Synthetic and biological approaches to map substrate specificities of proteases

2019 ◽  
Vol 401 (1) ◽  
pp. 165-182 ◽  
Author(s):  
Shiyu Chen ◽  
Joshua J. Yim ◽  
Matthew Bogyo
Keyword(s):  
Substrate Specificity ◽  
Antigen Processing ◽  
Therapeutic Agents ◽  
Protein Catabolism ◽  
Parasite Invasion ◽  
Peptide Substrates ◽  
Protein Substrates ◽  
Putative Protein ◽  
Natural Protein ◽  
Biological Methods

Abstract Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various disease conditions, such as malignant metastasis, viral infection and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed.

scholarly journals Serpin-derived Peptide Substrates for Investigating the Substrate Specificity of Human Tissue Kallikreins hK1 and hK2

1997 ◽  
Vol 272 (47) ◽  
pp. 29590-29595 ◽  
Author(s):  
Luc Bourgeois ◽  
Michèle Brillard-Bourdet ◽  
David Deperthes ◽  
Maria A. Juliano ◽  
Luiz Juliano ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Tissue ◽  
Peptide Substrates

A Proteasome Specific Binding Assay for Quantitation of Constitutive and Immunoproteasome Active Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2472-2472
Author(s):  
Mark K. Bennett ◽  
Monette A. Aujay ◽  
Tonia J. Buchholz ◽  
Susan D. Demo ◽  
Guy J. Laidig ◽  
...  
Keyword(s):  
Active Sites ◽  
Antigen Processing ◽  
Specific Binding ◽  
Peripheral Blood Mononuclear ◽  
Peptide Substrates ◽  
Enzyme Preparations ◽  
Proteolytic Activities ◽  
Fluorogenic Peptide

Abstract The ubiquitin-proteasome pathway constitutes a major intracellular system for protein degradation. Substrates for this pathway include misfolded or unassembled proteins as well as short-lived regulatory proteins that play key roles in signaling and proliferative pathways. The majority of cell types express the standard, or “constitutive”, form of the proteasome, while cells of the immune system also express the immunoproteasome, a form of the proteasome that contributes to class I major histocompatibility complex restricted antigen processing. Non-immune cells can also express immunoproteasome in response to interferon gamma exposure. The immunoproteasome retains the same structural subunits as the constitutive proteasome but has three different catalytic subunits. The catalytic activities of both forms of the proteasome have been traditionally characterized with purified enzyme preparations and fluorogenic peptide substrates. Such fluorogenic peptide substrates suffer from two characteristics that limit their utility in measuring proteasome activities in complex cell or tissue lysates: 1) they cannot distinguish proteasome activities from other proteolytic activities within the lysate; and 2) they can not distinguish between constitutive and immunoproteasome activities. We have developed an ELISA-based proteasome-specific binding (PSB) assay that can detect and quantify the chymotryptic-like proteasome active sites of the beta-5 constitutive proteasome subunit and the LMP7 immunoproteasome subunit. The assay utilizes a biotin-modified peptide epoxyketone probe that covalently and irreversibly interacts with the active site threonine present in catalytic proteasome subunits. Once bound to the probe, the labeled subunits are recovered on streptavidin-conjugated beads and detected with subunit-specific antibodies. The PSB assay is both quantitative and sensitive. We have demonstrated that the assay is capable of measuring constitutive proteasome and immunoproteasome binding activity in human whole blood and peripheral blood mononuclear cell preparations, respectively. In experiments with the epoxyketone-based proteasome inhibitor PR-171, the dose response for inhibition of the PSB assay is equivalent to that measured with a conventional fluorogenic peptide proteasome substrate. In addition, the PSB assay can effectively measure the level of PR-171 mediated inhibition of both the constitutive and immunoproteasome in the RPMI-8226 multiple myeloma cell line that co-expresses both proteasome types. Thus, the PSB assay overcomes the limitations of conventional fluorogenic substrate-based proteasome activity assays when applied to cell or tissue lysates that contain multiple proteolytic activities or mixtures of constitutive and immunoproteasomes. Potential applications of the PSB assay include the measurement of the pharmacodynamic response to proteasome inhibitors and the evaluation of constitutive vs. immunoproteasome selectivity of inhibitors both in vitro and in vivo.

Burn injury upregulates the activity and gene expression of the 20 S proteasome in rat skeletal muscle

2000 ◽  
Vol 99 (3) ◽  
pp. 181-187 ◽  
Author(s):  
Cheng-Hui FANG ◽  
Bing-Guo LI ◽  
David R. FISCHER ◽  
Jing Jing WANG ◽  
Herbert A. RUNNELS ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Burn Injury ◽  
Catalytic Core ◽  
Peptide Substrates ◽  
Protein Substrates ◽  
Proteolytic Pathway ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
Dependent Protein ◽  
Increased Activity

There is evidence that burn injury stimulates ubiquitin–proteasome-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, protein substrates are conjugated to multiple molecules of ubiquitin, whereafter they are recognized, unfolded and degraded by the multicatalytic 26 S protease complex. The 20 S proteasome is the catalytic core of the 26 S protease complex. The influence of burn injury on the expression and activity of the 20 S proteasome has not been reported. We tested the hypothesis that burn injury increases 20 S proteasome activity and the expression of mRNA for the 20 S proteasome subunits RC3 and RC7. Proteolytic activity of isolated 20 S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from burned rats. Northern-blot analysis revealed that the expression of mRNA for RC3 and RC7 was increased by 100% and 80% respectively following burn injury. Increased activity and expression of the 20 S proteasome in muscles from burned rats support the concept that burn-induced muscle cachexia is at least, in part, regulated by the ubiquitin–proteasome proteolytic pathway.

scholarly journals Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170078 ◽  
Author(s):  
Karim Rafie ◽  
Olawale Raimi ◽  
Andrew T. Ferenbach ◽  
Vladimir S. Borodkin ◽  
Vaibhav Kapuria ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Substrate Specificity ◽  
Active Site ◽  
Target Site ◽  
Post Translational Modification ◽  
Tetratricopeptide Repeats ◽  
Protein Substrates ◽  
Nucleocytoplasmic Proteins ◽  
Glcnac Transferase ◽  
Single Enzyme

O-linked N -acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.

scholarly journals Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10

2009 ◽  
Vol 424 (1) ◽  
pp. 79-88 ◽  
Author(s):  
Cristina I. Caescu ◽  
Grace R. Jeschke ◽  
Benjamin E. Turk
Keyword(s):  
Cleavage Site ◽  
Active Sites ◽  
Aromatic Amino Acids ◽  
Peptide Substrates ◽  
Protein Substrates ◽  
Site Selectivity ◽  
A Disintegrin And Metalloproteinase ◽  
Factor Α ◽  
Sequence Requirements

The metalloproteinases TACE [tumour necrosis factor α-converting enzyme; also known as ADAM17 (a disintegrin and metalloproteinase 17)] and ADAM10 are the primary enzymes responsible for catalysing release of membrane-anchored proteins from the cell surface in metazoan organisms. Although the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1′ position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, whereas ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identified three residues in the S1′ pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites.

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

2008 ◽  
Vol 416 (1) ◽  
pp. 109-116 ◽  
Author(s):  
Yoshikuni Goto ◽  
Hiroe Tanji ◽  
Akira Hattori ◽  
Masafumi Tsujimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enzymatic Activity ◽  
Basic Amino Acid ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Peptide Substrates ◽  
Endoplasmic Reticulum Aminopeptidase 1 ◽  
Natural Peptides

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.

scholarly journals Histone H4-based peptoids are inhibitors of protein arginine methyltransferase 1 (PRMT1)

2020 ◽  
Vol 477 (16) ◽  
pp. 2971-2980 ◽  
Author(s):  
Sarah A. Mann ◽  
Megan K. DeMart ◽  
Braidy May ◽  
Corey P. Causey ◽  
Bryan Knuckley
Keyword(s):  
Transcriptional Activation ◽  
Amino Acid Side Chain ◽  
Histone H4 ◽  
Peptide Substrates ◽  
Protein Arginine Methyltransferases ◽  
Protein Substrates ◽  
Arginine Residues ◽  
Natural Peptide ◽  
Protein Arginine Methyltransferase 1

Methylation of arginine residues occurs on a number of protein substrates, most notably the N-terminal tails of histones, and is catalyzed by a family of enzymes called the protein arginine methyltransferases (PRMTs). This modification can lead to transcriptional activation or repression of cancer-related genes. To date, a number of inhibitors, based on natural peptide substrates, have been developed for the PRMT family of enzymes. However, because peptides are easily degraded in vivo, the utility of these inhibitors as potential therapeutics is limited. The use of peptoids, which are peptide mimetics where the amino acid side chain is attached to the nitrogen in the amide backbone instead of the α-carbon, may circumvent the problems associated with peptide degradation. Given the structural similarities, peptoid scaffolds may provide enhanced stability, while preserving the mechanism of action. Herein, we have identified that peptoids based on natural peptide substrates are not catalyzed to the product by PRMT1, but instead are inhibitors of this enzyme. Reducing the length of the peptoid reduces inhibition and suggest the residues distal from the site of modification are important for binding. Furthermore, a positive charge on the N-terminus helps promote binding and improves inhibition. Selectivity among family members is likely possible based on inhibition being moderately selective for PRMT1 over PRMT5 and provides a scaffold that can be used to develop pharmaceuticals against this class of enzymes.

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