Daniella Ben-Meir

Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction.

A sensitive two‐stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3‐carboxypropanoyl‐alanyl‐alanyl‐leucine‐4‐nitroanilide supplemented with Streptomyces griseus amino‐peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25°C) with a catalytic efficiency (k cat=1.2 × 102 s−1, K m=0. 15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (k cat=1.2 × 103 s−1) but the overall efficiency is diminished by a higher K m (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidase and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.

Protein phosphatase 2Cα expression in normal human tissues: an immunohistochemical study

Abstract. Protein phosphatase (PP2Cα) is a member of the mammalian serine threonine-specific protein phosphatases family. We produced monoclonal antibodies against the recombinant PP2Cα and evaluated the immunoreactivity of normal human tissues. The reactivity was strong in normal skin, the digestive tract, lung, kidney, breast, prostate, endocrine glands, and brain, while it was moderate in the ovary, testis, and liver. Epithelial cells revealed high levels of PP2Cα expression, but stromal cells, including fibroblasts and endothelial cells, showed no or little PP2Cα expression. Given the broad reactivity in endocrine and secreting epithelial cells, we propose that PP2Cα expression might contribute to secretory cell function.

Protein Phosphatase Magnesium Dependent 1A (PPM1A) Plays a Role in the Differentiation and Survival Processes of Nerve Cells

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.

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases.

Tripeptide derivatives like 3‐carboxypropanoylalanyl‐alanyl‐leucine 4‐nitroanilide or 3‐carboxypropanoylalanyl‐alanyl‐phenylalanine 4‐nitroanilide are very sensitive substrates for neprilysin (k cat > 102 s−1; k cat/K m ≥ 106 s−1 · M−1) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k cat ∼ 1s−34 s−1). By substituting the N‐terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2–3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub‐site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

De novo SYNTHESIS OF PROTEIN PHOSPHATASE 1A, MAGNESIUM DEPENDENT, ALPHA ISOFORM (PPM1A) DURING OOCYTE MATURATION

Oocyte maturation in mammals is a multiple-stage process that generates fertilizable oocytes. Ovarian oocytes are arrested at prophase of the first meiotic division characterized by the presence of a germinal vesicle. Towards ovulation, the oocytes resume meiosis and proceed to the second metaphase in a process known as maturation; they undergo nuclear and cytoplasmic changes that are accompanied by translation and degradation of mRNA. Protein phosphatase 1A, magnesium dependent, alpha isoform (PPM1A), which belongs to the metal-dependent serine/threonine protein phosphatase family, is highly conserved during evolution. PPM1A plays a significant role in many cellular functions such as cell cycle progression, apoptosis and cellular differentiation. It works through diverse signaling pathways, including p38 MAP kinase JNK and transforming growth factor beta (TGF-β). Herein we report that PPM1A is expressed in mouse oocytes and that its mRNA level rises during oocyte maturation. Using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that PPM1A mRNA is synthesized at the beginning of the maturation process and remains elevated in the mature oocytes, promoting the accumulation of PPM1A protein. Since PPM1A function is mainly affected by its level, we propose that it might have an important role in oocyte maturation.

An inducible system to study the growth arrest properties of protein phosphatase 2C.

Publisher Summary The chapter presents a system that enables to study properties of protein phosphatase 2Cα (PP2Cα) expression in 293 cells.PP2Cα (also referred as PPM1A) is the most characterized member of the PP2C group. It is a monomeric enzyme of about 42 kDa that shows broad substrate specificity. The catalytic domain, of 300 amino acid residues, in the N terminus, consists of 6 α-helices and 11 β-sheets, and is common to all enzymes that belong to the PP2C family. The C-terminal domain includes a sequence of 80 amino acids that forms three α-helices. This domain determines the substrate specificity of the enzyme and displays no similarity to the other paralogs except for the closely related PP2C β. Growing list of substrates, specifically dephosphorylated by PP2C α in eukaryotic cells has been suggested. In human embryo kidney (HEK) 293 cells over expression of PP2C α and PP2C β were highly toxic to the cells and it is not possible to obtain stable cell lines overexpressing PP2C α. The chapter discusses several methods including establishment of inducible PP2C α cells; plating efficiency; antibodies; cell extracts and western blot analysis; XTT assay; and malachite-green assay of protein phosphatase activity.