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Proteases☆
Proteases☆ M Kaiser, Centre of Medical Biotechnology, University Duisburg-Essen, Essen, Germany R Huber, Centre of Medical Biotechnology, University Duisburg-Essen, Essen, Germany; Max-Planck-Institute for Biochemistry, Martinsried, Germany; and Cardiff University, School of Biosciences, Cardiff, United Kingdom M Ehrmann, Centre of Medical Biotechnology, University Duisburg-Essen, Essen, Germany and Cardiff University, School of Biosciences, Cardiff, United Kingdom r 2017 Elsevier Inc. All rights reserved.
Glossary Photoactivatable tag A chemical moiety that upon light irradiation turns into a highly reactive chemical species that irreversible crosslinks with nearby binding partners. Post-translational modifications A chemical protein modification of fully translated proteins, often associated with a switching of activity state, structure, etc., of the protein. Proenzyme The inactive precursor of an enzyme.
Proteasome A multicatalytic, oligomeric protease complex that plays a key role in intracellular protein degradation. SILAC A versatile approach for mass spectrometry-based protein quantification, using isotope-labeled amino acid precursors. Ubiquitin A small, highly-conserved and ubiquitous protein of eukaryotes that, among other functions, is used to tag proteins for proteasomal degradation.
Proteases and peptidases catalyze the hydrolysis of amide bonds in peptides and proteins, a protein-degradative process known as proteolysis. They are classified as either endo- or exopeptidases, depending on their preferred site of action: while exopeptidases cleave at either the N-terminal (aminopeptidases) or the C-terminal (carboxypeptidases) ends of a substrate, endopeptidases perform cuts within substrates. To date, six major protease classes are known. These are serine, threonine, cysteine, metallo, aspartate, and glutamate proteases with the amino acid or ion denoting the active site residue/ion that performs the hydrolytic reaction. These proteases follow two biochemically distinct reaction pathways of amide bond hydrolysis. In serine, threonine or cysteine proteases, a covalent acyl intermediate via a nucleophilic addition/elimination sequence of the hydroxyl or thiol active site residues is formed between substrate and protease that in a second, independent step is hydrolyzed by water. Metallo, aspartate and glutamate proteases, however, perform amide bond hydrolysis with a different mechanism. These proteases polarize via their active site residues a water molecule, which then directly carries out hydrolysis of the peptide bond. Thus, amide bond hydrolysis in these protease classes proceeds without the generation of a covalent protease–substrate intermediate. Nowadays, proteases belong to the best-studied enzymes in biochemistry and the structural basis of the underlying hydrolytic mechanisms can be considered as well established for all major protease families. This has led to a detailed view of the individual contributions and conformational changes of active site residues during catalysis at up to atomic resolution. A complementary and evolutionary meaningful classification of proteases (and their inhibitors) into clans and families has been established by Rawlings, Barrett and co-workers. They assembled and continuously update the MEROPS database of known protease sequences and diagnose their statistically relevant similarities in sequence and three-dimensional structure, using bioinformatic algorithms and structural data. Proteases are classified into the same clan if they show a similar catalytic mechanism and three-dimensional fold of the catalytic domain while their assignment into distinct protease families is carried out according to evolutionary ancestry.
Regulatory Mechanisms and Biological Function of Proteases As the proteolytic activity of proteases is inherently destructive for proteins, their action needs to be tightly controlled in all living systems. Consequently, distinct regulatory mechanisms have evolved, ranging from spatial confinement strategies to subtle biochemical control mechanisms. Spatial confinement is implemented in various ways, ranging from a defined subcellular location of proteases in specific cell organelles or at defined cellular structures such as membranes to tissue-specific expression. While these regulatory mechanisms are applicable to all proteases, a particular class of proteases, known as self-compartimentalized proteases, have evolved a structurallyencoded “self-confinement” to regulate their proteolytic activities. In these proteases, the active sites are located inside a central cavity where the access of substrates is strictly regulated. Substrate selection is mediated, for example, by additional regulator ☆
Change History: March 2016. M. Kaiser. Title was previously Proteolysis – it has been changed Proteases to more closely fit the content of the article. Text revised and updated throughout. Further Reading updated.
Reference Module in Life Sciences
doi:10.1016/B978-0-12-809633-8.06970-3
1
2
Proteases
proteins, as observed in the paradigmal ubiquitin–proteasome system (UPS). The UPS features an impressively complex substrate selection protein network, in which proteins destined for degradation are labeled with polyubiquitin chains before they are recognized, deubiquitinylated, unfolded and ultimately translocated into the self-compartmentalized protease known as the 20S proteasome. Remarkably, the whole UPS encompasses several hundred different proteins. It should however be noted that the UPS is an exceptional complex degradation system and much “simpler” selection strategies for limiting access to selfcompartmentalized proteases are also known. For example, compartmentalized proteases can have spatially restricted entrance gates to ensure that only unfolded substrates reach the active sites within the proteolytic chamber while native, folded proteins are excluded. Such “structural” selection criteria are, for example, often found in proteases involved in protein quality control. Some protein quality control proteases employ tethered PDZ domains that bind peptides and mediate allosteric activation as well as positive cooperativity. Recent evidence suggests that these PDZ domains can also be involved in disaggregation of amyloid fibrils to allow efficient proteolytic degradation of individual peptide strands. Alternatively, post-translational modifications (PTMs) are crucial for the regulation of proteases. PTMs are, for example, critical elements for the conversion of inactive proenzymes into active proteases. Conversion of the proenzymes into active enzymes requires a proteolytic processing. Besides this protease-“specific” PTM, also “classical” PTMs such as phosphorylation have been demonstrated to regulate protease activity in vivo. Inhibition of the proteolytic activity, either by direct binding of small molecule or proteinaceous inhibitors at the active site or at allosteric sites of the protease represents another biochemical regulatory mechanism. Living organisms possess a whole set of endogenous protein inhibitors for scavenging and deactivating proteolytically active proteases, for example, in the blood stream where the serpins (serine protease inhibitors) are controlled independently by switching between active, latent, and inactive forms. To date, it seems as most endogenous inhibitors act directly at the active site, but the identification of allosteric modulators of protease activity that – in contrast to active site inhibitors – sometimes also mediate an activation of the protease is currently under intense investigation. While peptide bond hydrolysis is the unifying feature in all proteolytic events, the extent of substrate degradation often various significantly, ranging from extensive protein degradation into amino acids and/or small peptide fragments to highly selective protein processing. The first case is realized, for example, during food digestion which serves to refill the amino acid pool of living organisms or for elimination of misfolded proteins during protein quality control. It is also employed in a process known as regulatory proteolysis which serves to irreversible remove proteins from the cells, thereby regulating cellular processes. In eukaryotic cells, this task is a prominent element in the above-mentioned UPS. In contrast to “bulk” degradation of substrates, finely balanced proteolytic events occur. Such “limited proteolysis” is usually performed by proteases with very strict cleavage preferences, thereby ensuring a highly sequence-specific proteolysis at a welldefined site in a substrate. Consequently, limited proteolysis events are equipollent to irreversible PTMs. Limited proteolysis is again a key process in innumerable biological processes, including the prominent examples of the proteolytic regulation of blood clotting or apoptosis. Proteases play also key roles in the regulation of gene expression. For example, proteolysis of proteins involved in signaling cascades often leads to enhanced or repressed gene expression. While such modulation is more indirect, proteolysis also interferes directly with gene expression, for example, via proteolytic inactivation of transcriptional repressors or the oppositional activation of transcriptional activators. These findings pinpoint that proteases are powerful enzymes that link biochemistry and genetics within cells and – in principle – could be exploited as tools in genetic studies.
Tools for Studying Proteases In order to identify the biological function of a protease of interest, an elucidation of its active state under various physiological conditions as well as its potential substrates is essential. For determining the active state of proteases, activity-based protein profiling (ABPP) has proven to be a particular potent methodology. In ABPP, small molecule “activity-based probes” (ABPs) are used to monitor the active state of proteases. ABPs consist of an inhibitor residue (also often referred to as a “warhead”) that binds to the protease of interest in an irreversible, covalent manner, a tag such as the fluorophore rhodamine or the small molecule biotin for visualization and/or purification of the labeled protease, and a linker moiety for the connection of both moieties. As binding and thus labeling of the protease of interest can only occur if the protease is in its active state, a subsequent protein gel or mass spectrometric analysis reveals if the protease was active in the biological process under investigation. While initially ABPP was limited to proteases that proceed via a covalent intermediate, recent technological advances such as the incorporation of photoactivatable tags have expanded the application range of ABPP also to metallo or aspartate proteases. Additionally, in vivo activity-imaging of proteases by application of modified ABPs has recently been established, offering novel and exciting experimental setups for protease research. Furthermore, the elucidation of “substrate/protease matches” is of crucial importance. Practically, two different scientific challenges can be differentiated. First, the identification of a protease that cleaves a particular substrate in vivo. Second, the identification of the in vivo substrates and thus biological function of a given protease. Again, a vast array of approaches and
Proteases
3
techniques are available. LC–MS-based proteomics methodologies, often coupled with gel-based separation approaches, isotope mass tag systems such as SILAC and/or cell biological knockout approaches are due to their robustness and wide applicability often employed. Nevertheless, further expansions of existing and new methodologies are constantly developed as to date no unifying approach for all scientific challenges have yet been established.
Proteases as Tools for Cell Biology Research The proteolytic activity of proteases can also be exploited to elucidate the biological function of target proteins in cells. These approaches are based on a targeted and thus specific degradation of proteins. Consequently, such an approach is comparable and complementary to a conditional genetic knockout of the corresponding target protein. The obvious prerequisite is a selective degradation at a given point-in-time. To achieve this challenging task, various approaches have been implemented. First, specificity can be achieved by a fusing an engineered destabilizing domain to the target protein. The presence of a destabilizing domain induces degradation of the hybrid construct under normal conditions. The destabilizing domain, however, can be engineered to bind a specific cell-permeable small molecule ligand that inactivates destabilizing domain and therefore no longer promotes proteolysis. Therefore, proteolytic degradation depends on the presence or absence of the small molecule ligand that serves as the on/off-switch of the system. In the complementary PROTACs approach, targeted degradation is initiated upon addition of a special small molecule ligand (PROTAC). PROTACS are modular chemical compounds that consist of a small molecule portion destined to bind the target protein, a second, often peptidic residue that serves as an E3 ubiquitin-ligase recognition element and a linker that connects both moieties. The E3-Ubiquitin-ligase mediates polyubiquitinylation and subsequent proteasomal degradation of the target protein. Therefore, selective degradation of is achieved by a specific small molecule ligand while control over degradation is achieved via addition of the PROTAC to the biological system. Advantageously, this approach requires no prior genetic engineering. However, it depends on selective POI small molecule ligands that need to be designed and chemically synthesized. A third approach is the use of Tobacco Etch Virus (TEV) protease that targets a 7 residues consensus cleavage site. Engineering of such TEV protease sites into the target protein allows in combination with regulated coexpression of TEV protease rapid processing in vivo. The pronounced specificity of TEV and related proteases even allows the cleavage of nascent chains emerging from the ribosome with ribosome-attached protease.
Proteases as Drug Targets Proteases have a long-lasting history as drug targets and several small molecule inhibitors of proteases have already entered the market. In the last decades, medicinal basic research efforts has led to the elucidation of proteases that can serve as drug targets due to their direct implication in the pathogenesis of various disorders. These implications can range from a disease-causing dysregulation of their proteolytic activities to proteases of viral or microbial origin that play a key role during microbial or viral infections. Consequently, their targeted inhibition often exerts a positive therapeutic effect and is therefore a frequent approach in the discovery of novel drugs. For example, inhibitors of the viral HIV protease have become one of the most important drugs for combating the human immune disease AIDS. As proteases are well-described druggable targets, they are also often targeted to alleviate disease symptoms which leads to a therapeutic effect in a more indirect manner. The most prominent examples of such drug discovery strategies are the development of blood pressure lowering drugs such as captopril or enalapril. These small molecules effectively inhibit the blood pressure regulation protease angiotensin-converting enzyme (ACE), thereby lightening the impact of high blood pressure without however addressing its primary cause. While several mechanism-based strategies for the successful rational development of protease inhibitors have evolved in the last decades, the major challenge for drug discovery is no longer the identification of potent protease inhibitors but the elucidation of inhibitor structures with suitable pharmacokinetic properties and safety profile. Due to the wealth of structural information on proteases structure-based drug discovery has thereby played a major role in the last years.
Further Readings Banaszynski, L.A., Chen, L.-C., Maynard-Smith, L.A., Ooi, A.G.L., Wandless, T.J., 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004. Barrett, A.J., 2000. Proteases. In: Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., Wingfield, P.T. (Eds.), Current Protocols in Protein Science. New York, NY: John Wiley & Sons, Inc, pp. 21.1.1–21.1.12. Bhattacharyya, S., Yu, H., Mim, C., Matouschek, A., 2014. Regulated protein turnover: Snapshots of the proteasome in action. Nat. Rev. Mol. Cell Biol. 15, 122–133. Bode, W., Huber, R., 1991. Ligand binding: Proteinase–protein inhibitor interactions. Curr. Opin. Struct. Biol. 1, 45–52. Clausen, T., Kaiser, M., Huber, R., Ehrmann, M., 2011. HtrA proteases: Regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12, 152–162.
4
Proteases
Ehrmann, M., Clausen, T., 2004. Proteolysis as a regulatory mechanism. Annu. Rev. Genet. 38, 709–724. Gallastegui, N., Groll, M., 2010. The 26S proteasome: Assembly and function of a destructive machine. Trends Biochem. Sci. 35, 634–642. Groll, M., Bochtler, M., Brandstetter, H., Clausen, T., Huber, R., 2005. Molecular machines for protein degradation. ChemBioChem 6, 222–256. Hauske, P., Ottmann, C., Meltzer, M., Ehrmann, M., Kaiser, M., 2008. Allosteric regulation of proteases. ChemBioChem 9, 2920–2928. Henrichs, T., Mikhaleva, N., Conz, C., et al., 2005. Target-directed proteolysis at the ribosome. Proc. Natl. Acad. Sci. USA 102, 4246–4251. Huber, E.M., Groll, M., 2012. Inhibitors for the immuno- and constitutive proteasome: Current and future trends in drug development. Angew. Chem. Int. Ed. Engl. 51, 8708–8720. Poepsel, S., Sprengel, A., Sacca, B., et al., 2015. Determinants of amyloid fibril degradation by the PDZ protease HTRA1. Nat. Chem. Biol. 11, 862–869. Schneekloth, J.S., Crews, C.M., 2005. Chemical approaches to controlling intracellular protein degradation. ChemBioChem 6, 40–46.
Relevant Website http://merops.sanger.ac.uk/ MEROPS.
Glossary Photoactivatable tag A chemical moiety that upon light irradiation turns into a highly reactive chemical species that irreversible crosslinks with nearby binding partners. Post-translational modifications A chemical protein modification of fully translated proteins, often associated with a switching of activity state, structure, etc., of the protein. Proenzyme The inactive precursor of an enzyme.
Proteasome A multicatalytic, oligomeric protease complex that plays a key role in intracellular protein degradation. SILAC A versatile approach for mass spectrometry-based protein quantification, using isotope-labeled amino acid precursors. Ubiquitin A small, highly-conserved and ubiquitous protein of eukaryotes that, among other functions, is used to tag proteins for proteasomal degradation.
Proteases and peptidases catalyze the hydrolysis of amide bonds in peptides and proteins, a protein-degradative process known as proteolysis. They are classified as either endo- or exopeptidases, depending on their preferred site of action: while exopeptidases cleave at either the N-terminal (aminopeptidases) or the C-terminal (carboxypeptidases) ends of a substrate, endopeptidases perform cuts within substrates. To date, six major protease classes are known. These are serine, threonine, cysteine, metallo, aspartate, and glutamate proteases with the amino acid or ion denoting the active site residue/ion that performs the hydrolytic reaction. These proteases follow two biochemically distinct reaction pathways of amide bond hydrolysis. In serine, threonine or cysteine proteases, a covalent acyl intermediate via a nucleophilic addition/elimination sequence of the hydroxyl or thiol active site residues is formed between substrate and protease that in a second, independent step is hydrolyzed by water. Metallo, aspartate and glutamate proteases, however, perform amide bond hydrolysis with a different mechanism. These proteases polarize via their active site residues a water molecule, which then directly carries out hydrolysis of the peptide bond. Thus, amide bond hydrolysis in these protease classes proceeds without the generation of a covalent protease–substrate intermediate. Nowadays, proteases belong to the best-studied enzymes in biochemistry and the structural basis of the underlying hydrolytic mechanisms can be considered as well established for all major protease families. This has led to a detailed view of the individual contributions and conformational changes of active site residues during catalysis at up to atomic resolution. A complementary and evolutionary meaningful classification of proteases (and their inhibitors) into clans and families has been established by Rawlings, Barrett and co-workers. They assembled and continuously update the MEROPS database of known protease sequences and diagnose their statistically relevant similarities in sequence and three-dimensional structure, using bioinformatic algorithms and structural data. Proteases are classified into the same clan if they show a similar catalytic mechanism and three-dimensional fold of the catalytic domain while their assignment into distinct protease families is carried out according to evolutionary ancestry.
Regulatory Mechanisms and Biological Function of Proteases As the proteolytic activity of proteases is inherently destructive for proteins, their action needs to be tightly controlled in all living systems. Consequently, distinct regulatory mechanisms have evolved, ranging from spatial confinement strategies to subtle biochemical control mechanisms. Spatial confinement is implemented in various ways, ranging from a defined subcellular location of proteases in specific cell organelles or at defined cellular structures such as membranes to tissue-specific expression. While these regulatory mechanisms are applicable to all proteases, a particular class of proteases, known as self-compartimentalized proteases, have evolved a structurallyencoded “self-confinement” to regulate their proteolytic activities. In these proteases, the active sites are located inside a central cavity where the access of substrates is strictly regulated. Substrate selection is mediated, for example, by additional regulator ☆
Change History: March 2016. M. Kaiser. Title was previously Proteolysis – it has been changed Proteases to more closely fit the content of the article. Text revised and updated throughout. Further Reading updated.
Reference Module in Life Sciences
doi:10.1016/B978-0-12-809633-8.06970-3
1
2
Proteases
proteins, as observed in the paradigmal ubiquitin–proteasome system (UPS). The UPS features an impressively complex substrate selection protein network, in which proteins destined for degradation are labeled with polyubiquitin chains before they are recognized, deubiquitinylated, unfolded and ultimately translocated into the self-compartmentalized protease known as the 20S proteasome. Remarkably, the whole UPS encompasses several hundred different proteins. It should however be noted that the UPS is an exceptional complex degradation system and much “simpler” selection strategies for limiting access to selfcompartmentalized proteases are also known. For example, compartmentalized proteases can have spatially restricted entrance gates to ensure that only unfolded substrates reach the active sites within the proteolytic chamber while native, folded proteins are excluded. Such “structural” selection criteria are, for example, often found in proteases involved in protein quality control. Some protein quality control proteases employ tethered PDZ domains that bind peptides and mediate allosteric activation as well as positive cooperativity. Recent evidence suggests that these PDZ domains can also be involved in disaggregation of amyloid fibrils to allow efficient proteolytic degradation of individual peptide strands. Alternatively, post-translational modifications (PTMs) are crucial for the regulation of proteases. PTMs are, for example, critical elements for the conversion of inactive proenzymes into active proteases. Conversion of the proenzymes into active enzymes requires a proteolytic processing. Besides this protease-“specific” PTM, also “classical” PTMs such as phosphorylation have been demonstrated to regulate protease activity in vivo. Inhibition of the proteolytic activity, either by direct binding of small molecule or proteinaceous inhibitors at the active site or at allosteric sites of the protease represents another biochemical regulatory mechanism. Living organisms possess a whole set of endogenous protein inhibitors for scavenging and deactivating proteolytically active proteases, for example, in the blood stream where the serpins (serine protease inhibitors) are controlled independently by switching between active, latent, and inactive forms. To date, it seems as most endogenous inhibitors act directly at the active site, but the identification of allosteric modulators of protease activity that – in contrast to active site inhibitors – sometimes also mediate an activation of the protease is currently under intense investigation. While peptide bond hydrolysis is the unifying feature in all proteolytic events, the extent of substrate degradation often various significantly, ranging from extensive protein degradation into amino acids and/or small peptide fragments to highly selective protein processing. The first case is realized, for example, during food digestion which serves to refill the amino acid pool of living organisms or for elimination of misfolded proteins during protein quality control. It is also employed in a process known as regulatory proteolysis which serves to irreversible remove proteins from the cells, thereby regulating cellular processes. In eukaryotic cells, this task is a prominent element in the above-mentioned UPS. In contrast to “bulk” degradation of substrates, finely balanced proteolytic events occur. Such “limited proteolysis” is usually performed by proteases with very strict cleavage preferences, thereby ensuring a highly sequence-specific proteolysis at a welldefined site in a substrate. Consequently, limited proteolysis events are equipollent to irreversible PTMs. Limited proteolysis is again a key process in innumerable biological processes, including the prominent examples of the proteolytic regulation of blood clotting or apoptosis. Proteases play also key roles in the regulation of gene expression. For example, proteolysis of proteins involved in signaling cascades often leads to enhanced or repressed gene expression. While such modulation is more indirect, proteolysis also interferes directly with gene expression, for example, via proteolytic inactivation of transcriptional repressors or the oppositional activation of transcriptional activators. These findings pinpoint that proteases are powerful enzymes that link biochemistry and genetics within cells and – in principle – could be exploited as tools in genetic studies.
Tools for Studying Proteases In order to identify the biological function of a protease of interest, an elucidation of its active state under various physiological conditions as well as its potential substrates is essential. For determining the active state of proteases, activity-based protein profiling (ABPP) has proven to be a particular potent methodology. In ABPP, small molecule “activity-based probes” (ABPs) are used to monitor the active state of proteases. ABPs consist of an inhibitor residue (also often referred to as a “warhead”) that binds to the protease of interest in an irreversible, covalent manner, a tag such as the fluorophore rhodamine or the small molecule biotin for visualization and/or purification of the labeled protease, and a linker moiety for the connection of both moieties. As binding and thus labeling of the protease of interest can only occur if the protease is in its active state, a subsequent protein gel or mass spectrometric analysis reveals if the protease was active in the biological process under investigation. While initially ABPP was limited to proteases that proceed via a covalent intermediate, recent technological advances such as the incorporation of photoactivatable tags have expanded the application range of ABPP also to metallo or aspartate proteases. Additionally, in vivo activity-imaging of proteases by application of modified ABPs has recently been established, offering novel and exciting experimental setups for protease research. Furthermore, the elucidation of “substrate/protease matches” is of crucial importance. Practically, two different scientific challenges can be differentiated. First, the identification of a protease that cleaves a particular substrate in vivo. Second, the identification of the in vivo substrates and thus biological function of a given protease. Again, a vast array of approaches and
Proteases
3
techniques are available. LC–MS-based proteomics methodologies, often coupled with gel-based separation approaches, isotope mass tag systems such as SILAC and/or cell biological knockout approaches are due to their robustness and wide applicability often employed. Nevertheless, further expansions of existing and new methodologies are constantly developed as to date no unifying approach for all scientific challenges have yet been established.
Proteases as Tools for Cell Biology Research The proteolytic activity of proteases can also be exploited to elucidate the biological function of target proteins in cells. These approaches are based on a targeted and thus specific degradation of proteins. Consequently, such an approach is comparable and complementary to a conditional genetic knockout of the corresponding target protein. The obvious prerequisite is a selective degradation at a given point-in-time. To achieve this challenging task, various approaches have been implemented. First, specificity can be achieved by a fusing an engineered destabilizing domain to the target protein. The presence of a destabilizing domain induces degradation of the hybrid construct under normal conditions. The destabilizing domain, however, can be engineered to bind a specific cell-permeable small molecule ligand that inactivates destabilizing domain and therefore no longer promotes proteolysis. Therefore, proteolytic degradation depends on the presence or absence of the small molecule ligand that serves as the on/off-switch of the system. In the complementary PROTACs approach, targeted degradation is initiated upon addition of a special small molecule ligand (PROTAC). PROTACS are modular chemical compounds that consist of a small molecule portion destined to bind the target protein, a second, often peptidic residue that serves as an E3 ubiquitin-ligase recognition element and a linker that connects both moieties. The E3-Ubiquitin-ligase mediates polyubiquitinylation and subsequent proteasomal degradation of the target protein. Therefore, selective degradation of is achieved by a specific small molecule ligand while control over degradation is achieved via addition of the PROTAC to the biological system. Advantageously, this approach requires no prior genetic engineering. However, it depends on selective POI small molecule ligands that need to be designed and chemically synthesized. A third approach is the use of Tobacco Etch Virus (TEV) protease that targets a 7 residues consensus cleavage site. Engineering of such TEV protease sites into the target protein allows in combination with regulated coexpression of TEV protease rapid processing in vivo. The pronounced specificity of TEV and related proteases even allows the cleavage of nascent chains emerging from the ribosome with ribosome-attached protease.
Proteases as Drug Targets Proteases have a long-lasting history as drug targets and several small molecule inhibitors of proteases have already entered the market. In the last decades, medicinal basic research efforts has led to the elucidation of proteases that can serve as drug targets due to their direct implication in the pathogenesis of various disorders. These implications can range from a disease-causing dysregulation of their proteolytic activities to proteases of viral or microbial origin that play a key role during microbial or viral infections. Consequently, their targeted inhibition often exerts a positive therapeutic effect and is therefore a frequent approach in the discovery of novel drugs. For example, inhibitors of the viral HIV protease have become one of the most important drugs for combating the human immune disease AIDS. As proteases are well-described druggable targets, they are also often targeted to alleviate disease symptoms which leads to a therapeutic effect in a more indirect manner. The most prominent examples of such drug discovery strategies are the development of blood pressure lowering drugs such as captopril or enalapril. These small molecules effectively inhibit the blood pressure regulation protease angiotensin-converting enzyme (ACE), thereby lightening the impact of high blood pressure without however addressing its primary cause. While several mechanism-based strategies for the successful rational development of protease inhibitors have evolved in the last decades, the major challenge for drug discovery is no longer the identification of potent protease inhibitors but the elucidation of inhibitor structures with suitable pharmacokinetic properties and safety profile. Due to the wealth of structural information on proteases structure-based drug discovery has thereby played a major role in the last years.
Further Readings Banaszynski, L.A., Chen, L.-C., Maynard-Smith, L.A., Ooi, A.G.L., Wandless, T.J., 2006. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004. Barrett, A.J., 2000. Proteases. In: Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., Wingfield, P.T. (Eds.), Current Protocols in Protein Science. New York, NY: John Wiley & Sons, Inc, pp. 21.1.1–21.1.12. Bhattacharyya, S., Yu, H., Mim, C., Matouschek, A., 2014. Regulated protein turnover: Snapshots of the proteasome in action. Nat. Rev. Mol. Cell Biol. 15, 122–133. Bode, W., Huber, R., 1991. Ligand binding: Proteinase–protein inhibitor interactions. Curr. Opin. Struct. Biol. 1, 45–52. Clausen, T., Kaiser, M., Huber, R., Ehrmann, M., 2011. HtrA proteases: Regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12, 152–162.
4
Proteases
Ehrmann, M., Clausen, T., 2004. Proteolysis as a regulatory mechanism. Annu. Rev. Genet. 38, 709–724. Gallastegui, N., Groll, M., 2010. The 26S proteasome: Assembly and function of a destructive machine. Trends Biochem. Sci. 35, 634–642. Groll, M., Bochtler, M., Brandstetter, H., Clausen, T., Huber, R., 2005. Molecular machines for protein degradation. ChemBioChem 6, 222–256. Hauske, P., Ottmann, C., Meltzer, M., Ehrmann, M., Kaiser, M., 2008. Allosteric regulation of proteases. ChemBioChem 9, 2920–2928. Henrichs, T., Mikhaleva, N., Conz, C., et al., 2005. Target-directed proteolysis at the ribosome. Proc. Natl. Acad. Sci. USA 102, 4246–4251. Huber, E.M., Groll, M., 2012. Inhibitors for the immuno- and constitutive proteasome: Current and future trends in drug development. Angew. Chem. Int. Ed. Engl. 51, 8708–8720. Poepsel, S., Sprengel, A., Sacca, B., et al., 2015. Determinants of amyloid fibril degradation by the PDZ protease HTRA1. Nat. Chem. Biol. 11, 862–869. Schneekloth, J.S., Crews, C.M., 2005. Chemical approaches to controlling intracellular protein degradation. ChemBioChem 6, 40–46.
Relevant Website http://merops.sanger.ac.uk/ MEROPS.