Literature summary for 3.4.11.3 extracted from

Mpakali, A.; Saridakis, E.; Harlos, K.; Zhao, Y.; Papakyriakou, A.; Kokkala, P.; Georgiadis, D.; Stratikos, E.
Crystal structure of insulin-regulated aminopeptidase with bound substrate analogue provides insight on antigenic epitope precursor recognition and processing (2015), J. Immunol., 195, 2842-2851 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene LNPEP, recombinant expression of His-tagged wild-type and mutant enzymes in Spodoptera frugiperda Sf9 cells and Trichoplusia ni Hi5 cells via the baculovirus transfection system	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme free or in complex with inhibitory antigenic peptide precursor analogue N2-(2- 8[(1-amino-3-phenylpropyl)(hydroxy)phosphoryl]methyl]-4-methylpentanoyl)-L-lysyl-L-histidyl-L-histidyl-L-alanyl-L-phenylalanyl-L-seryl-L-phenylalanyl-L-lysine, sitting drop vapor diffusion, mixing of 100 nl of 10 mg/ml protein in 150 mM NaCl and 10 mM HEPES, pH 7.4, with 100 nl of crystallization solution containing 10% w/v PEG 4000, 20% v/v glycerol, 53.4 mM bicine, 46.6 mM Trizma base, pH 8.5, and 0.02 M each of sodium L-glutamate, DL-alanine, glycine, DL-lysine, and DL-serine, method optimization, X-ray diffraction structure determination and analysis at 3.31-3.37 A resolution, molecular replacement using the highly homologous ERAP1 open structure (PDB ID 3QNF) as a search model, and modelling	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant enzyme free or in complex with inhibitory antigenic peptide precursor analogue N2-(2- 8[(1-amino-3-phenylpropyl)(hydroxy)phosphoryl]methyl]-4-methylpentanoyl)-L-lysyl-L-histidyl-L-histidyl-L-alanyl-L-phenylalanyl-L-seryl-L-phenylalanyl-L-lysine, sitting drop vapor diffusion, mixing of 100 nl of 10 mg/ml protein in 150 mM NaCl and 10 mM HEPES, pH 7.4, with 100 nl of crystallization solution containing 10% w/v PEG 4000, 20% v/v glycerol, 53.4 mM bicine, 46.6 mM Trizma base, pH 8.5, and 0.02 M each of sodium L-glutamate, DL-alanine, glycine, DL-lysine, and DL-serine, method optimization, X-ray diffraction structure determination and analysis at 3.31-3.37 A resolution, molecular replacement using the highly homologous ERAP1 open structure (PDB ID 3QNF) as a search model, and modelling

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
A6909T	naturally occuing mutation, polymorphism A609T interacts with the hinge domain III of IRAP, similarly to SNP K528R in ERAP1, which has been repeatedly validated for disease association and effects on activity. The A609T mutation leads to an almost 2fold reduction in activity compared to wild-type	Homo sapiens
I166M	naturally occuing mutation, the I166M polymorphism is located in domain I of the enzyme at the interface with domain II and makes contact with loop 534-538 that is adjacent to the S1 pocket of the enzyme. The mutant enzyme activity is slightly reduced compared to the wild-type	Homo sapiens
additional information	mapping and functional characterization of IRAP disease-associated singel-nucleotide polymorphisms, SNPs. Two coding single-nucleotide polymorphisms in IRAP have been associated with predisposition to the autoimmune diseases psoriasis and ankylosing spondylitis, namely rs2303138 coding for the A609T change and rs61752351 coding for the I166M change	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
N2-(2- 8[(1-amino-3-phenylpropyl)(hydroxy)phosphoryl]methyl]-4-methylpentanoyl)-L-lysyl-L-histidyl-L-histidyl-L-alanyl-L-phenylalanyl-L-seryl-L-phenylalanyl-L-lysine	i.e. DG025, an antigenic peptide precursor analogue, enzyme binding structure determination and analysis. The canonical orientation of the two N-terminal residues of DG025 is defined by the transition state nature of the ligand and the hydrophobic and aromatic interactions of the hPhe residue in the S1 specificity pocket of the enzyme (and in particular with Phe544). The remaining of the peptide extends toward the base of the cavity, where it is sandwiched between domains II and IV	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9UIQ6	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Trichoplusia ni Hi5 cells by dialysis and nickel affinity chromatography	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
dendritic cell	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-leucine-7-amido-4-methylcoumarin + H2O	-	Homo sapiens	L-leucine + 7-amino-4-methylcoumarin	-	?
LGGGGGL + H2O	hydrolysis of poly-glycine peptides, overview	Homo sapiens	L-Leu + GGGGGL	-	?

Synonyms

Synonyms	Comment	Organism
insulin-regulated aminopeptidase	-	Homo sapiens
IRAP	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Homo sapiens

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.0000077	-	pH 7.0, 37°C, recombinant wild-type enzyme	Homo sapiens	N2-(2- 8[(1-amino-3-phenylpropyl)(hydroxy)phosphoryl]methyl]-4-methylpentanoyl)-L-lysyl-L-histidyl-L-histidyl-L-alanyl-L-phenylalanyl-L-seryl-L-phenylalanyl-L-lysine

General Information

General Information	Comment	Organism
physiological function	aminopeptidases generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. These enzymes efficiently process a vast number of different long peptide substrates. Insulin-regulated aminopeptidase prepares antigenic epitopes for crosspresentation in dendritic cells	Homo sapiens