BackgroundEntry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.Methodology/Principal FindingsPurified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.Conclusions/SignificanceThese results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70–77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenicThe impairment of growth factor signaling is a near-universal feature of skeletal myopathies induced by unloading (6, 13). Clinical trials have established that during unloading, muscle tissue fails to respond to IGF-1, a dominant myotrophic hormone (7,19,34). Under normal conditions and in response to hypertrophic stimuli, IGF-1 promotes muscle growth and suppresses muscle loss largely through the Akt-dependent phosphorylation and cytosolic sequestration of FOXO transcription factors in skeletal myocytes, which leads to the inhibition of FOXO-dependent gene expression (38, 41). In contrast, IGF-1-dependent Akt signaling is impaired during muscle atrophy, which decreases the phosphorylation and increases the transactivation of FOXO target genes. In particular, FOXO regulates the expression of atrophy-related genes (atrogenes) that encode atrogin-1/MAFbx and MuRF-1, which are RING-type ubiquitin ligases that are critical mediators of atrophic myopathies in vivo (3,14). Atrogin-1 and MuRF-1 regulate the degradation of key proteins involved in striated muscle growth and differentiation, including MyoD, calcineurin, and troponin-I (24, 27, 47). Although diminished growth factor responsiveness and enhanced proteolysis both are major atrophyrelated processes, the mechanisms by which skeletal muscle becomes refractory to the trophic actions of muscle growth factors during unloading are not well defined.In a previous study designed to evaluate changes in skeletal muscle gene expression in rats exposed to a 16-day spaceflight (30), we identified novel potential atrogenes (37) using microarray analysis. The response of skeletal muscle to mechanical stress is accompanied by marked alterations in atrogene expression, and we showed that microgravity induces Siah-1A, MuRF-1 (30), and atrogin-1 (see Table S1 in the supplemental material). Microgravity also resulted in the increased expression of Cbl-b (greater than eightfold). Cbl-b is another RING-type ubiquitin ligase previously established as a negative regulator of receptor tyrosine kinase signaling in a variety of cells (23,45). These results complement our recent finding that Cbl-b downregulates bone formation

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance. Vitronectin (VN)1 is a 75-kDa adhesive glycoprotein. It circulates in blood in a monomeric ("closed," "native") form, but is converted into a multimeric ("extended," "opened," "denatured") form when incorporated into the extracellular matrix or treated with urea (1, 2). The extended form of VN binds to specific receptors on cells (3, 4) and to various other molecules such as the C5b-9 complement complex (5), the thrombin-antithrombin III complex (6, 7), plasminogen activator inhibitor 1 (PAI-1) (8 -10), uPAR (11), heparin (1, 2, 12, 13), collagen (14 -16), plasminogen (13, 17), and ␤-endorphin (18). These interactions not only promote the attachment, spreading, and growth of cells (19 -21) but also influence the coagulation, fibrinolytic, and complement systems (22,23).Although a number of investigators have attempted to identify the binding site(s) in VN for these molecules, the literature remains somewhat controversial. For example, three different regions of the VN molecule have been proposed to contain the binding sites for uPAR and PAI-1. The first region, the somatomedin B (SMB) domain (residues 1-44) was identified from direct binding studies (9, 24 -27) and from studies showing that soluble SMB competes with uPAR and PAI-1 for binding to denatured VN (9). The second region in VN that has been implicated in uPAR and PAI-1 binding is the heparin binding (HB) domain (residues 348 -370). Thus, synthetic peptides from this domain interfere with both uPAR (21) and PAI-1 (28) binding to VN. Moreover, mAb 8E6 (which has been mapped to the HB domain (Ref. 13)) also inhibits the binding of PAI-1 to VN. Although a third region in VN (residues 115-121) was shown to have weak PAI-...

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Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the shorttailed shrew Blarina brevicauda. Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L 106 TFFYKTFLG 115 . BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts.