwanted damage on folded polypeptides within the cytosol (
      • Groll M.
      • Ditzel L.
      • Löwe J.
      • Stock D.
      • Bochtler M.
      • Bartunik H.D.
      • Huber R.
      Structure of 20S proteasome from yeast at 2.4 A resolution.
      ,
      • Brandstetter H.
      • Kim J.S.
      • Groll M.
      • Huber R.
      Crystal structure of the tricorn protease reveals a protein disassembly line.
      ,
      • O'Farrell P.A.
      • Gonzalez F.
      • Zheng W.
      • Johnston S.A.
      • Joshua-Tor L.
      Crystal structure of human bleomycin hydrolase, a self-compartmentalizing cysteine protease.
      ). Oligomerization has also been shown to control different aspects of the peptidase functions. In the 20 S proteasome and its bacterial homologue ClpP, the priming of the peptidase enzymatic activity is coupled with subunit association (
      • Thompson M.W.
      • Miller J.
      • Maurizi M.R.
      • Kempner E.
      Importance of heptameric ring integrity for activity of Escherichia coli ClpP.
      ). In ATP-independent peptidases, such as protease 1 and Pab87, the active sites are formed at the subunit junctions (
      • Du X.
      • Choi I.G.
      • Kim R.
      • Wang W.
      • Jancarik J.
      • Yokota H.
      • Kim S.H.
      Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution.
      ,
      • Delfosse V.
      • Girard E.
      • Birck C.
      • Delmarcelle M.
      • Delarue M.
      • Poch O.
      • Schultz P.
      • Mayer C.
      Structure of the archaeal pab87 peptidase reveals a novel self-compartmentalizing protease family.
      ). In tripeptidyl peptidase II, a giant aminopeptidase found in eukaryotes, the activity increases in a nonlinear fashion with the oligomerization rate (
      • Geier E.
      • Pfeifer G.
      • Wilm M.
      • Lucchiari-Hartz M.
      • Baumeister W.
      • Eichmann K.
      • Niedermann G.
      A giant protease with potential to substitute for some functions of the proteasome.
      ), and in the case of bovine lens leucyl aminopeptidase, it has been suggested that the activity depends on the stabilization of each monomer catalytic site by the structure of the oligomer (
      • Burley S.K.
      • David P.R.
      • Taylor A.
      • Lipscomb W.N.
      Molecular structure of leucine aminopeptidase at 2.7-A resolution.
      ). On the contrary, in α-1 tryptase or kallikrein-related peptidases, self-oligomerization provides an inhibition of the enzyme activity (
      • Marquardt U.
      • Zettl F.
      • Huber R.
      • Bode W.
      • Sommerhoff C.
      The crystal structure of human α1-tryptase reveals a blocked substrate-binding region.
      ,
      • Debela M.
      • Magdolen V.
      • Grimminger V.
      • Sommerhoff C.
      • Messerschmidt A.
      • Huber R.
      • Friedrich R.
      • Bode W.
      • Goettig P.
      Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc-binding site.
      ). With respect to these findings, it was surprising to find out that the dimeric form of the TET complex still carries out the same catalytic activity on small peptides as the 12-subunit complex. This indicates that, in the TET dimers, the catalytic sites and pockets are already positioned in proteolytically active conformations. The SAXS structure of the free dimer is consistent with these findings, because it shows that the relative positions of the two monomers of each dimer within the tetrahedral complex are already imposed by the interactions between dimerization domains. In the case of this small peptide, the calculated Km values are similar, meaning that the substrate is recognized by the dimer and the dodecamer equally well. The similar calculated kcat value reflects that the active site remains unchanged upon oligomerization. Thus, in the case of PhTET2, the oligomerization does not affect the active site and catalytic pocket configurations and has little effect on the recognition and trimming of N-terminal amino acids. However, when studying the catalytic activity of TET dimers and dodecamers as a function of substrate length, we found that the dodecamers are more efficient in hydrolyzing long polypeptide substrates as compared with the dimers. The kinetic parameters indicated that the dodecamer possesses a better efficiency than the dimer toward long substrates as follows: the kcat value of the amidohydrolytic reaction, reflecting the catalytic efficiency of the system, is considerably reduced for the free dimer, although the Km value, reflecting the affinity of the active site and the catalytic pocket for the N-terminal amino acid, remains the same between the dimer and the dodecamer. Thus, during the reaction, the same number of peptidase-substrate complexes are formed, but these complexes are more productive in the case of the dodecamer. In contrast, the peptidase-substrate complexes formed by the dimer are unproductive and are not true Michaelis-Menten complexes and therefore cannot be turned over. Therefore, in the case of the PhTET complex, the self-compartmentalization provides a way to enhance the enzyme efficiency when the substrate size increase. This represents another type of peptidase functional regulation driven by self-oligomerization. The interior of the TET peptidases consists of four polypeptide navigation channels that cross the particles from the entry pores situated on the facets of the tetrahedrons toward the catalytic chambers located within each apex (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ). A model for polypeptide processing was proposed based on the structural and enzymatic comparisons of the three TET enzymes from P. horikoshii. In this model, a series of mobile loops and electrostatic attractions/repulsions in the entry channels and in the catalytic chambers would orient the N terminus of the polypeptides toward the negatively charged active sites (
      • Schoehn G.
      • Vellieux F.M.
      • Asunción Durá M.
      • Receveur-Bréchot V.
      • Fabry C.M.
      • Ruigrok R.W.
      • Ebel C.
      • Roussel A.
      • Franzetti B.
      An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron.
      ,
      • Durá M.A.
      • Rosenbaum E.
      • Larabi A.
      • Gabel F.
      • Vellieux F.M.
      • Franzetti B.
      The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea.
      ). In unassembled TET dimers, the polypeptide navigation system is not present. This would explain why the TET dimer is disabled to break down long polypeptides.
      P. horikoshii is a hyperthermophilic microorganism that grows optimally at 95 °C (
      • González J.M.
      • Masuchi Y.
      • Robb F.T.
      • Ammerman J.W.
      • Maeder D.L.
      • Yanagibayashi M.
      • Tamaoka J.
      • Kato C.
      Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough.
      ). In this work, it has also been shown that, in vitro, the free dimers are as stable at physiological extreme temperatures as the assembled TET particles, with half-lives of several hours at 80 °C. Different strategies are adopted by thermozymes to stabilize their native conformation at extreme temperatures (
      • Jaenicke R.
      • Böhm G.
      The stability of proteins in extreme environments.
      ). Among these, oligomerization has been proposed to represent an important determinant (
      • Clantin B.
      • Tricot C.
      • Lonhienne T.
      • Stalon V.
      • Villeret V.
      Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.
      ). In the case of the TET system, our results show that the dodecameric quaternary structure has little role in the high thermal stability of the enzyme. In fact, the monomer-monomer interfaces in the TET dimer consist of extended networks of ionic bonds that are likely to contribute to the high thermal stability of the enzyme as a free dimer. Therefore, dimers can be accumulated as stable precursors of the TET dodecamers in vivo.
      There is little information available about the in vivo oligomeric states of large energy-independent peptidase complexes. We showed here that the PhTET2 dimer co-exists with the dodecamer in cellular extracts. A similar observation was also made under hypersaline conditions in the extreme halophilic archaeon H. salinarum. Unlike Pyrococcus, the Halobacterium genome contains only one copy of the TET peptidase. Thus, it is reasonable to propose that the dimer-dodecamer equilibrium is a hallmark for TET peptidases and that a specific regulation occurs in vivo to control the oligomerization state of TET. Because oligomerization affects PhTET2 activity with respect to the size of the polypeptidic substrates, a regulated oligomerization would therefore allow the TET particle to process a broad variety of peptides, from dipeptides to long polypeptides, in response to varying physiological demands; although the TET dimer would act preferentially on small peptides for energetic and anabolic purposes, its assembly into dodecamers and the concomitant formation of the polypeptide navigation system would trigger the intracellular aminopeptidase activity toward longer peptides such as those produced by the 20 S proteasome or toward peptides possessing specific biological activities. Thus, the control of the amount of assembled TET in vivo may represent an important regulatory step in protein degradation and in many specific biological functions based on polypeptide activity. Regarding that, in vitro, the TET oligomerization appears to be a rapid process, and given that the assembled TET particles are extremely robust, it is likely that the in vivo regulatory mechanisms involve the stabilization of the dimeric species. Studies are in progress in our laboratory to identify this mechanism.

      Acknowledgments

      We thank A. Le Roy and C. Ebel from the PSB/IBS platform for the assistance and access to the instrument of analytical ultracentrifugation. We thank D. Fenel and C. Moriscot from the PSB/IBS for the electron microscopy. We thank J. Le Bars for help in P. horikoshii cultivation. This work used the platforms of the Grenoble Instruct Center, UMS 3518 CNRS-CEA-UJF-EMBL.

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