Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currently no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) glycoprotein, and infection is sensitive to lysosomotropic agents that perturb endosomal pH. We demonstrate here that the lysosomotropic-agent-mediated block to SARSCoV infection is overcome by protease treatment of target-cellassociated virus. In addition, SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. A cell-free membrane-fusion system demonstrates that engagement of receptor followed by proteolysis is required for SARS-CoV membrane fusion and indicates that cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These results suggest that SARS-CoV infection results from a unique, three-step process: receptor binding and induced conformational changes in S glycoprotein followed by cathepsin L proteolysis within endosomes. The requirement for cathepsin L proteolysis identifies a previously uncharacterized class of inhibitor for SARSCoV infection.SARS ͉ viral entry ͉ proteolysis ͉ membrane fusion ͉ viral envelope S evere acute respiratory syndrome (SARS) is an acute respiratory illness caused by a newly described coronavirus (SARS-CoV) (1), the result of a zoonosis of a highly related animal coronavirus (2). There continues to be potential for further zoonotic transmission events, leading to the reintroduction of SARS-CoV into the human population. No effective antiviral treatments have been described for SARS, and, although several promising studies are ongoing, there is currently no licensed protective vaccine.SARS-CoV entry into target cells is initiated by engagement of its cellular receptor, angiotensin-converting enzyme 2 (ACE2) by spike (S) glycoprotein (3). Subsequent infection is sensitive to inhibitors of endosomal acidification such as ammonium chloride (4-6), suggesting that SARS-CoV requires a low-pH milieu for infection. On the other hand, S protein can mediate cell-cell fusion at neutral pH (3, 4), indicating that S protein-mediated fusion does not include an absolute requirement for an acidic environment. Given these discordant findings, we hypothesized that cellular factors sensitive to ammonium chloride, such as pH-dependent endosomal proteins, may play a role in mediating SARS-CoV entry. In this study, the requirements for proteases in the activation of viral infectivity and the effect of protease inhibitors on SARS-CoV infection are examined. Our results are consistent with a model in which SARS-CoV employs a unique three-step method for membrane fusion, involving receptorbinding and induced conformational changes in S glycoprotein followed by cathepsin L (CTSL) proteolysis and activation of membrane fusion within endosomes. Cells were maintained in DMEM10 (DMEM supplemented with 10% FBS). A HeLa͞Tva cell line was produced by using pcDNA6-Tva and selection with blasticidin. The 293T cells were transiently transfected wit...
Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots͞cm 2 . Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and highthroughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 10 3 -10 4 smaller than current high-throughput methods. DNA chips and microarrays for genotyping and expression profiling give no information about the activities of enzymes that can be regulated by posttranslational modifications or cleavage state. Protein microarrays have used the capture of proteins to libraries of immobilized DNA sequences, peptides, antibodies, chemical motifs, or other proteins(1-6). The three major formats for protein arrays currently use plain glass slides (1), 3D gel pad chips (7) (''matrix'' chips), or nanowell chips (2, 3). All of the formats require pretreatment for immobilization, which is a laborious and potentially protein-altering process. Also, none of these three formats have used soluble fluorogenic substrates to quantify multicomponent reactions common to high-throughput screening or to quantify numerous enzymes in a sample like plasma. Microarray-based activity assays of kinases require the use of immobilized substrates (2, 6) and cannot be easily implemented for the direct screening of soluble compounds from a combinatorial library. Assays performed in well plates before arraying onto chips for detection do not exploit the scale down or liquid handling power of microarray printing (6). Thus, a need exists to create localized reaction volumes in an array-based format as well as to create a method of rapidly delivering small volumes of fluid to each reaction. Additionally, evaporation effects typically prevent the extreme scale down of well-plate reactions to nanoliter volumes (8). We have developed a technique that performs enzymatic assays at nanoliter volumes in the liquid phase with minimal evaporation, no crosscontamination, and high reproducibility, and that has the capability to rapidly assemble multicomponent reactions with minimal sample usage in a microarray format. Methods. An OmniGrid Accent and OmniGrid contact microarrayers (Gene Machines, San Carlos, CA) were used for arraying. Arrays were printed on the OmniGrid Accent by using a 1 ϫ 1 pin protocol or on the OmniGrid with a 4 ϫ 8 pin protocol. All glass slides (Erie Scientific, Portsmouth, NH) were washed in dry ethanol and vacuum dried before arra...
A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.
Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ؍ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ؍ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...
A microarray presenting glycerol nanodroplets of fluorogenic peptide substrates was used as a biosensor for the detection of multiple enzyme activities within human plasma. Using 10 different plasma proteases (kallikrein, factor XIIa, factor XIa, factor IXa, factor VIIa, factor Xa, thrombin, activated protein C, uPA and plasmin) and a 361-compound fluorogenic substrate library (Ac-Ala-P3-P2-Arg-coumarin for P = all amino acids except Cys), a database was created for deconvoluting the relative activity of each individual enzyme signal in human plasma treated with various activators (calcium, kaolin, or uPA). Three separate deconvolution protocols were tested: searching for "optimal" sensing substrate sequences for a set of 5 enzymes and using these substrates to detect protease signals in plasma; ranking the "optimal" sensing substrates for 10 proteases using local error minimization, resulting in a set of substrates which were bundled via weighted averaging into a super-pixel that had biosensing properties not obtainable by any individual fluorogenic substrate; and treating each 361-element map measured for each plasma preparation as a weighted sum of the 10 maps obtained for the 10 purified enzymes using a global error minimization. The similarity of the results from these latter two protocols indicated that a small subset of <90 substrates contained the majority of biochemical information. The results were consistent with the state of the coagulation cascade expected when treated with the given activators. This method may allow development of future biosensors using minimal and non-specific markers. These substrates can be applied to real-time diagnostic biosensing of complex protease mixtures.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.