An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy.

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Cathepsin L-mediated EGFR cleavage affects intracellular signalling pathways in cancer.

Proteolytic activity in the tumour microenvironment is an important factor in cancer development since it can also affect intracellular signalling pathways via positive feedback loops that result in either increased tumour growth or resistance to anticancer mechanisms. In this study, we demonstrated extracellular cathepsin L-mediated cleavage of epidermal growth factor receptor (EGFR) and identified the cleavage site in the extracellular domain after R224. To further evaluate the relevance of this cleavage, we cloned and expressed a truncated version of EGFR, starting at G225, in HeLa cells. We confirmed the constitutive activation of the truncated protein in the absence of ligand binding and determined possible changes in intracellular signalling. Furthermore, we determined the effect of truncated EGFR protein expression on HeLa cell viability and response to the EGFR inhibitors, tyrosine kinase inhibitor (TKI) erlotinib and monoclonal antibody (mAb) cetuximab. Our data reveal the nuclear localization and phosphorylation of EGFR and signal trancducer and activator of transcription 3 (STAT3) in cells that express the truncated EGFR protein and suggest that these phenomena cause resistance to EGFR inhibitors.

The Early Iron Age elite grave from the Kapiteljska njiva barrow cemetery (Novo mesto, Slovenia): an approach to dental profiling through non-invasive and minimally-invasive techniques.

Kapiteljska njiva is a prehistoric cemetery located in the town of Novo mesto in southern Slovenia. There is a long history of archaeological research at this site, as the first investigations date back to the end of the 19th century. In 2004, an Early Iron Age barrow XVI was investigated. The oldest surviving grave in the barrow is the central grave, numbered XVI/34, which according to its position and the richness of the grave goods, belongs to a woman of higher status. Most likely, she was the first member or initiator of a family that continued to bury its dead in the barrow for the next 300 years. There were no preserved skeletal elements; however, in the head part of the grave, remains of human teeth, mostly the tooth crowns and the shells of enamel from the tooth crowns, were found among the scattered amber beads of a necklace. Moreover, these tooth remains are one of the few human biological materials (mostly fragments of skull and long limb bones) unearthed in this cemetery, reflecting the influence of acidic soil from the burial site. This study aimed to create a dental profile of the deceased in a similar way as during a forensic investigation. The remains were examined macroscopically and stereo-microscopically. The morphological traits were scored following the ASUDAS protocol. Ancestry was estimated by entering these scores into a beta version of the web-based application rASUDAS. Brothwell's system was used for age at death estimation from occlusal attrition. Identification of sex was based on the analysis of sex-specific amelogenin isoforms in dental enamel by nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The present study reveals that the dental remains from Kapiteljska njiva belong to the permanent dentition of a single individual and that only the right upper central incisor and third molar are completely absent. The remains of teeth exhibit a simple external morphology, characterised by the absence of morphological dental traits, with a notable exception of the two-rooted left lower canine. The probability of assigning this individual to the Western Eurasian ancestry group is 98%. According to the degree of dentine exposure on the occlusal surfaces of molars, the estimated age range is 17-25 years. Another line of evidence comes from the observation that the first signs of approximal attrition are present in lower third molars but absent in the only preserved upper counterpart, indicating that the age at which the lower third molars entered into occlusion represents a proxy of the individual's age at death. Data on the chronology of the lower third molar development and eruption in present-day European populations from forensic literature (Brkic et al. 2011; Olze et al. 2008; Selmanagic et al. 2013) and The London Atlas confirm the above age-range estimate. Caution is, however, needed in the interpretation of results, because reference data are not based on the population of the individual's origin. Proteomic analysis classified the individual as a female, which is in line with the archaeological evidence. No pathological lesions or indicators of systemic stress were identified; however, the absence of approximal attrition facets in upper anterior teeth indicates interdental spacing. The results of dental profiling are discussed in the context of historical background, today's clinical knowledge, and epidemiological data.

Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins.

Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.

Circular RNA hsa_circ_0062682 Binds to YBX1 and Promotes Oncogenesis in Hepatocellular Carcinoma.

Circular RNAs (circRNAs) have been shown to play an important role in the pathogenesis of hepatocellular carcinoma (HCC). By implementing available transcriptomic analyses of HCC patients, we identified an upregulated circRNA hsa_circ_0062682. Stable perturbations of hsa_circ_0062682 in Huh-7 and SNU-449 cell lines influenced colony formation, migration, cell proliferation, sorafenib sensitivity, and additionally induced morphological changes in cell lines, indicating an important role of hsa_circ_0062682 in oncogenesis. Pathway enrichment analysis and gene set enrichment analysis of the transcriptome data from hsa_circ_0062682 knockdown explained the observed phenotypes and exposed transcription factors E2F1, Sp1, HIF-1α, and NFκB1 as potential downstream targets. Biotinylated oligonucleotide pulldown combined with proteomic analyses identified protein interaction partners of which YBX1, a known oncogene, was confirmed by RNA immunoprecipitation. Furthermore, we discovered a complex cell-type-specific phenotype in response to the oncogenic potential of hsa_circ_0062682. This finding is in line with different classes of HCC tumours, and more studies are needed to shed a light on the molecular complexity of liver cancer.

Identification of Desmoglein-2 as a novel target of Helicobacter pylori HtrA in epithelial cells.

BACKGROUND: High temperature requirement A (HtrA) is an active serine protease secreted by the group-I carcinogen Helicobacter pylori (H. pylori). The human cell adhesion protein and tumor suppressor E-cadherin (hCdh1) expressed on the surface of gastric epithelial cells was identified as the first HtrA substrate. HtrA-mediated hCdh1 cleavage and subsequent disruption of intercellular adhesions are considered as important steps in H. pylori pathogenesis. In this study, we performed a proteomic profiling of H. pylori HtrA (HpHtrA) to decipher the complex mechanism of H. pylori interference with the epithelial barrier integrity. RESULTS: Using a proteomic approach we identified human desmoglein-2 (hDsg2), neuropilin-1, ephrin-B2, and semaphorin-4D as novel extracellular HpHtrA substrates and confirmed the well characterized target hCdh1. HpHtrA-mediated hDsg2 cleavage was further analyzed by in vitro cleavage assays using recombinant proteins. In infection experiments, we demonstrated hDsg2 shedding from H. pylori-colonized MKN28 and NCI-N87 cells independently of pathogen-induced matrix-metalloproteases or ADAM10 and ADAM17. CONCLUSIONS: Characterizing the substrate specificity of HpHtrA revealed efficient hDsg2 cleavage underlining the importance of HpHtrA in opening intercellular junctions. Video Abstract.

Modulated Electro-Hyperthermia Induces a Prominent Local Stress Response and Growth Inhibition in Mouse Breast Cancer Isografts.

Modulated electro-hyperthermia (mEHT) is a selective cancer treatment used in human oncology complementing other therapies. During mEHT, a focused electromagnetic field (EMF) is generated within the tumor inducing cell death by thermal and nonthermal effects. Here we investigated molecular changes elicited by mEHT using multiplex methods in an aggressive, therapy-resistant triple negative breast cancer (TNBC) model. 4T1/4T07 isografts inoculated orthotopically into female BALB/c mice were treated with mEHT three to five times. mEHT induced the upregulation of the stress-related Hsp70 and cleaved caspase-3 proteins, resulting in effective inhibition of tumor growth and proliferation. Several acute stress response proteins, including protease inhibitors, coagulation and heat shock factors, and complement family members, were among the most upregulated treatment-related genes/proteins as revealed by next-generation sequencing (NGS), Nanostring and mass spectrometry (MS). pathway analysis demonstrated that several of these proteins belong to the response to stimulus pathway. Cell culture treatments confirmed that the source of these proteins was the tumor cells. The heat-shock factor inhibitor KRIBB11 reduced mEHT-induced complement factor 4 (C4) mRNA increase. In conclusion, mEHT monotherapy induced tumor growth inhibition and a complex stress response. Inhibition of this stress response is likely to enhance the effectiveness of mEHT and other cancer treatments.

The Structure of Clostridioides difficile SecA2 ATPase Exposes Regions Responsible for Differential Target Recognition of the SecA1 and SecA2-Dependent Systems.

SecA protein is a major component of the general bacterial secretory system. It is an ATPase that couples nucleotide hydrolysis to protein translocation. In some Gram-positive pathogens, a second paralogue, SecA2, exports a different set of substrates, usually virulence factors. To identify SecA2 features different from SecA(1)s, we determined the crystal structure of SecA2 from Clostridioides difficile, an important nosocomial pathogen, in apo and ATP-γ-S-bound form. The structure reveals a closed monomer lacking the C-terminal tail (CTT) with an otherwise similar multidomain organization to its SecA(1) homologues and conserved binding of ATP-γ-S. The average in vitro ATPase activity rate of C. difficile SecA2 was 2.6 ± 0.1 µmolPi/min/µmol. Template-based modeling combined with evolutionary conservation analysis supports a model where C. difficile SecA2 in open conformation binds the target protein, ensures its movement through the SecY channel, and enables dimerization through PPXD/HWD cross-interaction of monomers during the process. Both approaches exposed regions with differences between SecA(1) and SecA2 homologues, which are in agreement with the unique adaptation of SecA2 proteins for a specific type of substrate, a role that can be addressed in further studies.

Time-Dependent miRNA Profile during Septic Acute Kidney Injury in Mice.

(1) Background: Lipopolysaccharide (LPS)-induced systemic inflammation is associated with septic acute kidney injury (AKI). We investigated the time-dependent miRNA expression changes in the kidney caused by LPS. (2) Methods: Male outbred NMRI mice were injected with LPS and sacrificed at 1.5 and 6 h (40 mg/kg i.p., early phase, EP) or at 24 and 48 h (10 mg/kg i.p., late phase, LP). The miRNA profile was established using miRCURY LNA™ microarray and confirmed with qPCR. Total renal proteome was analyzed by LC-MS/MS (ProteomeXchange: PXD014664). (3) Results: Septic AKI was confirmed by increases in plasma urea concentration and in renal TNF-α and IL-6 mRNA expression. Most miRNAs were altered at 6 and 24 h and declined by 48 h. In EP miR-762 was newly identified and validated and was the most elevated miRNA. The predicted target of miR-762, Ras related GTPase 1B (Sar1b) was downregulated. In LP miR-21a-5p was the most influenced miRNA followed by miR-451a, miR-144-3p, and miR-146a-5p. Among the potential protein targets of the most influenced miRNAs, only aquaporin-1, a target of miR-144-3p was downregulated at 24 h. (4) Conclusion: Besides already known miRNAs, septic AKI upregulated miR-762, which may regulate GTP signaling, and miR-144-3p and downregulated its target, aquaporin-1.

Degradomics in Biomarker Discovery.

The upregulation of protease expression and proteolytic activity is implicated in numerous pathological conditions such as neurodegeneration, cancer, cardiovascular and autoimmune diseases, and bone degeneration. During disease progression, various proteases form characteristic patterns of cleaved proteins and peptides, which can affect disease severity and course of progression. It has been shown that qualitative and quantitative monitoring of cleaved protease substrates can provide relevant prognostic, diagnostic, and therapeutic information. As proteolytic fragments and peptides generated in the affected tissue are commonly translocated to blood, urine, and other proximal fluids, their possible application as biomarkers is the subject of ongoing research. The field of degradomics has been established to enable the global identification of proteolytic events on the organism level, utilizing proteomic approaches and sample preparation techniques that facilitate the detection of proteolytic processing of protease substrates in complex biological samples. In this review, some of the latest developments in degradomic methodologies used for the identification and validation of biologically relevant proteolytic events and their application in the search for clinically relevant biomarker candidates are presented. The current state of degradomics in clinics is discussed and the future perspectives of the field are outlined.

Characterization of Legumain Degradome Confirms Narrow Cleavage Specificity.

Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. It is ubiquitously expressed in various tissues and besides its involvement in immune response and other physiological processes, it was also shown to play a role in pathological states such as inflammation and cancer. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins derived from MDA-MB-231 cells using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed narrow cleavage specificity for P1 position combined with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events.

The Acute Phase Response Is a Prominent Renal Proteome Change in Sepsis in Mice.

(1) Background: Sepsis-induced acute kidney injury (AKI) is the most common form of acute kidney injury (AKI). We studied the temporal profile of the sepsis-induced renal proteome changes. (2) Methods: Male mice were injected intraperitoneally with bacterial lipopolysaccharide (LPS) or saline (control). Renal proteome was studied by LC-MS/MS (ProteomeXchange: PXD014664) at the early phase (EP, 1.5 and 6 h after 40 mg/kg LPS) and the late phase (LP, 24 and 48 h after 10 mg/kg LPS) of LPS-induced AKI. Renal mRNA expression of acute phase proteins (APP) was assessed by qPCR. (3) Results: Renal proteome change was milder in EP vs. LP. APPs dominated the proteome in LP (proteins upregulated at least 4-fold (APPs/all): EP, 1.5 h: 0/10, 6 h: 1/10; LP, 24 h: 22/47, 48 h: 17/44). Lipocalin-2, complement C3, fibrinogen, haptoglobin and hemopexin were the most upregulated APPs. Renal mRNA expression preceded the APP changes with peak effects at 24 h, and indicated renal production of the majority of APPs. (4) Conclusions: Gene expression analysis revealed local production of APPs that commenced a few hours post injection and peaked at 24 h. This is the first demonstration of a massive, complex and coordinated acute phase response of the kidney involving several proteins not identified previously.

Protease Specificity: Towards In Vivo Imaging Applications and Biomarker Discovery.

Proteases are considered of major importance in biomedical research because of their crucial roles in health and disease. Their ability to hydrolyze their protein and peptide substrates at single or multiple sites, depending on their specificity, makes them unique among the enzymes. Understanding protease specificity is therefore crucial to understand their biology as well as to develop tools and drugs. Recent advancements in the fields of proteomics and chemical biology have improved our understanding of protease biology through extensive specificity profiling and identification of physiological protease substrates. There are growing efforts to transfer this knowledge into clinical modalities, but their success is often limited because of overlapping protease features, protease redundancy, and chemical tools lacking specificity. Herein, we discuss the current trends and challenges in protease research and how to exploit the growing information on protease specificities for understanding protease biology, as well as for development of selective substrates, cleavable linkers, and activity-based probes and for biomarker discovery.

Cystatin C deficiency suppresses tumor growth in a breast cancer model through decreased proliferation of tumor cells.

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro, indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-ß pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

Protease cleavage site fingerprinting by label-free in-gel degradomics reveals pH-dependent specificity switch of legumain.

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.

Salivary Tick Cystatin OmC2 Targets Lysosomal Cathepsins S and C in Human Dendritic Cells.

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

FPPS: Fast Profiling of Protease Specificity.

Profiling of protease specificity is crucial for characterization of these important enzymes that play numerous roles in health and disease. In the past, several proteomic methods have been developed that enable profiling of protease specificities. Although able to identify thousands of protease cleavage events, these degradomics approaches are often time consuming and methodologically challenging, which limits their application to specialized proteomic groups or the laboratories that initially introduced them. The FPPS approach described here (i.e., fast proteomic profiling of protease specificity) is simple and straightforward and produces reliable results comparable to other more elaborate procedures. It employs labeling the novel N-termini generated by the protease under investigation and subsequent peptide fractionation on SAX-C18 Stage Tips. The procedure can be performed in 2 days, does not require peptide enrichment steps, and can thus be implemented in any proteomic laboratory.

Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea.

BACKGROUND: Omics approaches have significantly increased our understanding of biological systems. However, they have had limited success in explaining the dramatically increased productivity of commercially important natural products by industrial high-producing strains, such as the erythromycin-producing actinomycete Saccharopolyspora erythraea. Further yield increase is of great importance but requires a better understanding of the underlying physiological processes. RESULTS: To reveal the mechanisms related to erythromycin yield increase, we have undertaken an integrated study of the genomic, transcriptomic, and proteomic differences between the wild type strain NRRL2338 (WT) and the industrial high-producing strain ABE1441 (HP) of S. erythraea at multiple time points of a simulated industrial bioprocess. 165 observed mutations lead to differences in gene expression profiles and protein abundance between the two strains, which were most prominent in the initial stages of erythromycin production. Enzymes involved in erythromycin biosynthesis, metabolism of branched chain amino acids and proteolysis were most strongly upregulated in the HP strain. Interestingly, genes related to TCA cycle and DNA-repair were downregulated. Additionally, comprehensive data analysis uncovered significant correlations in expression profiles of the erythromycin-biosynthetic genes, other biosynthetic gene clusters and previously unidentified putative regulatory genes. Based on this information, we demonstrated that overexpression of several genes involved in amino acid metabolism can contribute to increased yield of erythromycin, confirming the validity of our systems biology approach. CONCLUSIONS: Our comprehensive omics approach, carried out in industrially relevant conditions, enabled the identification of key pathways affecting erythromycin yield and suggests strategies for rapid increase in the production of secondary metabolites in industrial environment.

Current trends and challenges in proteomic identification of protease substrates.

Proteolytic cleavage is a ubiquitous, irreversible, posttranslational modification that changes protein structure and function and plays an important role in numerous physiological and pathological processes. Over the last decade, proteases have become increasingly important clinical targets because many of their inhibitors are already used in the clinic or in various stages of clinical testing. Therefore, a better understanding of protease action and their repertoires of physiological substrates can not only provide an important insight into their mechanisms of action but also open a path toward novel drug design. Historically, proteases and their substrates were mainly studied on a case-by-case basis, but recent advancements in mass spectrometry-based proteomics have enabled proteolysis studies on a global scale. Because there are many different types of proteases that can operate in various cellular contexts, multiple experimental approaches for their degradomic characterization had to be developed. The present paper reviews the mass spectrometry-based approaches for determining the proteolytic events in complex biological samples. The methodologies for substrate identification and the determination of protease specificity are discussed, with a special focus on terminomic strategies, which combine peptide labeling and enrichment.

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.