Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 A resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like beta-structure domain; a thermolysin-like catalytic domain; a small barrel-like beta-structure domain; and an alpha-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn(2+)-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions.
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PMID:Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations. 1589 68

Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.
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PMID:Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics: new insights into relationship between conformational flexibility and hydrogen bonding. 1918 63

A total of 49 protein sequences of alkaline proteases retrieved from GenBank representing different species of Aspergillus have been characterized for various physiochemical properties, homology search, multiple sequence alignment, motif, and super family search and phylogenetic tree construction. The sequence level homology was obtained among different groups of alkaline protease enzymes, viz alkaline serine protease, oryzin, calpain-like protease, serine protease, subtilisin-like alkaline proteases. Multiple sequence alignment of alkaline protease protein sequence of different Aspergillus species revealed a stretch of conserved region for amino acid residues from 69 to 110 and 130-204. The phylogenetic tree constructed indicated several Aspergillus species-specific clusters for alkaline proteases namely Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus. The distributions of ten commonly observed motifs were analyzed among these proteases. Motif 1 with a signature amino acid sequence of 50 amino acids, i.e., ASFSNYGKVVDIFAPGQDILSCWIGSTTATNTISGTSMATPHIVGLSCYL, was uniformly observed in proteases protein sequences indicating its involvement with the structure and enzymatic function. Motif analysis of acidic proteases of Aspergillus and bacterial alkaline proteases has revealed different signature amino acid sequences. The superfamily search for these proteases revealed the presence of subtilases, serine-carboxyl proteinase, calpain large subunit, and thermolysin-like superfamilies with 45 representing the subtilases superfamily.
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PMID:In silico characterization of alkaline proteases from different species of Aspergillus. 2207 40

Reversible as well as stereo- and chemoselective: various proteases such as thermolysin and chymotrypsin catalyze amine acyl exchange in peptides. This acyl exchange can be used to modify amino-functionalized surfaces under physiological reaction conditions and provides an alternative mechanism for posttranslational transpeptidation reactions such as peptide-splicing reactions in the proteasome.
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PMID:Enzymatic amine acyl exchange in peptides on gold surfaces. 2316 84

The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities.
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PMID:Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome. 3028 96


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