Gene/Protein Disease Symptom Drug Enzyme Compound
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 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. To study the maturation pathway of the S glycoprotein of the severe acute respiratory syndrome (SARS)-coronavirus (CoV) within the host cell, a T7/vaccinia virus-based expression system coupled to immunoprecipitation with anti-S antibodies was used to test and analyze different forms of the S glycoprotein. The state of maturity of the S glycoprotein can be deduced from its sensitivity to hydrolysis by endoglycosidase H (EndoH) or N-glycosidase F (N-Gly F). A fully matured S glycoprotein will be modified with complex oligosaccharides which makes it resistant to cleavage by EndoH but not by N-Gly F. By exploiting this characteristic, it is then possible to determine which forms of the immunoprecipitated S protein are properly processed by the host cell. With this system, many different constructs of the S glycoprotein can be analyzed in parallel thus providing another method by which to study the functional domains of S involved in membrane fusion event that occurs during viral infection.
Methods Mol Biol 2007
PMID:Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV. 1750 75

Proteolytic enzymes constitute some 2% of the human genome and provide important therapeutic targets in many diseases. The identification of many novel proteases from genome sequencing programmes provides both qualitative and quantitative challenges to target identification and validation. Some approaches to dealing with these questions and addressing functional correlates of proteolytic activity at the level of molecular cell biology are provided in this review focusing on two spheres of interest: neurodegeneration and cardiovascular regulation. The role and regulation of the neprilysin (NEP) family of metalloproteinases is highlighted in particular in the context of proteolytic events underlying the pathology of Alzheimer's disease. The second exemplar involves the newly appreciated complexity of the renin-angiotensin system as a regulator of the cardiovascular system. The application of functional genomics approaches to the discovery of angiotensin-converting enzyme-2 (ACE2) as a counterbalance to the well known hypertensive target ACE will be highlighted and their differential cellular targeting and enzymology addressed. Finally, the serendipitous discovery of ACE2 as the SARS virus receptor illustrates the surprises always in store from nature.
Cell Mol Biol (Noisy-le-grand) 2006 Dec 31
PMID:Proteinase dysbalance in pathology: the neprilysin (NEP) and angiotensin-converting enzyme (ACE) families. 1754 97

Angiotensin-converting enzyme (ACE) and ACE2 are highly homologous metalloproteases that provide essential catalytic functions in the renin-angiotensin system (RAS). Angiotensin II is one key effector peptide of the RAS, inducing vasoconstriction and exerting multiple biological functions. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 reduces angiotensin II levels. Accumulating evidence has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Intriguingly, the SARS coronavirus, the cause of severe acute respiratory syndrome (SARS), utilizes ACE2 as an essential receptor for cell fusion and in vivo infections. Moreover, recent studies have demonstrated that ACE2 protects murine lungs from acute lung injury as well as SARS-Spike protein-mediated lung injury, suggesting a dual role of ACE2 in SARS infections and protection from ARDS.
Cell Mol Life Sci 2007 Aug
PMID:Angiotensin-converting enzyme 2 in acute respiratory distress syndrome. 1755 69

The 3C-like main peptidase 3CL(pro) is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL(pro) and the structurally related 3C(pro) viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL(pro) in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.
J Mol Biol 2007 Aug 24
PMID:A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase. 1759 57

Continuous efforts have been made to develop a prophylactic vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, two recombinant baculoviruses, vAc-N and vAc-S, were constructed, which contained the mammalian-cell activate promoter element, human elongation factor 1alpha-subunit (EF-1alpha), the human cytomegalovirus (CMV) immediate-early promoter, and the nucleocapsid (N) or spike (S) gene of bat SARS-like CoV (SL-CoV) under the control of the CMV promoter. Mice were subcutaneously and intraperitoneally injected with recombinant baculovirus, and both humoral and cellular immune responses were induced in the vaccinated groups. The secretion level of IFN-gamma was much higher than that of IL-4 in vAc-N or vAc-S immunized groups, suggesting a strong Th1 bias towards cellular immune responses. Additionally, a marked increase of CD4 T cell immune responses and high levels of anti-SARS-CoV humoral responses were also detected in the vAc-N or vAc-S immunized groups. In contrast, there were significantly weaker cellular immune responses, as well as less antibody production than in the control groups. Our data demonstrates that the recombinant baculovirus can serve as an effective vaccine strategy. In addition, because effective SARS vaccines should act to not only prevent the reemergence of SARS-CoV, but also to provide cross-protection against SL-CoV, findings in this study may have implications for developing such cross-protective vaccines.
Mol Immunol 2008 Feb
PMID:Vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of SARS-like coronavirus generates humoral and cellular immune responses. 1790 35

The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires Mg2+ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.
J Biochem Mol Biol 2007 Sep 30
PMID:Biochemical characterization of exoribonuclease encoded by SARS coronavirus. 1792 96

Chemokines and their receptors function in the recruitment and activation of cells of the immune system to sites of inflammation. As such, chemokines play an important role in mediating pathophysiological events during microbial infection. In particular, CXCL9, CXCL10 and CXCL11 and their cognate receptor CXCR3 have been associated with the clinical course of several infectious diseases, including severe acute respiratory syndrome (SARS) and influenza. While CXCL9, CXCL10 and CXCL11 share the same receptor and have overlapping functions, each can also have unique activity in host defense. The lack of a preferred characterized animal model for SARS has brought our attention to ferrets, which have been used for years in influenza studies. The lack of immunological reagents for ferrets prompted us to clone CXCL9, CXCL10, CXCL11 and CXCR3 and, in the case of CXCL10, to express the gene as a recombinant protein. In this study we demonstrate that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the lungs of deceased SARS patients and ferrets experimentally infected with SARS coronavirus. This study therefore represents an important step towards development of the ferret as a model for the role of CXCL9, CXCL10 and CXCL11:CXCR3 axis in severe viral infections.
Mol Immunol 2008 Mar
PMID:Cloning, expression and characterization of ferret CXCL10. 1800 61

The epidemic outbreak of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus (CoV), designated SARS-CoV. The RNA genome of SARS-CoV is complexed by the nucleocapsid protein (N) to form a helical nucleocapsid. Besides this primary function, N seems to be involved in apoptotic scenarios. We show that upon infection of Vero E6 cells with SARS-CoV, which elicits a pronounced cytopathic effect and a high viral titer, N is cleaved by caspases. In contrast, in SARS-CoV-infected Caco-2 cells, which show a moderate cytopathic effect and a low viral titer, this processing of N was not observed. To further verify these observations, we transiently expressed N in different cell lines. Caco-2 and N2a cells served as models for persistent SARS-CoV infection, whereas Vero E6 and A549 cells did as prototype cell lines lytically infected by SARS-CoV. The experiments revealed that N induces the intrinsic apoptotic pathway, resulting in processing of N at residues 400 and 403 by caspase-6 and/or caspase-3. Of note, caspase activation is highly cell type specific in SARS-CoV-infected as well as transiently transfected cells. In Caco-2 and N2a cells, almost no N-processing was detectable. In Vero E6 and A549 cells, a high proportion of N was cleaved by caspases. Moreover, we examined the subcellular localization of SARS-CoV N in these cell lines. In transfected Vero E6 and A549 cells, SARS-CoV N was localized both in the cytoplasm and nucleus, whereas in Caco-2 and N2a cells, nearly no nuclear localization was observed. In addition, our studies indicate that the nuclear localization of N is essential for its caspase-6-mediated cleavage. These data suggest a correlation among the replication cycle of SARS-CoV, subcellular localization of N, induction of apoptosis, and the subsequent activation of caspases leading to cleavage of N.
J Mol Biol 2008 Feb 08
PMID:Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection. 1815 31

A new strain of coronavirus has caused an outbreak of severe acute respiratory syndrome (SARS) from 2002 to 2003 resulting in 774 deaths worldwide. By protein chip array profiling technology, a number of serum biomarkers that might be useful in monitoring the clinical course of SARS patients were identified. This book chapter describes how the protein chip array profiling was carried out for this study. Briefly, SARS patients' serum samples were first fractionated in Q Ceramic HyperD ion exchange sorbent beads by buffers at different pH. Serum protein fractions thus obtained were then bound onto a copper (II) immobilized metal affinity capture (IMAC30 Cu [II]) ProteinChip Array or a weak cation-exchange (CM10) ProteinChip Array. After washing and addition of sinapinic acid, the chips were read in a Protein Biological System (PBS) IIc mass spectrometer. Ions were generated by laser shots and flied in a time of flight mode to the ion detector according to their mass over charge (m/z) ratio. The serum profiling spectra in SARS patients were acquired, baseline subtracted and analyzed in parallel with those from the control subjects by Ciphergen ProteinChip Software 3.0.2 with their peak intensities compared by a nonparametric two sample Mann-Whitney-U test. More than twelve peaks were differentially expressed in SARS patients with one at m/z of 11,695 (later identified to be serum amyloid A protein), which had increase in peak intensity correlating with the extent of SARS-coronavirus induced pneumonia as defined by a serial chest X-ray opacity score. The remaining biomarkers could also be useful in the study of other clinical parameters in SARS patients.
Methods Mol Biol 2007
PMID:Application of ProteinChip array profiling in serum biomarker discovery for patients suffering from severe acute respiratory syndrome. 1822 Feb 40

Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of (125)I-amiodarone and (125)I-B2, an analog lacking the lateral group diethylamino-beta-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-beta-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.
Am J Respir Cell Mol Biol 2008 Aug
PMID:Amiodarone alters late endosomes and inhibits SARS coronavirus infection at a post-endosomal level. 1831 40


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