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Proteolysis-related genes have diverse functions across taxa and have long been considered as key players for intracellular protein turnover. Growing evidence indicates the biological significance of peptidases in degradation, maturation and modulation of bioactive peptides/proteins. By screening T-DNA tagged lines and functional analysis approaches we unraveled the Arabidopsis leucine aminopeptidase (AtLAP2) function in amino acid turnover. Transcriptomics and metabolomics profiling data suggested involvement of AtLAP2 in specific metabolic pathways. Loss-of-function of AtLAP2 resulted in early-leaf senescent and stress-sensitive phenotypes. Our work indicates an important in planta role for AtLAP2 contributing to a further understanding of the proteases having several implications in higher plants.

Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized. In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants. Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant. The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

The adipocyte-derived leucine aminopeptidase (A-LAP)/ER aminopeptidase-1 is a multi-functional enzyme belonging to the M1 family of aminopeptidases. It was reported that the polymorphism Lys528Arg in the human A-LAP gene is associated with essential hypertension. In this study, the role of Lys528 in the enzymatic activity of human A-LAP was examined by site-directed mutagenesis. Among non-synonymous polymorphisms tested, only Lys528Arg reduced enzymatic activity. The replacement of Lys528 with various amino acids including Ala, Met, His and Arg caused a significant decrease in the enzymatic activity. Molecular modeling of the enzyme suggested that Lys528 is located near the entrance of the substrate pocket. These results suggest that Lys528 is important for maximal activity of A-LAP by maintaining the appropriate structure of the substrate pocket of the enzyme. The reduced enzymatic activity of A-LAP may cause high blood pressure and the observed association between the polymorphism and hypertension.

Purpose: Angiotensin (Ang) II was reported to induce vascular endothelial growth factor (VEGF) expression in various cells. Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases. Enzymatic characterization demonstrated that A-LAP hydrolyzes Ang II. This study examined the role of A-LAP in angiogenesis of human endometrial carcinoma., Experimental Design: We investigated whether Ang II induces VEGF expression in human endometrial carcinoma cells. To investigate the possible function of A-LAP in angiogenesis of endometrial carcinoma, we transfected A-LAP cDNA into HEC-1A cells, showing the lowest expression of A-LAP., Results: In the present study, we showed that Ang II enhanced VEGF expression in a dose-dependent manner in endometrial carcinoma cells (HEC-1A cells). Overexpression of A-LAP attenuated Ang II-induced VEGF expression in HEC-1A cells. In addition, Human umbilical vascular endothelial cell migration was increased in conditioned media from Ang II-treated wild-type cells, but not in conditioned media from Ang II-treated A-LAP-overexpressing cells (HEC-1A-A-LAP cells). In an in vivo study, we showed that A-LAP overexpression in endometrial carcinoma cells results in a reduction of VEGF immunoreactivity and the number of blood vessels within tumors., Conclusions: Our study demonstrates that it is feasible to overexpress Ang II-degrading enzymes in cultured cells and that this overexpression attenuated some effects of exogenous and endogenous Ang II. These experiments are a first step toward the development of novel strategies to selectively antagonize locally generated Ang II and suppress VEGF-induced angiogenesis in endometrial carcinoma.

We recently identified two novel aminopeptidases, placental leucine aminopeptidase (P-LAP) and adipocyte-derived leucine aminopeptidase (A-LAP). Enzymatically, P-LAP degrades oxytocin, vasopressin, and angiotensin III, while A-LAP degrades angiotensin II and kallidin. In this study we investigated the expression and localization of P-LAP and A-LAP in human trophoblastic cells in the normal placenta (n = 26), gestational choriocarcinoma (n = 8), and placental site trophoblastic tumor (n = 3). On immunoblot analysis both P-LAP and A-LAP proteins were detected in normal placenta and five choriocarcinoma tissues, as well as in two choriocarcinoma cell lines. Immunohistochemical staining of normal placental tissues demonstrated that P-LAP was not only localized in villous syncytiotrophoblasts but also highly expressed in extravillous trophoblasts (EVTs) invading the decidua or maternal spiral arteries. The expression level of P-LAP on these invasive EVTs reached a maximum during the late first to second trimesters of pregnancy, and it decreased in the third trimester. Similarly, A-LAP was strongly expressed in EVTs invading the decidua or spiral arteries in the second trimester of pregnancy, while it was weakly or moderately expressed in villous cytotrophoblasts or EVTs located in the cell columns. These two aminopeptidases were more strongly expressed in all eight choriocarcinomas and three placental site trophoblastic tumors and mainly localized to the intermediate-type trophoblastic tumor cells invading the uterine myometrium or stromal vessels. In summary P-LAP and A-LAP were predominantly expressed in the invasive phenotype of EVTs during placentation, as well as in the invasive tumor cells of trophoblastic neoplasms. These results suggest the involvement of these aminopeptidases in invasiveness of both normal and malignant intermediate-type trophoblasts possibly through degradation of specific peptide substrates.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel zinc-metallopeptidase involved in angiotensin II (AngII) metabolism, cell migration and antigen presentation. These functions are implicated in the progression of cancer, whereas A-LAP expression and involvement have not been studied in any type of cancer. We investigated the expression of A-LAP in endometrial cancer as well as its association with angiogenesis and clinicopathological features. Immunohistochemical staining of 58 endometrial endometrioid adenocarcinoma specimens revealed that 37 were A-LAP immunoreactive. We also found that A-LAP staining correlated with histological tumor grade in a significant and reverse manner. In addition, serum CA-125 levels in patients with A-LAP positive cancers were significantly higher. However, contrary to our hypothesis that A-LAP suppresses angiogenic activity via AngII metabolism, A-LAP expression was not associated with the microvessel count determined by CD34 immunostaining. Our results suggest that A-LAP is involved in endometrial cancer cell growth and differentiation. However, further studies, especially of the biological roles of A-LAP, are required to confirm this notion., (Copyright 2003 S. Karger AG, Basel)

Precursors to major histocompatibility complex (MHC) class I-presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residues from many antigenic precursors. It was identified as a metallopeptidase named "adipocyte-derived leucine" or "puromycin-insensitive leucine-specific" aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER-aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-gamma. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.