Inorganic polyphosphate inhibits an aspartic protease-like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Laboratório de Entomologia Médica, Programa de Biologia Celular e Parasitologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, Brazil.
Journal of Cellular Physiology (Impact Factor: 3.84). 01/2009; 222(3):606-11. DOI: 10.1002/jcp.21975
Source: PubMed


Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus.


Available from: Ednildo A Machado
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    • "Here, we measured PolyP mobilization by following DAPI-PolyP fluorescence levels in samples containing PolyP extracted from eggs of the insect R. prolixus during the early stages of development. Results showed that a strong mobilization of PolyP takes place during this period (Figure 7C) and were validated after a comparison against the well-established protocol for PolyP quantification based on the PolyP binding to silica powder in suspension followed by enzymatic PolyP hydrolysis into Pi by a recombinant yeast exopolyphosphatase,14,26,39,53 which showed similar profiles of PolyP mobilization during the early days of development. "
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    ABSTRACT: Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4',6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multi-photon microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.
    European journal of histochemistry: EJH 10/2013; 57(4):e34. DOI:10.4081/ejh.2013.e34 · 2.04 Impact Factor
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    • "Although the first evidence for the presence of poly P in mammalian cells was obtained a long time ago 50, relatively few studies have addressed its physiological roles in animal cells 1, 9-12, 14, 18, 42, 51, 52. "
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    ABSTRACT: The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exopolyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos.
    International journal of biological sciences 08/2013; 9(8):842-52. DOI:10.7150/ijbs.6628 · 4.51 Impact Factor
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    • "Other classes of serpins have also been shown to inhibit cysteine and aspartic proteases (Kamada et al. 1997; Liu et al. 2003; Mathialagan and Hansen 1996; Silverman et al. 2004; Takeda et al. 1995) and to regulate cell death processes (Bird et al. 1998; Medema et al. 2001; Suminami et al. 2000; Tewari et al. 1995). We have previously described a role for PolyP in the regulation of an aspartic-like protease in the eggs of Rhodnius prolixus Stahl (Gomes et al. 2010). The role of PolyP regulation in protease-triggered events such as digestion and cell death in the midgut of Anticarsia is currently under investigation in our laboratory. "
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