A systems biology analysis of the Drosophila phagosome.

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.

A predicted alpha -helix mediates targeting of the proprotein convertase PC1 to the regulated secretory pathway.

The proprotein convertase PC1 is a protease whose activity is largely confined to the dense core secretory granules of neuroendocrine cells. Efficient processing of PC1 substrates in granules requires a mechanism that will both limit the activity of the enzyme to these organelles and promote its targeting to the nascent secretory granules. In the current study, we provide evidence that targeting of PC1 to secretory granules is mediated by alpha-helical structures in its C-terminal tail and, at least in part, is dependent on interactions with specific components of the secretory granule membrane.

Prorenin processing by cathepsin B in vitro and in transfected cells.

Renin, which catalyzes the initial proteolytic cleavage reaction in the production of angiotensins, is first synthesized as a zymogen, prorenin, and requires the proteolytic removal of an amino-terminal prosegment for activation in vivo. The lysosomal hydrolase cathepsin B has been proposed as a prorenin processing enzyme based on reports of its co-localization with renin in the secretory granules of certain tissues and its ability to activate prorenin in vitro. In the current study, scanning mutagenesis was used to identify the amino acids which determine the site selectivity of prorenin cleavage by human cathepsin B in vitro. Co-expression assays in AtT-20 cells were also used to test for the ability of cathepsin B to cleave human prorenin within cells. Our results suggest that a basic lysine residue at the -2 position from the cleavage site is required for cathepsin B cleavage of prorenin in vitro and that the structure of prorenin itself may account for the selection of the proper cleavage site. In addition, although cathepsin B appears to be correctly sorted to lysosomes, the enzyme exhibits prorenin processing activity in transfected AtT-20 cells, raising the question of the cellular localization in which the processing event occurs.

Two activation states of the prohormone convertase PC1 in the secretory pathway.

PC1, a neuroendocrine member of the prohormone convertase family of serine proteinases, is implicated in the processing of proproteins in the secretory pathway. PC1 is synthesized as a zymogen and cleaves not only its own profragment in the endoplasmic reticulum, but a subset of protein substrates in the Golgi apparatus and in the Golgi-distal compartments of the regulated secretory pathway. Likewise, mouse PC1 (mPC1) has previously been shown to cleave human prorenin in GH4 cells (that contain secretory granules) while being unable to cleave prorenin in cells, such as Chinese hamster ovary (CHO) or BSC-40, which are devoid of secretory granules. In the current study, we show that removal of a C-terminal tail of mPC1 allows the efficient cleavage of prorenin in the constitutive secretory pathway of CHO cells. The C-terminal tail thus appears to act as an inhibitor of PC1 activity against certain substrates in the endoplasmic reticulum and Golgi apparatus, and its removal, which occurs naturally in secretory granules, may explain the observed granule-specific processing of certain proproteins. These results also demonstrate that PC1 is present in a partially active state prior to the secretory granules where it is processed to a maximally active state.

Prorenin activation and prohormone convertases in the mouse As4.1 cell line.

The precise identification of prorenin-processing enzymes has been hampered by the very low abundance of juxtaglomerular cells in the kidney. Recently, an immortalized renin-producing renal tumor cell line (As4.1) has been proposed as a model to carry out such studies. Despite the fact that they contain secretory granules, we found no evidence (on the basis of enzymatic assays of renin activity in the supernatant of the cells and of immunoprecipitations experiments) that the As4.1 cells can secrete active renin through the regulated pathway. As4.1 cells produce only renin-1, as they derive from a strain of mice expressing only one renin gene. However, stable transfection of these cells with a renin-2 expression plasmid increased the capacity of this cell line to secrete active renin in the regulated pathway. Northern blot and reverse transcriptase-polymerase chain reaction amplification (RT-PCR) assays revealed that furin, PACE4 and PC5 were the only members of the proprotein convertase (PC) family to be present in these cells. As PC5 is the only such enzyme with the demonstrated ability to process mouse prorenin 2, it may constitute a candidate enzyme for the processing of prorenin-2 in mouse juxtaglomerular cells. However, it is not likely to be involved in the processing of mouse prorenin 1.

Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex.

We isolated a cDNA clone encoding the human prohormone convertase PC5 from human adrenal gland mRNA. The deduced protein sequence would encode a 915 amino acid preproPC5 that shares a very high degree of homology with previously cloned rat and mouse homologues. PC5 mRNA was detected in multiple human tissues, including the brain, adrenal and thyroid glands, heart, placenta, lung, and testes. PC5 mRNA was undetectable in the liver and was present at lower levels in skeletal muscle, kidney, pancreas, small intestine, and stomach. Co-transfection of human PC5 and human prorenin expression vectors in cultured GH4C1 cells led to secretion of active renin. The activation of human prorenin by PC5 depended on a pair of basic amino acids at positions 42 and 43 of the prorenin prosegment and occurred only in cells containing dense core secretory granules. Human PC5 was colocalized with renin by immunohistochemistry in the zona glomerulosa of the adrenal gland, suggesting that it could participate in the activation of a local renin-angiotensin system in the human adrenal cortex.

Stimulation of osteoclast-like cell formation by Pasteurella multocida toxin from hemopoietic progenitor cells in mouse bone marrow cultures.

The effects of purified Pasteurella multocida toxin (PMT) on osteoclast formation from hemopoietic progenitor cells were studied using an in vitro system. Mononuclear adherent mouse bone marrow cells were cultured for 7 or 14 days in the presence of PMT, or 1,25-dihydroxy-vitamin D3, or both. Mononuclear osteoclast-like cells, which are postmitotic osteoclast precursor cells, were identified as tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells possessing calcitonin receptors. Multinucleated osteoclast-like cells were TRAP-positive multinuclear cells with calcitonin receptors. The results demonstrate that, as does 1,25(OH)2D3, Pasteurella multocida toxin stimulates proliferation of adherent bone marrow mononuclear cells (progenitor cells), and their differentiation into postmitotic mononuclear osteoclast precursor cells. It also causes fusion of the latter into multinuclear osteoclasts; however, the number of osteoclasts obtained with PMT is smaller than with 1,25-dihydroxy-vitamin D3.

Effects of Pasteurella multocida toxin on the osteoclast population of the rat.

Pasteurella multocida type D toxin is a peptide shown to induce severe atrophic rhinitis in the pig as the result of an increased osteoclastic resorption of the ventral nasal turbinates. In the present study, the effects of the toxin on the histological, cytochemical and ultrastructural features of the osteoclast population of the rat were examined. Pasteurella multocida toxin induced atrophy of the ventral and dorsal nasal turbinates and thinning of the nasal bones. The number and size of the long bone metaphyseal osteoclasts were significantly increased, but not the number of nuclei per cell. Osteoclasts of toxin-treated rats had more developed clear zones and ruffled borders than those of the controls and their cytoplasmic vacuoles were more abundant and larger. We concluded that P. multocida toxin stimulates bone resorption by osteoclasts in the rat by increasing resorption activity and by increasing their number. Its action is not limited to the nasal turbinates but occurs also in the other bones, such as the long bones.