Arabidopsis thaliana Phytocystatin 6 Forms Functional Oligomer and Amyloid Fibril States.

Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.

Amyloid Formation of Stefin B Protein Studied by Infrared Spectroscopy.

BACKGROUND: Stefin B, an established model protein for studying the stability and mechanism of protein folding, was used for monitoring protein aggregation and formation of amyloid structure by infrared spectroscopy. METHODS: The analyses of the integral intensities of the low frequency part of the Amide I band, which is directly connected to the appearance of the cross-ß structure reveals the temperature but not pH dependence of stefin B structure. RESULTS: We show that pH value has significant role in the monomer stability of stefin B. Protein is less stable in acidic environment and becomes more stable in neutral or basic conditions. While in the case of the Amide I band area analysis we apply only spectral regions characteristic for only part of the protein in cross-ß structure, the temperature study using multivariate curve resolution (MCR) analysis contains also information about the protein conformation states which do not correspond to native protein nor protein in cross-ß structure. CONCLUSIONS: These facts results in the slightly different shapes of fitted sigmoid functions fitted to the weighted amount of the second basic spectrum (sc2), which is the closed approximation of the protein spectra with cross-ß structure. Nevertheless, the applied method detects the initial change of the protein structure. Upon the analysis of infrared data a model for stefin B aggregation is proposed.

Urea induced unfolding of rai seed cystatin: Influence of glycerol as a chemical chaperone.

Cystatin superfamily members, by virtue of their thiol protease regulatory properties, show involvement in myriad physiological processes important for survival and well-being. The current study involves urea-induced denaturation of a novel variant of the cystatin superfamily, rai seed cystatin (RSC), employing a variety of biophysical assays in order to characterize different folding intermediates generated on unfolding. Urea as a denaturant presented the passage of RSC through a series of events resulting in the loss of RSC functional capability, accompanied by changes in the archetype at secondary and tertiary structural levels, as evident from protease inhibitory, UV absorption, and intrinsic fluorescence assays, respectively. ANS fluorescence also revealed routing of RSC through discrete multiple sub-states thus presenting the generation of intermediate states somewhat close to the pre-molten globule and/or molten globule forms of RSC. Furthermore, far-UV circular dichroism analysis revealed a concentration-dependent gradual loss in typical -helical RSC peaks, indicating a nearly 50 % loss in secondary structural elements around 5 M urea treatment. The study also reports the possible role of glycerol in the refolding and/or reactivation of the urea unfolded RSC form. Glycerol presented itself as a potent structural stabilizer as it assisted in the refolding and reactivation of the unfolded RSC in a dosage-dependent manner, concomitantly paving the way for unravelling the mechanistic approach involved in the phenomenon, which can facilitate future studies.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily.

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

A brief review on oryzacystatin: a potent phytocystatin for crop management.

Phytocystatins are a type of proteinase inhibitor which are extensively studied for their specific inhibitory action against cysteine protease enzymes (CP) of insects and pathogens. Oryzacystatins (OC), a phytocystatin from rice inhibits CP in a reversible manner with its conserved tripartite wedge. OCs have important role in plant innate defense mechanism through phytohormonal signalling pathways. OC are induced in response to both biotic and abiotic stress conditions and are used to develop transgenic plants exhibiting resistance against stress conditions. In this review, we focus on the structure and mechanism of action of oryzacystatins, their possible role in plant physiology, biotic and abiotic stress tolerance mechanism in plants and their potential application strategies for future crop management studies.

Review: Unraveling the origin of the structural and functional diversity of plant cystatins.

The regulation of protease activity is a critical factor for the physiological balance during plant growth and development. Among the proteins involved in controlling protease activity are the cystatins, well-described inhibitors of cysteine proteases present in viruses, bacteria and most Eukaryotes. Plant cystatins, commonly called phytocystatins, display unique structural and functional diversity and are classified according to their molecular weight as type-I, -II, and -III. Their gene structure is highly conserved across Viridiplantae and provides insights into their evolutionary relationships. Many type-I phytocystatins with introns share sequence similarities with type-II phytocystatins. New data shows that they could have originated from recent losses of the carboxy-terminal extension present in type-II phytocystatins. Intronless type-I phytocystatins originated from a single event shared by flowering plants. Pieces of evidence show multiple events of gene duplications, intron losses, and gains throughout the expansion and diversity of the phytocystatin family. Gene duplication events in Gymnosperms and Eudicots resulted in inhibitors with amino acid substitutions that may modify their interaction with target proteases and other proteins. This review brings a phylogenomic analysis of plant cystatin evolution and contributes to a broader understanding of their origins. A complete functional genomic analysis among phytocystatins and their roles in plant development and responses to abiotic and biotic stresses remains a question to be fully solved.

Loop replacement design: a new way to improve potency of plant cystatins.

Plant cystatins function as competitive inhibitors of cysteine proteases. Similar to other defence proteins, cystatins include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Protein engineering approaches, such as point mutations, at these functionally relevant amino acid sites have already been found to be a powerful tool in improving the inhibitory properties of cystatins. Such engineered cystatins not only better protect against digestive proteases of herbivorous arthropods but also against cysteine proteases of several other plant pests as well as against cysteine proteases produced in plant during stress-induced senescence. Despite previous engineering successes, an urgent need still exists to further improve both plant cystatin potency and specificity. Tremblay and colleagues propose in this issue a new cystatin engineering strategy to substitute the function-related structural elements (SEs) of a cystatin by the corresponding elements of an alternative cystatin. This strategy, possibly combined with direct cystatin gene editing in a target plant, might provide an innovative way to control cysteine protease activity. Comment on https://doi.org/10.1111/febs.16288.

Amyloid aggregation and secondary structure changes of liver cystatin: Acidic denaturation and TFE induced studies.

A cysteine proteinase inhibitor has been purified by affinity chromatography from the liver of buffalo. Liver cystatin is subjected to incubation at low pH with co-solvent TFE, where we have studied the effect on the conformation, activity and tendency to form aggregates or fibrils. ANS fluorescence was used to study conformational changes. The fibril formation and aggregation was studied using ThT assay, CD, FTIR and fluorescence spectroscopy. At pH 3.0 there was no fibril formation though aggregates were formed but in presence of TFE fibrils appeared. At pH 2.0 and 1.0, TFE induced rapid fibril formation compared to only acid induced state as assessed by Thioflavin T (ThT) fluorescence.TFE stabilized each of the three acid induced intermediates at predenaturational concentrations (20%) and accelerated fibril formation. Solvent conditions had a profound effect on the tendency of liver cystatin to produce fibrils and aggregation.Communicated by Ramaswamy H. Sarma.

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization.

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

NAC blocks Cystatin C amyloid complex aggregation in a cell system and in skin of HCCAA patients.

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disease caused by a leucine to glutamine variant of human cystatin C (hCC). L68Q-hCC forms amyloid deposits in brain arteries associated with micro-infarcts, leading ultimately to paralysis, dementia and death in young adults. To evaluate the ability of molecules to interfere with aggregation of hCC while informing about cellular toxicity, we generated cells that produce and secrete WT and L68Q-hCC and have detected high-molecular weight complexes formed from the mutant protein. Incubations of either lysate or supernatant containing L68Q-hCC with reducing agents glutathione or N-acetyl-cysteine (NAC) breaks oligomers into monomers. Six L68Q-hCC carriers taking NAC had skin biopsies obtained to determine if hCC deposits were reduced following NAC treatment. Remarkably, ~50-90% reduction of L68Q-hCC staining was observed in five of the treated carriers suggesting that L68Q-hCC is a clinical target for reducing agents.

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via ß-Catenin involvement.

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.

Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Cross-seeding between the functional amyloidogenic CRES and CRES3 family members and their regulation of Aß assembly.

Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-ß reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aß by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aß assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.

Crystal structure and physicochemical characterization of a phytocystatin from Humulus lupulus: Insights into its domain-swapped dimer.

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Sugarcane cystatins: From discovery to biotechnological applications.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Maturation of the functional mouse CRES amyloid from globular form.

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming ß-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid ß-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.

Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro.

BACKGROUND: Naegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells. METHODS: Recombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: Fowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1. CONCLUSIONS: Fowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.

Small protease inhibitors in tick saliva and salivary glands and their role in tick-host-pathogen interactions.

Ticks must durably suppress vertebrate host responses (hemostasis, inflammation, immunity) to avoid rejection and act as vectors of many pathogenic microorganisms that cause disease in humans and animals. Transcriptomics and proteomics studies have been used to study tick-host-pathogen interactions and have facilitated the systematic characterization of salivary composition and molecular dynamics throughout tick feeding. Tick saliva contains a complement of protease inhibitors that are differentially produced during feeding, many of which inhibit blood coagulation, platelet aggregation, vasodilation, and immunity. Here we focus on two major groups of protease inhibitors, the small molecular weight Kunitz inhibitors and cystatins. We discuss their role in tick-host-pathogen interactions, how they mediate the interaction between ticks and their hosts, and how they might be exploited both by pathogens to invade hosts and as candidates for the treatment of various human pathologies.

The first characterization of a cystatin and a cathepsin L-like peptidase from Aedes aegypti and their possible role in DENV infection by the modulation of apoptosis.

Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.