SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/significanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy

Biochemical Characterization of Proteolytic Activities and Substrate Specificities

Proteases are crucial components of life. They regulate numerous biological pathways, such blood coagulation, neurotransmission, cell proliferation and apoptosis. They are also found to be involved in the development cancer, Alzheimer’s and many infectious diseases. Proteases, as a post-translational modification enzyme, regulate biological processes through proteolysis. And like many other enzymes, one of the key features of proteases is their substrate specificity, which is tightly controlled by specific interactions between the amino acid sequences of the substrates and their binding pockets. It’s important to characterize proteases’ specificities as it is a key to understand their biological functions. This work focused on the development and applications of a mass spectrometry-based technology, named Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), to characterize the specificities of proteases, and the design of fluorescent substrates and inhibitors based on protease specificity profiles.Chapter II demonstrates the application of MSP-MS in uncovering the substrate specificity of a bacterial protease, Pd_dinase, which is a putative C1B-like cysteine protease commonly secreted by the human gut commensal Parabacteroidetes distasonis. We designed and synthesized a potent protease inhibitor glycine-arginine-AOMK based on its specificity profile. Furthermore, we revealed that Pd_dinase hydrolyzes several human antimicrobial peptides, such as β-defensin 2 and keratin-derived antimicrobial peptides, indicating that it may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.Chapter III presents the study to extend the application of the MSP-MS technology in studying complex biological samples. In this study, we isolated bovine chromaffin granules (CGs) and quantified the endogenous proteins and peptides through proteomics and peptidomics. In addition, we performed degradation assays to profile proteolytic processing of proneuropeptides and MSP-MS assays to characterize proteas activities. With comprehensive profile of proteases, substrates and proteolytic specificities, we were able to discover six catalytically active proteases and assign their activities to some of the cleavages on proneuropeptides. Chapter IV presents the study of developing quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) method by combining the quantitative power of tandem mass tags (TMTs) with our previously established peptide cleavage assay, MSP-MS. We validated the method with papain, a well-characterized cysteine peptidase and uncovered the substrate specificity of two minimally characterized intramembrane rhomboid proteases. We further showed that activity from multiple peptidases in complex biological samples can be deciphered, including secretions from lung cancer cell lines. Discovery of the protease specificity at the site of the disease highlights the potential for qMSP-MS to guide the development of protease-activating drugs for cancer and infectious disease

Biochemical Characterization of Proteolytic Activities and Substrate Specificities

Proteases are crucial components of life. They regulate numerous biological pathways, such blood coagulation, neurotransmission, cell proliferation and apoptosis. They are also found to be involved in the development cancer, Alzheimer’s and many infectious diseases. Proteases, as a post-translational modification enzyme, regulate biological processes through proteolysis. And like many other enzymes, one of the key features of proteases is their substrate specificity, which is tightly controlled by specific interactions between the amino acid sequences of the substrates and their binding pockets. It’s important to characterize proteases’ specificities as it is a key to understand their biological functions. This work focused on the development and applications of a mass spectrometry-based technology, named Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), to characterize the specificities of proteases, and the design of fluorescent substrates and inhibitors based on protease specificity profiles.Chapter II demonstrates the application of MSP-MS in uncovering the substrate specificity of a bacterial protease, Pd_dinase, which is a putative C1B-like cysteine protease commonly secreted by the human gut commensal Parabacteroidetes distasonis. We designed and synthesized a potent protease inhibitor glycine-arginine-AOMK based on its specificity profile. Furthermore, we revealed that Pd_dinase hydrolyzes several human antimicrobial peptides, such as β-defensin 2 and keratin-derived antimicrobial peptides, indicating that it may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.Chapter III presents the study to extend the application of the MSP-MS technology in studying complex biological samples. In this study, we isolated bovine chromaffin granules (CGs) and quantified the endogenous proteins and peptides through proteomics and peptidomics. In addition, we performed degradation assays to profile proteolytic processing of proneuropeptides and MSP-MS assays to characterize proteas activities. With comprehensive profile of proteases, substrates and proteolytic specificities, we were able to discover six catalytically active proteases and assign their activities to some of the cleavages on proneuropeptides. Chapter IV presents the study of developing quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) method by combining the quantitative power of tandem mass tags (TMTs) with our previously established peptide cleavage assay, MSP-MS. We validated the method with papain, a well-characterized cysteine peptidase and uncovered the substrate specificity of two minimally characterized intramembrane rhomboid proteases. We further showed that activity from multiple peptidases in complex biological samples can be deciphered, including secretions from lung cancer cell lines. Discovery of the protease specificity at the site of the disease highlights the potential for qMSP-MS to guide the development of protease-activating drugs for cancer and infectious disease

Biochemical Characterization of Proteolytic Activities and Substrate Specificities

Proteases are crucial components of life. They regulate numerous biological pathways, such blood coagulation, neurotransmission, cell proliferation and apoptosis. They are also found to be involved in the development cancer, Alzheimer’s and many infectious diseases. Proteases, as a post-translational modification enzyme, regulate biological processes through proteolysis. And like many other enzymes, one of the key features of proteases is their substrate specificity, which is tightly controlled by specific interactions between the amino acid sequences of the substrates and their binding pockets. It’s important to characterize proteases’ specificities as it is a key to understand their biological functions. This work focused on the development and applications of a mass spectrometry-based technology, named Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), to characterize the specificities of proteases, and the design of fluorescent substrates and inhibitors based on protease specificity profiles. Chapter II demonstrates the application of MSP-MS in uncovering the substrate specificity of a bacterial protease, Pd_dinase, which is a putative C1B-like cysteine protease commonly secreted by the human gut commensal Parabacteroidetes distasonis. We designed and synthesized a potent protease inhibitor glycine-arginine-AOMK based on its specificity profile. Furthermore, we revealed that Pd_dinase hydrolyzes several human antimicrobial peptides, such as β-defensin 2 and keratin-derived antimicrobial peptides, indicating that it may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides. Chapter III presents the study to extend the application of the MSP-MS technology in studying complex biological samples. In this study, we isolated bovine chromaffin granules (CGs) and quantified the endogenous proteins and peptides through proteomics and peptidomics. In addition, we performed degradation assays to profile proteolytic processing of proneuropeptides and MSP-MS assays to characterize proteas activities. With comprehensive profile of proteases, substrates and proteolytic specificities, we were able to discover six catalytically active proteases and assign their activities to some of the cleavages on proneuropeptides. Chapter IV presents the study of developing quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) method by combining the quantitative power of tandem mass tags (TMTs) with our previously established peptide cleavage assay, MSP-MS. We validated the method with papain, a well-characterized cysteine peptidase and uncovered the substrate specificity of two minimally characterized intramembrane rhomboid proteases. We further showed that activity from multiple peptidases in complex biological samples can be deciphered, including secretions from lung cancer cell lines. Discovery of the protease specificity at the site of the disease highlights the potential for qMSP-MS to guide the development of protease-activating drugs for cancer and infectious disease

Targeting proteasomes in infectious organisms to combat disease.

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents

Targeting proteasomes in infectious organisms to combat disease.

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents

Stent graft coverage of dual-stent strategy in the management of abdominal aortic aneurysms

Abstract Treating an abdominal aortic aneurysm (AAA) with a stent graft (SG) and a multilayer stent (MS) is a key technology in isolating flow fields. Clinically, dual stents (an SG in the proximal and an MS in the distal of AAA) are used for treatment of AAA, but only a few studies have examined the relationship between SG coverage and treatment effects. Through numerical simulation of the hemodynamics after SG and MS implantation, the SG coverage and position were simulated at 0% (0 mm), 25% (13.75 mm), 50% (27.5 mm), and 75% (41.25 mm). With increasing SG coverage, the pressure on the aneurysm sac wall and the flow of branch vessels gradually decreased, and the lower wall shear stress (WSS) gradually increased. The changes in pressure, lower WSS, and the mass flow rate of the branch vessels did not change significantly. The coverage of the SG has a nonsignificant effect on hemodynamics in the treatment of AAA; the implantation position need not be very precise. This research can provide theoretic support for clinicians’ decision-making

Multiplex substrate profiling by mass spectrometry for proteases

Proteolysis is a central regulator of many biological pathways and the study of proteases has had a significant impact on our understanding of both native biology and disease. Proteases are key regulators of infectious disease and misregulated proteolysis in humans contributes to a variety of maladies, including cardiovascular disease, neurodegeneration, inflammatory diseases, and cancer. Central to understanding a protease's biological role, is characterizing its substrate specificity. This chapter will facilitate the characterization of individual proteases and complex, heterogeneous proteolytic mixtures and provide examples of the breadth of applications that leverage the characterization of misregulated proteolysis. Here we present the protocol of Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), a functional assay that quantitatively characterizes proteolysis using a synthetic library of physiochemically diverse, model peptide substrates, and mass spectrometry. We present a detailed protocol as well as examples of the use of MSP-MS for the study of disease states, for the development of diagnostic and prognostic tests, for the generation of tool compounds, and for the development of protease-targeted drugs

An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases

Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.status: publishe