Proteases: Pivot Points in Functional Proteomics (original) (raw)
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Proteolysis to Identify Protease Substrates: Cleave to Decipher
Proteomics, 2018
Proteolysis is an irreversible post-translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding of the mechanism of proteolytic activity, the identity and role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, we recapitulate the library screening, proteomics and bioinformatics based approaches that have been employed for identification of protease substrates.
Biochemical Journal, 2011
Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, o...
Protease signalling: the cutting edge
The EMBO Journal, 2012
Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.
Methods in Molecular Biology, 2009
The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.
A plasma proteolysis pathway comprising blood coagulation proteases
Oncotarget, 2016
Coagulation factors are essential for hemostasis. Here, we show that these factors also team up to degrade plasma proteins that are unrelated to hemostasis. Prolidase, SRC and amyloid β1-42 (Aβ1-42) are used as probes. Each probe, upon entering the blood circulation, binds and activates factor XII (FXII), triggering the intrinsic and common coagulation cascades, which in turn activate factor VII, a component of the extrinsic coagulation cascade. Activated factor VII (FVIIa) rapidly degrades the circulating probes. Therefore, FXII and FVIIa serve as the sensor/initiator and executioner, respectively, for the proteolysis pathway. Moreover, activation of this pathway by one probe leads to the degradation of all three probes. Significant activation of this pathway follows tissue injury and may also occur in other disorders, e.g., Alzheimer's disease, of which Aβ1-42 is a key driver. However, enoxaparin, a clinically used anticoagulant, inhibits the proteolysis pathway and elevates p...
Mass Spectrometry-Assisted Protease Substrate Screening
Analytical Chemistry, 2007
Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture.
Proteomic discovery of protease substrates
Current Opinion in Chemical Biology, 2007
Elucidation of in vivo substrate degradomes of a protease is a daunting endeavor because of the large number of proteins in a proteome and often minute and transient amounts of key substrates. Proteomic substrate screens for proteases are currently experiencing impressive progress. Mass spectrometry-based global proteome analysis, interfaced with liquid-chromatography and together with stable isotope labeling strategies, has provided increased coverage and sensitivity for quantitative proteomics. ICAT and iTRAQ labeling have been used to identify a plethora of new matrix metalloproteinase substrates. Emerging techniques focus on the quantitative analysis of proteolytically generated neo amino-termini, which we call terminopes, on a system-wide basis. In vivo SILAC pulse-chase experiments have also enabled the study of individual protein turnover and global proteome dynamics in cells and whole organisms. Together with activity-based probes for the profiling of functional proteases, there is now in place an array of complementary technologies to dissect the 'protease web' and its distortion in pathology.
Proteases A Beneficial Degradative Enzyme in Therapeutic Applications
International Journal of Scientific Research in Biological Sciences
Proteases probably arose at the initial phases of protein development as simple degradative enzymes essential for protein degradation into its simpler form as amino acids in prokaryotic organisms. Proteases are an increasing group of enzymes that hold great potential. Proteases are produced by animals, plants, and microorganisms. Microorganisms play a dominant role in the field of proteases production. Their contribution in the life cycle of numerous pathogenic organisms has led them to become a probable target for emerging therapeutic agents in contradiction of life-threatening diseases. These enzymes are being utilized in the cure of respiratory tract issues, cardiovascular disease, inflammation, and cancer. Proteases promote healing process of tissue damage; it may be surgical or accidental damages, fractures or burns. Due to these special features, proteases are an emerging class of enzymes that possess diverse medical applications. Along with proteases, protease inhibitors are also played a very important role in disease control. Therefore protease inhibitors have drawn the attention towards their crucial role in the therapeutic application.