Substrate Library Research Articles

Profiling of Microbial Colonies for High-Throughput Engineering of Multistep Enzymatic Reactions via Optically Guided Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.

Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates.

House dust mite (HDM) protease allergens, through cleavages of critical surface proteins, drastically influence the initiation of the Th2 type immune responses. However, few human protein substrates for HDM proteases have been identified so far, mainly by applying time-consuming target-specific individual studies. Therefore, the identification of substrate repertoires for HDM proteases would represent an unprecedented key step toward a better understanding of the mechanism of HDM allergic response. In this study, phage display screenings using totally or partially randomized nonameric peptide substrate libraries were performed to characterize the extended substrate specificities (P5–P4′) of the HDM proteases Der p 1, Der p 3 and Der p 6. The bioinformatics interface PoPS (Prediction of Protease Specificity) was then applied to define the proteolytic specificity profile of each protease and to predict new protein substrates within the human cell surface proteome, with a special focus on immune receptors. Specificity profiling showed that the nature of residues in P1 but also downstream the cleavage sites (P′ positions) are important for effective cleavages by all three HDM proteases. Strikingly, Der p 1 and Der p 3 display partially overlapping specificities. Analysis with PoPS interface predicted 50 new targets for the HDM proteases, including 21 cell surface receptors whose extracellular domains are potentially cleaved by Der p 1, Der p 3 and/or Der p 6. Twelve protein substrate candidates were confirmed by phage ELISA (enzyme linked immunosorbent assay). This extensive study of the natural protein substrate specificities of the HDM protease allergens unveils new cell surface target receptors for a better understanding on the role of these proteases in the HDM allergic response and paves the way for the design of specific protease inhibitors for future anti-allergic treatments.

Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration

Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-dl-Trp. The production of both 4-Me-5-NO2-l-Trp and 4-Me-7-NO2-l-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

Building a Synthetic Library of Fluorogenic Ester Substrates to Analyze Serine Hydrolases

Mycobacterium tuberculosis, the organism responsible for the respiratory infection tuberculosis, has evolved multi‐drug resistance strains, introducing the need for novel drug targets. Serine hydrolases with diverse roles in lipid metabolism and dormant growth have been proposed as novel targets for tuberculosis. Although many serine hydrolases from TB have been identified by proteomics and activity based protein profiling, the natural substrates and biological reactions for these serine hydrolases are still unknown. To globally investigate the natural substrates for mycobacterial serine hydrolases, we synthesized a structure activity library of 32 fluorogenic ester substrates to screen against mycobacterial hydrolases. The library contains acyloxy methyl ester derivatives of fluorescein with small variations in ester functionalities to mimic mycobacterial lipids. Synthesis of novel compounds required separation techniques including silica‐gel column chromatography, along with characterization techniques including 1HNMR and 13CNMR and high‐resolution mass spectroscopy. To confirm the utility of the fluorogenic library for characterizing serine hydrolases, two structurally homologous bacterial serine hydrolases, Rv0045c from M. tuberculosis and ybfF from V. cholera, were screened against the library of fluorogenic substrates to determine the catalytic efficiency of these enzymes on each substrate. Rv0045c and ybfF both favor short, 3‐atom, ether chains with the heteroatom in the b‐position. ybfF with the most favored substrates showed catalytic efficiencies of high activity (kcat/kM > 104 M−1s−1), while Rv0045c did not perform as well (kcat/kM 103–104 M−1s−1). In addition, comparative kinetic analysis of hydrolase variants with substitutions in their binding pocket provided insight into the substrate specificity of the enzymes, and thereby the natural substrate. Catalytic efficiency deviations from the WT, could be a result of steric hindrance, hydrogen bond interactions, and localized acid‐base chemistry. Future work would be aimed at making more variants and determining the structural features, which contribute to their substrate specificity.Support or Funding InformationFunded by NIH 1R15GM110641‐01A1

Real-Time Screening of Biocatalysts in Live Bacterial Colonies.

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold.

Neutrophils are directly responsible for destroying invading pathogens via reactive oxygen species, antimicrobial peptides, and neutrophil serine proteases (NSPs). Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders, and engineered inhibitors of NSPs are a potential therapeutic pathway. In this study we characterized the extended substrate specificity (P4-P1) of the NSP cathepsin G using a peptide substrate library. Substituting preferred cathepsin G substrate sequences into sunflower trypsin inhibitor-1 (SFTI-1) produced a potent cathepsin G inhibitor (Ki = 0.89 nM). Cathepsin G's P2' preference was determined by screening against a P2' diverse SFTI-based library, and the most preferred residue at P2' was combined in SFTI-1 with a preferred substrate sequence (P4-P2) and a nonproteinogenic P1 residue (4-guanidyl-l-phenylalanine) to produce a potent (Ki = 1.6 nM) and the most selective (≥360-fold) engineered cathepsin G inhibitor reported to date. This compound is a promising lead for further development of cathepsin G inhibitors targeting chronic inflammatory disorders.

Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER. Multicolor FACS screening is used to isolate cells labeled with fluorophore-conjugated antibodies, followed by NGS to profile the cleaved substrates. The YESS-NGS method was successfully applied to profile the sequence specificity of the wild-type and an engineered variant of the tobacco etch mosaic virus protease. Proteolysis in the yeast secretory pathway was also mapped for the first time in vivo revealing a major cleavage pattern of Ali/Leu-X-Lys/Arg-Arg. Here Ali is any small aliphatic residue, but especially Leu. This pattern was verified to be due to the well-known endogenous protease Kex2 after comparison to a newly generated Kex2 knockout strain as well as cleavage of peptides with recombinant Kex2 in vitro. This information is particularly important for those using yeast display technology, as library members with Ali/Leu-X-Lys/Arg-Arg patterns are likely being removed from screens via Kex2 cleavage without the researcher's knowledge.

Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease

Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease.

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

ABSTRACTThe nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat/K m values >10,000 M−1 s−1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat/K m values exhibited against these canonical substrates are well under 105 M−1 s−1. By contrast, several Nudix enzymes show much larger k cat/K m values (in the range of 105 to >107 M−1 s−1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

A Semiautomated Structure-Based Method To Predict Substrates of Enzymes via Molecular Docking: A Case Study with Candida antarctica Lipase B.

The discovery of unique substrates is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming, and expensive. Although in silico structure-based approaches show great promise, recent extensive studies have shown that these approaches remain a formidable challenge for current biocomputational methodologies. Here we present an open-source, extensible, and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of an enzyme on the basis of the enzyme's catalysis mechanism. On the basis of a generalized transition state theory of enzyme catalysis, we introduce a modified docking procedure called "mechanism-based restricted docking" (MBRD) for novel substrate recognition from molecular docking. Comprising a series of utilities written in C/Python, THEMIS automatically executes parallel-computing MBRD tasks and evaluates the results with various molecular mechanics (MM) criteria such as energy, distance, angle, and dihedral angle to help identify desired substrates. Exhaustive sampling and statistical measures were used to improve the robustness and reproducibility of the method. We used Candida antarctica lipase B (CALB) as a test system to demonstrate the effectiveness of our computational prediction of (non)substrates. A novel MM score function for CALB substrate identification derived from the near-attack conformation was used to evaluate the possibility of chemical transformation. A highly positive rate of 93.4% was achieved from a CALB substrate library with 61 known substrates and 35 nonsubstrates, and the screening rate has reached 103 compounds/day (96 CPU cores, 100 samples/compound). The performance shows that the present method is perhaps the first reported scheme to meet the requirement for practical applicability to enzyme studies. An additional study was performed to validate the universality of our method. In this verification we employed two distinct enzymes, nitrilase Nit6803 and SDR Gox2181, where the correct rates of both enzymes exceeded 90%. The source code used will be released under the GNU General Public License (GPLv3) and will be free to download. We believe that the present method will provide new insights into enzyme research and accelerate the development of novel enzyme applications.

Phage display and structural studies reveal plasticity in substrate specificity of caspase-3a from zebrafish.

The regulation of caspase-3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase-3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase-3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5-P1' or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X-ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub-site by increasing flexibility of one active site loop and by affecting hydrogen-bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase-6 in zebrafish development.

A Spectral Emissivity Library of Spoil Substrates

Post-mining sites have a significant impact on surrounding ecosystems. Afforestation can restore these ecosystems, but its success and speed depends on the properties of the excavated spoil substrates. Thermal infrared remote sensing brings advantages to the mapping and classification of spoil substrates, resulting in the determination of its properties. A library of spoil substrates containing spectral emissivity and chemical properties can facilitate remote sensing activities. This study presents spectral library of spoil substrates’ emissivities extracted from brown coal mining sites in the Czech Republic. Extracted samples were homogenized by drying and sieving. Spectral emissivity of each sample was determined by spectral smoothing algorithm applied to data measured by a Fourier transform infrared (FTIR) spectrometer. A set of chemical parameters (pH, conductivity, Na, K, Al, Fe, loss on ignition and polyphenol content) and toxicity were determined for each sample as well. The spectral library presented in this paper also offers valuable information in the form of geographical coordinates for the locations where samples were obtained. Presented data are unique in nature and can serve many remote sensing activities in longwave infrared electromagnetic spectrum.

Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

Legumain (AEP) is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP upregulation is linked to a number of diseases including inflammation, arteriosclerosis, and tumorigenesis. Thus this protease is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.

The unusual substrate specificity of a virulence associated serine hydrolase from the highly toxic bacterium, Francisella tularensis

Francisella tularensis is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of F. tularensis was the proposed serine hydrolase FTT0941c. Herein, we purified FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent Francisella subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in F. tularensis.

Human 20S proteasome activity towards fluorogenic peptides of various chain lengths

The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

Transition Metal Cation Inhibition of Mycobacterium tuberculosis Esterase Rv0045c

The survival of Mycobacterium tuberculosis within its human host is highly dependent upon the lipid metabolism performed by the numerous serine hydrolase enzymes at the bacterium's disposal. Among these serine hydrolases, Rv0045c is a mycobacterial‐specific serine hydrolase that has been characterized for global substrate specificity by scanning the hydrolase against a library of fluorogenic hydrolase substrates. Rv0045c expresses strong selectivity for short, straight‐chained alkyl esters, but the relative enzyme kinetic rate is slow in comparison to a majority of bacterial hydrolases. Herein, we determined the transition metal ion dependence of Rv0045c on hydrolysis of the four‐carbon methoxyacetic fluorogenic substrate. The catalytic activity of Rv0045c was significantly inhibited by low concentrations of Ni+2, Cu+2, and Zn+2 metal cations while other 3d metal cations displayed no effects on the activity of the hydrolase. The low inhibition constants for these three metals on Rv0045c suggest a likely physiological relevance for the metal dependent inhibition of Rv0045c.Support or Funding InformationFunding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.

PEGylated substrates of NSP4 protease: A tool to study protease specificity.

Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

Barrel-shaped ClpP Proteases Display Attenuated Cleavage Specificities.

ClpP is a self-compartmentalizing protease with crucial roles in bacterial and mitochondrial protein quality control. Although the ClpP homocomplex is composed of 14 equivalent active sites, it degrades a multitude of substrates to small peptides, demonstrating its capability to carry out diverse cleavage reactions. Here, we show that ClpP proteases from E. coli, S. aureus, and human mitochondria exhibit preferences for certain amino acids in the P1, P2, and P3 positions using a tailored fluorogenic substrate library. However, this high specificity is not retained during proteolysis of endogenous substrates as shown by mass spectrometric analysis of peptides produced in ClpXP-mediated degradation reactions. Our data suggest a mechanism that implicates the barrel-shaped architecture of ClpP not only in shielding the active sites to prevent uncontrolled proteolysis but also in providing high local substrate concentrations to enable efficient proteolytic processing. Furthermore, we introduce customized fluorogenic substrates with unnatural amino acids that greatly surpass the sensitivity of previously used tools. We used these to profile the activity of cancer-patient- and Perrault-syndrome-derived ClpP mutant proteins.

Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.