Tricorn Protease Research Articles

Capsids of Tricorn Protease Studied by Electron Cryomicroscopy

Tricorn protease from the archaeon Thermoplasma acidophilum acts “downstream” of the proteasome; in conjunction with its aminopeptidase cofactors it converts peptides generated by the proteasome into free amino acids. The basic functional unit of Tricorn is a homohexamer of the 121-kDa subunit, 20 of which can assemble further to form an icosahedral capsid with a molecular mass of 14.6 MDa. We have used electron cryomicroscopy to determine the structure of the Tricorn capsids to a resolution of 1.3 nm.

β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain β-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed β-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the β-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.

Beta-propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection.

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.

The Role of Tricorn Protease and Its Aminopeptidase-Interacting Factors in Cellular Protein Degradation

Tricorn protease was previously described as the core enzyme of a modular proteolytic system displaying multicatalytic activity. Here we elucidate the mode of cooperation between Tricorn and its interacting factors, and we identify two additional factors, F2 and F3, closely related aminopeptidases of 89 kDa. In conjunction with these three factors, Tricorn degrades oligopeptides in a sequential manner, yielding free amino acids. We have been able to reconstitute a proteolytic pathway comprising the proteasome, Tricorn, and its interacting factors, F1, F2, and F3, which converts proteins efficiently into amino acids. Therefore, it is quite likely that Tricorn also acts in vivo downstream of the proteasome and, in cooperation with its interacting factors, completes protein catabolic pathways.

A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast

One eukaryotic proteolytic complex – the proteasome – is classed as the major nonlysosomal protease, by its known and suspected functions, its size and its complexity [1,2]. It seems improbable that other enzymes may be capable of substituting, even partially, for the potent proteasome, as this complex has a vital role in many cellular processes [1–3]. Nevertheless, it is possible to adapt cultured EL-4 mouse lymphoma cells to survive in the presence of a specific inhibitor of the proteasome [4]. The inhibition of the proteasome in these adapted EL-4 cells is accompanied by a dramatic increase in the activity of a new, as yet uncharacterized, large proteolytic complex [4]. Here, we have presented evidence that a similar proteolytic activity is constitutively present in fission yeast, Schizosaccharomyces pombe, and that the yeast and mouse enzymes share basic physicochemical properties. We have shown that the S. pombe protease is found in two stable oligomeric forms, both of which are peptidases, although only the larger form acts as a proteinase. The relative amounts of the large and the small forms of the protease in the complex depended on the growth phase of the yeast culture and affected enzyme activity, suggesting that the activity of the enzyme is regulated by its oligomerization status. We refer to the new proteolytic complex as the ‘multicorn’ to indicate its analogy to the archaebacterial tricorn protease [5].

Tricorn Protease Exists as an Icosahedral Supermolecule In Vivo

Tricorn protease is the core enzyme of a recently discovered modular proteolytic system. We present evidence that tricorn protease exists in vivo in the form of a higher-order assembly, namely as an icosahedral capsid. Its size exceeds that of many virus particles and represents by far the largest known homooligomeric enzyme complex. Each capsid is built from 20 copies of the tricorn hexameric toroid and thus has a molecular weight of 14.6 MDa. Three-dimensional reconstructions of ice-embedded capsids from electron micrographs show that it is hollow and has large void volumes in its wall. We suggest that the tricorn capsid, in addition to its intrinsic proteolytic activity, serves as the organizing center of a multienzyme complex.

Tricorn protease (TRI) interacting factor 1 from Thermoplasma acidophilum is a proline iminopeptidase

Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H-Pro-AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N-terminus. The pip gene encodes a 293 amino acid residue protein with a molecular mass of 33487 Da. By means of site-directed mutagenesis we identified Ser 105 and His 271 as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.

Tricorn protease--the core of a modular proteolytic system.

Large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. A tricorn-shaped protease was isolated from the archaeon Thermoplasma and was shown to form a multisubunit proteolytic complex. The 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities.