Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass Eur. J. Biochem. 183, 471 -4771. A study of the specificity of S. griseus aminopeptidase using amino acid 4-nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4-nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa-proline N-terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate-dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1 ,lo-phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4-nitroanilide at a 10-fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4-nitroanilide at a more than 20-fold faster rate than the zinc or manganese enzymes.Streptomyces griseus aminopeptidase (S. griseus AP) is a heat-stable and a low-molecular-mass extracellular bacterial enzyme, distinct from the high-molecular-mass cytosolic (e. g. leucine aminopeptidase) or membrane-bound (e. g. aminopeptidase N) mammalian aminopeptidases [l -51. The complete amino acid sequence and three-dimensional structure of S. griseus AP are still not known. The enzyme is a zinc metalloenzyme inhibitable by 1 ,lo-phenanthroline, EDTA or EGTA and it can bind CaZ+ with a concomitant increase in its activity [4]. S. griseus AP rapidly hydrolyzes N-terminal hydrophobic amino acids like Leu. Due to the unique physicochemical properties of S. griseus AP and its high enzymic reactivity, it has been used as an effective additive in twostage assays of yet other peptidases, neutral endopeptidase and thermolysin [6, 71. In this communication, we investigated the specificity of S. griseus AP and the modulation of its activity by Ca2+ binding and by transition-metal-ion substitution. The spectrum of activities observed with the different substrates and the way by which these activities are influenced by metal ions

Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2C␣, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2C␣ expression is regulated by a tetracycline-inducible promoter, PP2C␣ overexpression led to G 2 /M cell cycle arrest and apoptosis. Furthermore, PP2C␣ induced the expression of endogenous p53 and the p53-responsive gene p21. Activation of the p53 pathway by PP2C␣ took place both in cells harboring endogenous p53, as well as in p53-null cells transfected with exogenous p53. Induction of PP2C␣ resulted in an increase in the overall levels of p53 protein as well as an augmentation of p53 transcription activity. The dephosphorylation activity of PP2C␣ is essential to the described phenomena, as none of these effects was detected when an enzymatically inactive PP2C␣ mutant was overexpressed. p53 plays an important role in PP2C␣-directed cell cycle arrest and apoptosis because perturbation of p53 expression in human 293 cells by human papillomavirus E6 led to a significant increase in cell survival. The role of PP2C␣ in p53 activation is discussed.

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca 2ϩ -binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca 2ϩ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner. Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k cat / K m). Large enhancements in affinity (1/Ki) upon Ca 2ϩ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

The serine/threonine phosphatase type 2C (PPM1A) has a broad range of substrates, and its role in regulating stress response is well established. We have investigated the involvement of PPM1A in the survival and differentiation processes of PC6-3 cells, a subclone of the PC12 cell line. This cell line can differentiate into neuron like cells upon exposure to nerve growth factor (NGF). Overexpression of PPM1A in naive PC6-3 cells caused cell cycle arrest at the G2/M phase followed by apoptosis. Interestingly, PPM1A overexpression did not affect fully differentiated cells. Using PPM1A overexpressing cells and PPM1A knockdown cells, we show that this phosphatase affects NGF signaling in PC6-3 cells and is engaged in neurite outgrowth. In addition, the ablation of PPM1A interferes with NGF-induced growth arrest during differentiation of PC6-3 cells.

Protein phosphatase magnesium dependent 1A (PPM1A) has been implicated in fibrosis and skin wounding. We generated PPM1A knockout mice to study the role of PPM1A in the wound healing-inflammation-angiogenesis cross talk. The role of PPM1A in these processes was studied using the ocular alkali burn model system. In the injured cornea the absence of PPM1A led to enhanced inflammatory response, stromal keratocyte transactivation, fibrosis, increased p38 mitogen-activated protein kinase phosphorylation, elevated expression of transforming growth factor-β-related genes (including Acta2, TGF-β, Col1, MMP9, and VEGF) and subsequently to neovascularization. Augmented angiogenesis in the absence of PPM1A is a general process occurring in vivo in PPM1A knockout mice upon subcutaneous Matrigel injection and ex vivo in aortic ring Matrigel cultures. Using primary keratocyte cultures and various experimental approaches, we found that phospho-p38 is a favored PPM1A substrate and that by its dephosphorylation PPM1A participates in the regulation of the transforming growth factor-β signaling cascade, the hallmark of inflammation and the angiogenic process. On the whole, the studies presented here position PPM1A as a new player in the wound healing-inflammation-angiogenesis axis in mouse, reveal its crucial role in homeostasis on injury, and highlight its potential as a therapeutic mediator in pathologic conditions, such as inflammation and angiogenesis disorders, including cancer.