M1 Family Of Aminopeptidases Research Articles

Asparatic Acid 221 Is Critical in the Calcium-induced Modulation of the Enzymatic Activity of Human Aminopeptidase A

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.

Essential role of placental leucine aminopeptidase in gynecologic malignancy

The M1 aminopeptidase family is important for the maturation or degradation of bioactive peptides by hydrolyzing their N-terminal amino acids. Some investigators have studied aminopeptidase in the maintenance of homeostasis including maintenance of normal pregnancy, memory retention, blood pressure regulation and antigen presentation. However, there are a few reports on the relation between the M1 aminopeptidase family and carcinoma. In addition to its capacity to degrade a range of peptides, placental-leucine aminopeptidase (P-LAP) has novel functions that impact on normal cells and neoplastic cells. P-LAP is the focus of this review.

A phase 1 dose finding study of CHR-2797, an inhibitor of M1 aminopeptidases, in patients with advanced solid tumours

3053 Background: CHR-2797 is a novel, orally bioavailable inhibitor of the M1 family of aminopeptidases, in particular PuSA, and LTA4 hydrolase. Exposure of cancer cells to CHR-2797 results in the generation of the active metabolite CHR-79888 which is poorly membrane-permeable, resulting in intracellular accumulation. CHR-2797 has shown anti-proliferative activity in syngeneic (rat) and xenograft (mouse) cancer models, and is anti-angiogenic in vitro. Methods: Patients (pts) (ECOG PS ≤ 2) with histologically confirmed advanced solid tumors resistant or refractory to standard therapy were eligible. The accelerated titration design of the trial involved two phases for evaluation of schedule and dose: 1) fixed dose with increasing duration, and 2) dose escalation over a fixed duration (28 days). Results: 16 pts (median age: 64.5 years [range 48.8–80.1], 13M/3F)were treated with once daily doses ranging from 10mg to 130mg. The first four patients received a 10mg dose for 7, 14, 21 or 28 days respectively. Five subsequent cohorts received 28 days continuous dosing, with dose doubling in single patient cohorts until drug-related toxicity ≥ Grade 2. Thereafter dose was escalated in ≤ 40% increments in 3-patient cohorts. The most frequent adverse events were: gr 1–2 thrombocytopenia (44%), gr 1 diarrhea (25%), gr 1–2 transaminitis (19%), gr 1–2 fatigue (13%), gr 1 hot flushes (13%), and gr 1 lightheadedness (13%). There were no DLTs. Five patients continued therapy after 28 days; stable disease has been achieved in 1 pt for 6 months with granulosa cell carcinoma of ovary, who was progressing prior to study entry. CHR-2797 and CHR-79888 demonstrate dose proportional increases in AUC and Cmax. The terminal half-life for CHR-2797 is around 1–2 hours, whereas it is between 9 and 11 hours for CHR-79888. Intracellular (packed blood cells) exposure to both CHR-2797 and CHR-79888 is good, with CHR-79888 accumulating over 28 days, such that by day 28 the intracellular levels are comparable to plasma. Conclusions: CHR-2797 is well tolerated and can be safely administered at doses that reach plasma concentrations associated with activity in pre-clinical models. Accrual into the study continues. No significant financial relationships to disclose.

Physiological roles of M1 aminopeptidase family

Physiological roles of M1 aminopeptidase family

Cloning and characterisation of a Heterodera glycines aminopeptidase cDNA

An aminopeptidase full-length cDNA ( Hg-amp-1) was cloned from the adult female soybean cyst nematode Heterodera glycines by heterologous screening of a cDNA library with a Caenorhabditis elegans EST sequence. The predicted open reading frame encoded an 882-amino acid protein containing the conserved zinc-binding domain and GAMEN motif that are characteristic of M1 family aminopeptidases. The putative protein lacks any subcellular targeting signals and displays strong similarity to puromycin-sensitive aminopeptidases from C. elegans, Drosophila and mammals. Hg-amp-1 is expressed in juvenile nematodes and both male and female adults, with highest expression in gravid females. In situ mRNA hybridisation localised the Hg-amp-1 transcript to the genital primordium of pre-parasitic juvenile nematodes and the reproductive tract of adult females. Suppression of Hg-amp-1 transcript level by RNA-interference led to a 61% reduction in the number of female nematodes parasitising soybean roots 21 days post infection with infective juvenile nematodes that had been exposed to double-stranded RNA.

Metal ion modulation of cystinyl aminopeptidase

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

Membrane Bound Members of the M1 Family: More Than Aminopeptidases

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.

A mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase is expressed in endothelial cells and plays an important role in angiogenesis.

Using polymerase chain reaction-coupled subtractive hybridization, we have isolated genes expressed during the in vitro differentiation of murine embryonic stem cells into endothelial cells (ECs). Among the genes obtained, we identified one gene that was inducible by vascular endothelial growth factor (VEGF) in the murine EC line MSS31. Analysis of the nucleotide and deduced amino acid sequences revealed that the protein was composed of 930 amino acids, including an HEXXH(X)18E consensus sequence of the M1 aminopeptidase family, and is thought to be a mouse orthologue of puromycin-insensitive leucyl-specific aminopeptidase (mPILSAP). The recombinant protein hydrolyzed N-terminal leucyl and methionyl residues from synthetic substrates. Immunohistochemical analysis revealed that mPILSAP was expressed in ECs during postnatal angiogenesis. Specific elimination of mPILSAP expression by antisense oligodeoxynucleotide (AS-ODN) attenuated VEGF-stimulated proliferation, migration, and network formation of ECs in vitro. Moreover, AS-ODN to mPILSAP inhibited angiogenesis in vivo. These results suggest a novel function of mPILSAP, which is expressed in ECs and plays an important role in angiogenesis.