Cell Death Proteases Research Articles

Calmodulin regulates protease versus co-chaperone activity of a metacaspase

Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both invitro and invivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.

Mechanisms controlling plant proteases and their substrates.

In plants, proteolysis is emerging as an important field of study due to a growing understanding of the critical involvement of proteases in plant cell death, disease and development. Because proteases irreversibly modify the structure and function of their target substrates, proteolytic activities are stringently regulated at multiple levels. Most proteases are produced as dormant isoforms and only activated in specific conditions such as altered ion fluxes or by post-translational modifications. Some of the regulatory mechanisms initiating and modulating proteolytic activities are restricted in time and space, thereby ensuring precision activity, and minimizing unwanted side effects. Currently, the activation mechanisms and the substrates of only a few plant proteases have been studied in detail. Most studies focus on the role of proteases in pathogen perception and subsequent modulation of the plant reactions, including the hypersensitive response (HR). Proteases are also required for the maturation of coexpressed peptide hormones that lead essential processes within the immune response and development. Here, we review the known mechanisms for theactivationof plant proteases, including post-translational modifications, together with the effects of proteinaceous inhibitors.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

<em>In Vitro</em> Cleavage Assays using Purified Recombinant <em>Drosophila</em> Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening.

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

Quantification of Immunostained Caspase-9 in Retinal Tissue.

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

A phase I study of safety and immunogenicity of survivin long peptide vaccine (SurVaxM) in patients (pts) with metastatic neuroendocrine tumors (NETs).

TPS375 Background: Metastatic neuroendocrine tumors (NETs) have a poor prognosis and there are limited options after first-line treatment with somatostatin analogues (SSA)/chemotherapy. Therefore, new therapies are urgently needed. Survivin is an intracellular protein that alters cell division, function of cell death proteases and inhibits apoptosis. It is undetectable in adult cells and expressed in many tumors, making it an ideal target. SurVaxM is a 15 amino acid synthetic peptide vaccine. It stimulates antigen presentation via intracellular cell-surface target recognition and induces CD8+ & CD4+ T cells and IgG production. It was well tolerated in a phase I trial in recurrent glioma pts (Fenstermaker et al., Neurooncol; 2014). Prelim analysis from a phase II study in glioma patients (pts) showed its efficacy (Ahluwalia et al., Neurooncol; 2018). Targeting survivin in NETs is based on our work with tissue microarrays from gastroenteropancreatic (GEP) and lung NETs that showed that its expression correlated with a worse overall survival (Hanif et al., Oncotarget; 2020). We have an ongoing single-arm phase I trial evaluating the safety and immunogenicity of SurVaxM in metastatic NETs. Methods: Ten eligible pts with any grade metastatic GEP/lung origin NETs that are survivin positive by immunohistochemistry and have progression on SSA within the last 6 months on two CT scans >4 weeks apart per RECIST v1.1 are being enrolled. Pts receive a fixed dose of 500 mcg of SurVaxM in Montanide ISA 51 subcutaneously along with 100 mcg of GM-CSF q2 weeks X 4 doses. SSA is continued at the same dose as before. Pts free of progression and toxicity at 6 months get extra doses of SurVaxM q12 weeks, up to 1 year. Subjects are assessed continuously for safety per NCI CTCAEv5.0. Response assessment via CT scans per RECIST v1.1 q12 weeks. Primary objective is to assess safety of SurVaxM +/- SSA. Secondary objectives are to assess overall response rate, progression free survival, duration of response and vaccine immunological response (anti-survivin antibody titers and survivin-specific CD8 T-cell responses). Safety analysis is per Pocock stopping boundary with the assumption of true toxicity of 0.2 and unacceptable toxicity of 0.3 at a significance level of 0.05.To this date, eight patients were screened out of which five patients were enrolled. One patient did not meet the eligibility criterion due to negative survivin staining and the other two did not meet the RECIST v1.1 progression criterion. Of the five pts enrolled, two were of lung origin, two of GEP, and the remainder one of thymic origin. Three pts had high grade neuroendocrine carcinoma, one patient had atypical bronchial tumor and one with low-grade NET. Currently, two pts remain on the study. The enrollment is ongoing. The trial is funded by The Neuroendocrine Tumor Research Foundation.

CTIM-10. PHASE II STUDY OF PEMBROLIZUMAB PLUS SurVaxM FOR GLIOBLASTOMA AT FIRST RECURRENCE

Abstract BACKGROUND Pembrolizumab is a potent humanized immunoglobulin G4 monoclonal antibody with high specificity of binding to the programmed cell death 1 (PD-1) receptor, thus inhibiting its interaction with programmed cell death ligand 2 (PD-L2). Survivin is a 16.5 kDa intracellular protein that belongs to the inhibitor of apoptosis protein (IAP) family. It acts in concert with the mitotic spindle apparatus to regulate cell division and localizes to the spindle microtubule organizing center (MTOC) during the G2/M phase of cell cycle progression. Survivin has also been shown to modulate the function of a number of terminal effector cell death proteases (caspases) leading to an inhibition of apoptosis. METHODS This is a Phase II study of two arms in patients with recurrent glioblastoma. Arm A is patients with first recurrence of glioblastoma who have failed prior chemotherapy and radiation but have not received any immunotherapy. Arm B is an exploratory arm of 10 patients who have failed prior anti-PD1 therapy. The ongoing study is a phase II clinical study with a 10 patient, toxicity run-in. All patients will receive the study drug combination consisting of SurVaxM and pembrolizumab with no randomization, stratification or dose escalation. RESULTS So far ten patients have been enrolled on the study as safety run in. Primary endpoint is Progression free survival at 6 months. Safety and tolerability of Pembrolizumab and SurVaxM, Response rates of Pembrolizumab and SurVaxM determined using RANO criteria are secondary endpoints. Additional secondary endpoints include Overall survival and Progression Free survival Exploratory endpoints include Cellular and humoral immune responses during concurrent administration of Pembrolizumab and SurVaxM. CONCLUSION This is an ongoing clinical trial.

Columbamine suppresses proliferation and invasion of melanoma cell A375 via HSP90-mediated STAT3 activation

Purpose The goal of this study is to explore the effects of columbamine in melanoma cells and the signaling pathway involved. Methods Human melanoma cell line A375 cells were used in this study. Cell proliferative ability was detected by MTT assay and clone formation assay. Cell migration and invasion were measured by wound healing assay and transwell assay, respectively. Protein expression was examined by Western blotting. Results Columbamine reduced cell proliferative ability and the number of clone spots in A375 cells. Western blotting results demonstrated that expression of cleaved caspase 3, an activated cell death protease, was upregulated by 20 and 50 µM of columbamine. Wound healing results showed that the scratch width was wider in cell treated with 20 and 50 µM of columbamine than that in cell treated with 0 and 10 µM of columbamine. Phosphorylation of STAT3 and expression of HSP90 was also repressed by columbamine in a concentration-dependent manner. Overexpression of HSP90 attenuated the inhibition of cell proliferation, migration and invasion induced by columbamine. Conclusion Columbamine inhibited melanoma cell proliferation, migration, and invasion in A375 cells through inactivation of STAT3, which is mediated by HSP90.

Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus

Abstract Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.

Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement.

Under stress, isolated microspores are reprogrammed in vitro towards embryogenesis, producing doubled haploid plants that are useful biotechnological tools in plant breeding as a source of new genetic variability, fixed in homozygous plants in only one generation. Stress-induced cell death and low rates of cell reprogramming are major factors that reduce yield. Knowledge gained in recent years has revealed that initiation and progression of microspore embryogenesis involve a complex network of factors, whose roles are not yet well understood. Here, I review recent findings on the determinant factors underlying stress-induced microspore embryogenesis, focusing on the role of autophagy, cell death, auxin, chromatin modifications, and the cell wall. Autophagy and cell death proteases are crucial players in the response to stress, while cell reprogramming and acquisition of totipotency are regulated by hormonal and epigenetic mechanisms. Auxin biosynthesis, transport, and action are required for microspore embryogenesis. Initial stages involve DNA hypomethylation, H3K9 demethylation, and H3/H4 acetylation. Cell wall remodelling, with pectin de-methylesterification and arabinogalactan protein expression, is necessary for embryo development. Recent reports show that treatments with small modulators of autophagy, proteases, and epigenetic marks reduce cell death and enhance embryogenesis initiation in several crops, opening up new possibilities for improving in vitro embryo production in breeding programmes.

Plant serpin protease inhibitors: specificity and duality of function.

The serpins are a family of structurally conserved protease inhibitors found in all animal and plant kingdoms. After interaction with their cognate substrate(s), their native energetically stressed state is relaxed by hydrolysis, resulting in a semi-stable covalent bond that disables the protease. The inherent flexible serpin structure supports additional non-inhibitory functions. This review will focus on several biological functions attributed to plant serpins, ranging from specific cell death protease inhibitors to a stabilizing role for β-amylase in seeds. Functional conservation of a particular serpin type, the LR serpins, is suggested by its compelling ubiquity throughout the plant kingdom. The multiple target specificity of plant serpins including the LR serpins enables them to perform dual functions that are not mutually exclusive both as a regulator of cell death and as a protective anti-pathogenic protein.

Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley

ABSTRACTMicrospore embryogenesis is a powerful biotechnological tool that is very useful in crop breeding for the rapid production of haploid and double-haploid embryos and plants. In this in vitro system, the haploid microspore is reprogrammed by the application of specific stress treatments. A high level of cell death after the stress is a major factor that greatly reduces embryogenesis yield at its initial stages. Autophagy is a degradation pathway that is present in all eukaryotes and plays key roles in a range of processes, including stress responses. Many proteases participate in autophagy and cell death; among them, cathepsins are the most abundant enzymes with a role in plant senescence and programmed cell death (PCD). Moreover, although plant genomes do not contain homologues of caspases, caspase 3-like activity (main executioner protease of animal cell death) has been detected in many plant PCD processes. Recent studies by our group in barley microspore cultures reported that the stress treatment required for inducing microspore embryogenesis (cold treatment), also produced reactive oxygen species (ROS) and cell death, concomitantly with the induction of autophagy, as well as cathepsin-like and caspase 3-like proteolytic activities. In the present study, we report new data on microspore embryogenesis of rapeseed that indicate, as in barley, activation of cell death and autophagy processes after the inductive stress. The results revealed that treatments modulating autophagy and proteases produced the same effect in the two plant systems, regardless of the stress applied, cold in barley or heat in rapeseed. Pharmacological treatments with small bioactive compounds that inhibit ROS, autophagy and specific cell death-proteases led to reduced cell death and an increased embryogenesis initiation rate in both, barley and rapeseed. Taken together, these findings open up new intervention pathways by modulating autophagy and proteases, which are very promising in terms of increasing the efficiency of in vitro microspore embryogenesis systems for biotechnological applications in crop breeding.

Apoptotic Molecules In Chronic Leukemia

Background: Chronic myeloid leukemia (CML) is characterized by the spread of malignant cells that exhibit resistance to caspase-mediated cell death (apoptosis) and this mechanism is proposed to play an important role in myeloid cell growth. However, the extent to which caspase-mediated cell death plays a crucial role in the regulation of myelopoiesis remains controversial.Objectives & Proceedure: The objectives of this study were to examine whether or not caspase-mediated cell death-related proteins take part in the development of CML and to also to detect the relationship between Fas, p53 and caspase-mediated cell death protease activating factor (Apaf-1) in 5 patients with CML using the real-time quantitative polymerase chain reaction. We demonstratedthatp53 and Apaf-1 messenger ribonucleic acid (mRNA) expression was moderately elevated (up to 5 fold, p<0.05) in 4 out of 5 CML patients. One patient with a p53 point mutation, exhibited a far greater elevation of p53 mRNA expression throughout their blast crisis, but in contrast, displayed a significant reduction in levels of Apaf-1 mRNA and Fas mRNA.Conclusion: Our results show in-vivo linkages between Fas, p53 and Apaf-1 transcription parameters suggesting that the key genes involved in the caspase-mediated cell death might contribute to CML disease development.Bangladesh Journal of Medical Science Vol.15(4) 2016 p.651-654

Caspase-2 deficiency accelerates chemically induced liver cancer in mice.

Aberrant cell death/survival has a critical role in the development of hepatocellular carcinoma (HCC). Caspase-2, a cell death protease, limits oxidative stress and chromosomal instability. To study its role in reactive oxygen species (ROS) and DNA damage-induced liver cancer, we assessed diethylnitrosamine (DEN)-mediated tumour development in caspase-2-deficient (Casp2(-/-)) mice. Following DEN injection in young animals, tumour development was monitored for 10 months. We found that DEN-treated Casp2(-/-) mice have dramatically elevated tumour burden and accelerated tumour progression with increased incidence of HCC, accompanied by higher oxidative damage and inflammation. Furthermore, following acute DEN injection, liver injury, DNA damage, inflammatory cytokine release and hepatocyte proliferation were enhanced in mice lacking caspase-2. Our study demonstrates for the first time that caspase-2 limits the progression of tumourigenesis induced by an ROS producing and DNA damaging reagent. Our findings suggest that after initial DEN-induced DNA damage, caspase-2 may remove aberrant cells to limit liver damage and disease progression. We propose that Casp2(-/-) mice, which are more susceptible to genomic instability, are limited in their ability to respond to DNA damage and thus carry more damaged cells resulting in accelerated tumourigenesis.

Programmed Cell Death During Caenorhabditis elegans Development.

Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role.

Protease signaling in animal and plant-regulated cell death.

This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom-specific with very little overlap.

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action.

Staphylococcus aureus uses the two-component regulatory system GraRS to sense and respond to host defense peptides (HDPs). However, the mechanistic impact of GraS or its extracellular sensing loop (EL) on HDP resistance is essentially unexplored. Strains with null mutations in the GraS holoprotein (ΔgraS) or its EL (ΔEL) were compared for mechanisms of resistance to HDPs of relevant immune sources: neutrophil α-defensin (human neutrophil peptide 1 [hNP-1]), cutaneous β-defensin (human β-defensin 2 [hBD-2]), or the platelet kinocidin congener RP-1. Actions studied by flow cytometry included energetics (ENR); membrane permeabilization (PRM); annexin V binding (ANX), and cell death protease activation (CDP). Assay conditions simulated bloodstream (pH 7.5) or phagolysosomal (pH 5.5) pH contexts. S. aureus strains were more susceptible to HDPs at pH 7.5 than at pH 5.5, and each HDP exerted a distinct effect signature. The impacts of ΔgraS and ΔΕL on HDP resistance were peptide and pH dependent. Both mutants exhibited defects in ANX response to hNP-1 or hBD-2 at pH 7.5, but only hNP-1 did so at pH 5.5. Both mutants exhibited hyper-PRM, -ANX, and -CDP responses to RP-1 at both pHs and hypo-ENR at pH 5.5. The actions correlated with ΔgraS or ΔΕL hypersusceptibility to hNP-1 or RP-1 (but not hBD-2) at pH 7.5 and to all study HDPs at pH 5.5. An exogenous EL mimic protected mutant strains from hNP-1 and hBD-2 but not RP-1, indicating that GraS and its EL play nonredundant roles in S. aureus survival responses to specific HDPs. These findings suggest that GraS mediates specific resistance countermeasures to HDPs in immune contexts that are highly relevant to S. aureus pathogenesis in humans.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.