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PubMed1989

Molecular analysis of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger.

Nucleotide sequence of a 906-bp fragment of Streptomyces alboniger DNA containing the gene (pac), which encodes a puromycin N-acetyltransferase (PAC), has been determined. The pac gene contains a 600-nt open reading frame, starting with an ATG codon, which encodes a polypeptide of Mr 21,531; this is consistent with the 23 +/- 1.5 kDa size of the PAC enzyme. High-resolution S1 mapping indicates that transcription starts at or next to a C residue 35 bp upstream from the putative ATG start codon. A 263-bp DNA fragment from the 5' region of the pac gene has promoter activity in the promoter-probe plasmid pIJ486. Its -35 and -10 regions show significant structural homology to the corresponding regions of the hyg gene promoter, but they are different from the promoter sequences of other Streptomyces and Escherichia coli genes.

Acetyltransferases↗
PubMed1992

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.

Acetyltransferases↗
PubMed2004

Transcriptional regulation of angiogenesis-related puromycin-insensitive leucyl-specific aminopeptidase in endothelial cells.

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) was expressed in endothelial cells (ECs) and played an important role in angiogenesis. Here, we characterized its transcriptional regulation. Mouse PILSAP gene contained 19 exons and located in the chromosome 13C1-C2. We identified two transcripts; one transcribed from exon 1 and the other from exon 2. Mouse ECs expressed dominantly the one from exon 1. The promoter analysis using 5' upstream region of exon 1 revealed that -1868 to -1812 was critical for its transcription in mouse ECs. We identified a motif of the transcription factor PEBP2 in this region, and the deletion or mutation of this motif decreased promoter activity. Protein extracted from mouse ECs bound specifically to this motif. AML1/Runx1/PEBP2alphaB increased PILSAP mRNA in mouse ECs, whereas dominant interfering chimerical PEBP2beta-MYH11 decreased it. These results indicate that the expression of PILSAP in mouse ECs is regulated, at least in part, by PEBP2.

Animals↗
PubMed2006

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

Aminopeptidases↗
PubMed2003

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain.

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.

Aminopeptidases↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1997

Cloning of the human puromycin-sensitive aminopeptidase and evidence for expression in neurons.

The puromycin-sensitive aminopeptidase (PSA) is thought to contribute to the degradation of enkephalins. Besides being the most abundant aminopeptidase in the brain, PSA is expressed in other organs as well. From a human fetal brain cDNA library, we have isolated a cDNA encoding the human PSA (huPSA) protein. The isolated cDNA gave rise to a protein with a molecular mass of 99 kDa. Compared with mouse PSA, homology at the amino acid and cDNA level was 98 and 93%, respectively. Translation of the huPSA was found to be initiated at the second of two possible start codons, as shown by studies with antibodies directed against peptide sequences of both potential N-terminal regions. Northern blot analysis with RNA isolated from different human organs demonstrated that the huPSA transcript is strongest but not exclusively expressed in the brain. Vesicular stomatitis virus epitope-tagged huPSA protein was expressed in HeLa cells and found to be localized in the cytoplasm, especially in the perinuclear region. By in situ hybridization, huPSA transcript could be identified in cortical and cerebellar neurons, whereas glial cells and blood vessels remained negative.

Amino Acid Sequence↗
PubMed2004

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana.

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.

Amino Acid Sequence↗
PubMed2006

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is required for the development of vascular as well as hematopoietic system in embryoid bodies.

We have shown that puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is required for regulation of angiogenesis. However, it remains unclear whether PILSAP plays a role in endothelial cell (EC) differentiation. We examined the role of PILSAP by using an embryoid bodies (EBs) culture system. Fms-like tyrosine kinase-1 (Flk-1) showed two expression peaks on days 4 and 10 of culture. These two peaks represent populations of mesodermal precursors and mature ECs, respectively. Endothelial markers such as VE-cadherin, CD34, CD31 and Tie2 followed the first peak of Flk-1. Interestingly, the expression of PILSAP showed a pattern similar to that of Flk-1. ES cells transfected with mutant PILSAP (mtPILSAP) cDNA of a dominant negative activity organized less vascular structure and showed decreased levels of vascular lineage markers. The similar results were obtained in EBs treated with leucinethiol, a specific inhibitor of leucine aminopeptidase or siRNA for PILSAP. However, Flk-1 expression was unaffected on day 4. The expression of markers for hematopoietic lineage and muscle cells in mtPILSAP-EBs was also reduced. These results suggest that although PILSAP may not function in the initial generation of Flk-1 positive mesodermal precursors, it dose play a role in growth of vascular, hematopoietic, and muscular lineage population from those precursors.

Animals↗
PubMed2001

Human puromycin-sensitive aminopeptidase: cloning of 3' UTR, evidence for a polymorphism at a.a. 140 and refined chromosomal localization to 17q21.

Puromycin-sensitive aminopeptidase is a predominantly cytoplasmatic zinc-dependent exopeptidase. Its physiological function is not known to date. Here we report data on tissue distribution, a polymorphism within the coding region and the complete 3' UTR. The gene (NPEPPS alias PSA) was physically mapped to chromosome 17q21.2-->q21.32 using fluorescence in situ hybridization.

3' Untranslated Regions↗
PubMedDate not supplied

Purification and immunohistochemical localization of a 17-kD porcine renal puromycin aminonucleoside binding protein.

We have purified a 17-kD puromycin aminonucleoside (PAN) binding protein from porcine kidney and identified it as the reported 17-kD protein kinase C inhibitor on the basis of its partial amino acid sequence. Of 54 determined amino acid sequences of the 17-kD porcine renal protein, 51 residues were identical to those of the 17-kD bovine brain protein kinase C inhibitor. However, our purified protein did not carry the inhibitory activity on protein kinase C. Immunohistochemical studies showed a unique intrarenal distribution of the 17-kD PAN-binding protein at the apical side of epithelial cells of Henle's loops in the inner medulla and of distal convoluted tubules. Immunoblot analysis revealed that the 17-kD PAN-binding protein was extractable by an isotonic buffer without sodium deoxycholate extraction. These results suggest that this protein binds loosely to the apical membranes of epithelial cells of Henle's loops and distal tubules and has specific functions related to tubular functions of these nephron segments at the apical side. Whether this protein is a real inhibitor of protein kinase C or not remains to be investigated.

Amino Acid Sequence↗
PubMed2004

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis.

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) plays an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs). Here we characterize the mechanism by which PILSAP regulates the vascular endothelial growth factor (VEGF)-stimulated proliferation of ECs. The specific elimination of PILSAP expression or its enzymatic activity inhibited VEGF-stimulated G1/S transition in ECs. This G1 arrest correlated with reduced cyclin dependent kinase 4/6 (CDK4/6) activity and retinoblastoma (Rb) protein phosphorylation. Analyses of signaling molecules upstream of CDK4/6 revealed that S6 kinase (S6K) activation was affected by PILSAP, whereas that of phosphatidylinositol-3 kinase (PI3K), Akt, and extracellular signal-related kinase 1/2 (ERK1/2) was not. We further demonstrated that PILSAP bound phosphatidylinositol-dependent kinase 1 (PDK1) and removed 9 amino acids from its N-terminus, which allowed S6K to associate with PDK1 and PILSAP upon VEGF stimulation. We constructed mutant PILSAP, which lacked the aminopeptidase activity but bound PDK1. Mutant PILSAP abrogated S6K activation upon VEGF stimulation in a dominant-negative manner. An N-terminal truncated form of PDK1 abolished the dominant-negative effect of mutant PILSAP. Finally, the introduction of a mutated PILSAP gene in ECs inhibited angiogenesis and retarded tumor growth in vivo. These results indicate that PILSAP plays a crucial role in the cell cycle progression of ECs and angiogenesis via the binding and modification of PDK1.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Male reproductive defects caused by puromycin-sensitive aminopeptidase deficiency in mice.

Male reproductive performance is composed of two principal elements, copulation and spermatogenesis. A wealth of literature has described the intricate web of endocrine events underlying these biological processes. In the present study we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice are infertile, lack copulatory behavior, and have impaired spermatogenesis. The reproductive deficits of the mutants are not restored by androgen administration, although no aberrant localization of the sex steroid receptors was detectable in their brains and testes. Considering the strong expression of the Psa gene in the brain and Sertoli cells and the degenerative morphology of Sertoli cells in Psa-deficient mice, Psa may participate in testosterone-mediated reproductive signal pathways in the brain and testis.

Aminopeptidases↗
PubMed2001

Puromycin-sensitive aminopeptidase is essential for the maternal recognition of pregnancy in mice.

Maternal recognition of pregnancy in rodents requires semicircadian surges of hypophyseal PRL secretion during early gestation, which are required for the formation of the corpus luteum of pregnancy (CLP). Here we show that puromycin-sensitive aminopeptidase (Psa)-deficient mice display female infertility that results from impaired formation of CLP. Transplantation of mutant ovaries into normal females restored fertility but not vice versa. Psa-deficient females revealed no semicircadian surges of PRL induced after mating stimuli. Pregnancy in the mutant females was restored by grafting intact pituitaries to elevate circulating levels of PRL. Psa is thus required for the appearance of the semicircadian surges of PRL secretion that are crucial for maintaining pregnancy in rodents.

Aminopeptidases↗
PubMed1999

Increased anxiety and impaired pain response in puromycin-sensitive aminopeptidase gene-deficient mice obtained by a mouse gene-trap method.

A mouse mutation, termed goku, was generated by a gene-trap strategy. goku homozygous mice showed dwarfism, a marked increase in anxiety, and an analgesic effect. Molecular analysis indicated that the mutated gene encodes a puromycin-sensitive aminopeptidase (Psa; EC 3. 4.11.14), whose functions in vivo are unknown. Transcriptional arrest of the Psa gene and a drastic decrease of aminopeptidase activity indicated that the function of Psa is disrupted in homozygous mice. Together with the finding that the Psa gene is strongly expressed in the brain, especially in the striatum and hippocampus, these results suggest that the Psa gene is required for normal growth and the behavior associated with anxiety and pain.

Aminopeptidases↗