Cathepsins in the extracellular space: Focusing on non-lysosomal proteolytic functions with clinical implications.

Cathepsins can be found in the extracellular space, cytoplasm, and nucleus. It was initially suspected that the primary physiological function of the cathepsins was to break down intracellular protein, and that they also had a role in pathological processes including inflammation and apoptosis. However, the many actions of cathepsins outside the cell and their complicated biological impacts have garnered much interest. Cathepsins play significant roles in a number of illnesses by regulating parenchymal cell proliferation, cell migration, viral invasion, inflammation, and immunological responses through extracellular matrix remodeling, signaling disruption, leukocyte recruitment, and cell adhesion. In this review, we outline the physiological roles of cathepsins in the extracellular space, the crucial pathological functions performed by cathepsins in illnesses, and the recent breakthroughs in the detection and therapy of specific inhibitors and fluorescent probes in associated dysfunction.

Potential Antagonistic Bacteria against Verticillium dahliae Isolated from Artificially Infested Nursery.

As an ecofriendly biocontrol agent, antagonistic bacteria are a crucial class of highly efficient fungicides in the field against Verticillium dahliae, the most virulent pathogen for cotton and other crops. Toward identifying urgently needed bacterial candidates, we screened bacteria isolated from the cotton rhizosphere soil for antagonisitic activity against V. dahliae in an artificially infested nursery. In preliminary tests of antagonistic candidates to characterize the mechanism of action of on culture medium, 88 strains that mainly belonged to Bacillus strongly inhibited the colony diameter of V. dahliae, with inhibiting efficacy up to 50% in 9 strains. Among the most-effective bacterial strains, Bacillus sp. ABLF-18, and ABLF-50 and Paenibacillus sp. ABLF-90 significantly reduced the disease index and fungal biomass of cotton to 40-70% that of the control. In further tests to elucidate the biocontrol mechanism (s), the strains secreted extracellular enzymes cellulase, glucanase, and protease, which can degrade the mycelium, and antimicrobial lipopeptides such as surfactin and iturin homologues. The expression of PAL, MAPK and PR10, genes related to disease resistance, was also elicited in cotton plants. Our results clearly show that three candidate bacterial strains can enhance cotton defense responses against V. dahliae; the secretion of fungal cell-wall-degrading enzymes, synthesis of nonribosomal antimicrobial peptides and induction of systemic resistance shows that the strains have great potential as biocontrol fungicides.

Isolation and Screening of Microorganisms for the Effective Pretreatment of Lignocellulosic Agricultural Wastes.

Lignocellulosic waste is the most abundant biorenewable biomass on earth, and its hydrolysis releases highly valued reducing sugars. However, the presence of lignin in the biopolymeric structure makes it highly resistant to solubilization thereby hindering the hydrolysis of cellulose and hemicellulose. Microorganisms are known for their potential complex enzymes that play a dominant role in lignocellulose conversion. Therefore, the current study was designed to isolate and screen potential microorganisms for their selective delignification ability for the pretreatment of lignocellulosic biomass. An extensive isolation and screening procedure yielded 36 desired isolates (22 bacteria, 7 basidiomycete fungi, and 7 filamentous fungi). Submerged cultivation of these desired microorganisms revealed 4 bacteria and 10 fungi with potent lignocellulolytic enzyme activities. The potent isolates were identified as Pleurotus, Trichoderma, Talaromyces, Bacillus, and Chryseobacterium spp. confirmed by morphological and molecular identification. The efficiency of these strains was determined through enzyme activities, and the degraded substrates were analyzed through scanning electron microscopy (SEM) and X-ray diffraction (XRD). Among all isolated microbes, Pleurotus spp. were found to have high laccase activity. The cellulose-decomposing and selective delignification strains were subjected to solid-state fermentation (SSF). SSF of field waste corn stalks as a single-carbon source provides Pleurotus spp. better condition for the secretion of ligninolytic enzymes. These isolated ligninolytic enzymes producing microorganisms may be used for the effective pretreatment of lignocellulosic agricultural wastes for the production of high value-added natural products by fermentation.

Elevated Extracellular Levels of Granzymes in Patients with Scrub Typhus.

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi, which is transmitted through chigger mites. Delayed treatment results in various complications and, in severe cases, death. Granzymes are secreted by cytotoxic T lymphocytes or natural killer cells and are known to play an important role in controlling intracellular pathogens. To date, few studies have been done on granzymes in patients with scrub typhus. In this study, granzymes A and B showed a significant increase during the acute stage of scrub typhus compared with healthy control subjects, and decreased sharply after treatment. In addition, granzymes A and B were significantly high in the moderately elevated liver enzyme group. In conclusion, it appears that the host during the acute phase of scrub typhus increases cytotoxic T-cell activity to control infection.

First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by Xylaria grammica.

BACKGROUND: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known. METHODS: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus. RESULTS: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center. CONCLUSIONS: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

Effect of earthworm Eisenia fetida epidermal mucus on the vitality and pathogenicity of Beauveria bassiana.

Beauveria bassiana is one of the most widely studied and used entomopathogenic fungus as biopesticide. In the biological control of pests, B. bassiana will persist in the soil after application, and will inevitably contact with earthworms, especially the epigeic earthworm species. So, what are the effects of earthworm and its epidermal mucus on the activity of B. bassiana? We employed the epigeic earthworm Eisenia fetida, B. bassiana TST05 strain, and the insect Atrijuglans hetaohei mature larvae to study the impact of earthworm epidermal mucus on the vitality and pathogenicity of B. bassiana to insect. Methods included scanning electron microscope observation, detection of spore germination, fungal extracellular enzyme activity, and infection testing to A. hetaohei. The results showed that the B. bassiana spores may attach to the cuticle of E. fetida but they could be covered by the epidermal mucus and became rough and shrunken. After treatment with the epidermal mucus, the spore germination and extracellular enzymes of B. bassiana was significantly inhibited. Inoculation of A. hetaohei larvae with a mixture of B. bassiana and mucus showed that the mucus could reduce the pathogenicity of B. bassiana to the insect, resulting in a slower disease course and lower mortality. It was concluded that the epidermal mucus of the earthworm E. fetida can inhibit the activity of B. bassiana, as well as the infectivity and pathogenicity of fungus to target insects. However, after treatment with epidermal mucus the surviving B. bassiana still had certain infectivity to insects. This is of great significance for the application of B. bassiana in biological control of pests.

The antagonistic Metschnikowia andauensis produces extracellular enzymes and pulcherrimin, whose production can be promoted by the culture factors.

Biological control against microbial infections has a great potential as an alternative approach instead of fungicidal chemicals, which can cause environmental pollution. The pigment producer Metschnikowia andauensis belongs to the antagonistic yeasts, but details of the mechanism by which it inhibits growth of other microbes are less known. Our results confirmed its antagonistic capacity on other yeast species isolated from fruits or flowers and demonstrated that the antagonistic capacity was well correlated with the size of the red pigmented zone. We have isolated and characterized its red pigment, which proved to be the iron chelating pulcherrimin. Its production was possible even in the presence of 0.05 mg/ml copper sulphate, which is widely used in organic vineyards because of its antimicrobial properties. Production and localisation of the pulcherrimin strongly depended on composition of the media and other culture factors. Glucose, galactose, disaccharides and the presence of pectin or certain amino acids clearly promoted pigment production. Higher temperatures and iron concentration decreased the diameter of red pigmented zones. The effect of pH on pigment production varied depending of whether it was tested in liquid or solid media. In addition, our results suggest that other mechanisms besides the iron depletion of the culture media may contribute to the antagonistic capacity of M. andauensis.

The extracellular HDAC6 ZnF UBP domain modulates the actin network and post-translational modifications of Tau.

BACKGROUND: Microtubule-associated protein Tau undergoes aggregation in Alzheimer`s disease (AD) and a group of other related diseases collectively known as Tauopathies. In AD, Tau forms aggregates, which are deposited intracellularly as neurofibrillary tangles. Histone deacetylase-6 (HDAC6) plays an important role in aggresome formation, where it recruits polyubiquitinated aggregates to the motor protein dynein. METHODS: Here, we have studied the effects of HDAC6 ZnF UBP on Tau phosphorylation, ApoE localization, GSK-3ß regulation and cytoskeletal organization in neuronal cells by immunocytochemical analysis. This analysis reveals that the cell exposure to the UBP-type zinc finger domain of HDAC6 (HDAC6 ZnF UBP) can modulate Tau phosphorylation and actin cytoskeleton organization. RESULTS: HDAC6 ZnF UBP treatment to cells did not affect their viability and resulted in enhanced neurite extension and formation of structures similar to podosomes, lamellipodia and podonuts suggesting the role of this domain in actin re-organization. Also, HDAC6 ZnF UBP treatment caused increase in nuclear localization of ApoE and tubulin localization in microtubule organizing centre (MTOC). Therefore, our studies suggest the regulatory role of this domain in different aspects of neurodegenerative diseases. Upon HDAC6 ZnF UBP treatment, inactive phosphorylated form of GSK-3ß increases without any change in total GSK-3ß level. CONCLUSIONS: HDAC6 ZnF UBP was found to be involved in cytoskeletal re-organization by modulating actin dynamics and tubulin localization. Overall, our study suggests that ZnF domain of HDAC6 performs various regulatory functions apart from its classical function in aggresome formation in protein misfolding diseases. Video abstract.

Deletion of Aspergillus nidulans cpsA/rseA induces increased extracellular hydrolase production in solid-state culture partly through the high osmolarity glycerol pathway.

Koji molds, such as Aspergillus oryzae and Aspergillus sojae, are used in the food industry in East Asia and have been explored for the large-scale production of extracellular hydrolases. We previously found that the deletion of a gene encoding a putative GT2 glycosyltransferase increased production of extracellular hydrolases in A. sojae. The gene was named rseA (regulator of the secretory enzyme A). We predicted that intracellular signaling pathways were involved in the increased production of hydrolases in the ΔrseA mutant of A. sojae. However, little has been reported on molecular biological knowledge about A. sojae. Hence, Aspergillus nidulans, a typical model organism used in molecular biology, was employed for the functional characterization of rseA in this study. Deletion of the rseA ortholog in A. nidulans induced increased extracellular production of hydrolases under the solid-state cultivation condition, similar to that in A. sojae. The involvement of the cell wall integrity pathway and the high osmolarity glycerol pathway in ΔrseA was further investigated. The results indicated that the HOG pathway played an important role in the increased extracellular production of hydrolases caused by the deletion of the rseA gene. rseA ortholog in A. nidulans was identical to cpsA, which was reported to function as a regulator of mycotoxin production, morphogenesis, and cell wall biosynthesis. However, this is the first study reporting that rseA/cpsA regulates extracellular hydrolase production in A. nidulans.

Induction of Extracellular Aminopeptidase Production by Peptides in Some Marine Bacterial Species.

Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.

Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase.

BACKGROUND: Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS: A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL-1 . CONCLUSION: A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

Recovery of motor function of chronic spinal cord injury by extracellular pyruvate kinase isoform M2 and the underlying mechanism.

In our previous study, we found that pyruvate kinase isoform M2 (PKM2) was secreted from the skeletal muscle and extended axons in the cultured neuron. Indirect evidence suggested that secreted PKM2 might relate to the recovery of motor function in spinal cord injured (SCI) mice. However, in vivo direct evidence has not been obtained, showing that extracellular PKM2 improved axonal density and motor function in SCI mice. In addition, the signal pathway of extracellular PKM2 underlying the increase in axons remained unknown. Therefore, this study aimed to identify a target molecule of extracellular PKM2 in neurons and investigate the critical involvement of extracellular PKM2 in functional recovery in the chronic phase of SCI. Recombinant PKM2 infusion to the lateral ventricle recovered motor function in the chronic phase of SCI mice. The improvement of motor function was associated with axonal increase, at least of raphespinal tracts connecting to the motor neurons directly or indirectly. Target molecules of extracellular PKM2 in neurons were identified as valosin-containing protein (VCP) by the drug affinity responsive target stability method. ATPase activation of VCP mediated the PKM2-induced axonal increase and recovery of motor function in chronic SCI related to the increase in axonal density. It is a novel finding that axonal increase and motor recovery are mediated by extracellular PKM2-VCP-driven ATPase activity.

Characterization of the Extracellular Fructanase FruA in Lactobacillus crispatus and Its Contribution to Fructan Hydrolysis in Breadmaking.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.

Extracellular Granzyme A Promotes Colorectal Cancer Development by Enhancing Gut Inflammation.

If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC.

Characterization of an efficient extracellular cyanophycinase and its encoding cphEStrept. gene from Streptomyces pratensis strain YSM.

Until now, no enzymes were described that hydrolyze cyanophycin granular protein (CGP) from a species of the genus Streptomyces. An isolate able to hydrolyze CGP was identified as Streptomyces pratensis strain YSM. The CGPase from S. pratensis strain YSM had an optimum activity at 42 °C and pH 8.5, and was able to degrade CGP at a rate of 12 ±â€¯0.3 µg/mL min. Additionally, this CGPase hydrolyzes water-soluble CGP significantly faster than water-insoluble CGP. The molecular mass of CGPase subunits from S. pratensis strain YSM as determined by SDS-PAGE was about 43 kDa, and the enzyme was entirely inhibited by serine-protease inhibitors. The CGPase coding gene (cphEStrept.) was amplified from genomic DNA using primers designed form consensus sequence of putative CGPase sequences. The cphEStrept. was 1427 bp encoding a CGPase of 420 amino acids that showed about 44% and 22% similarities to CGPase from Pseudomonas anguilliseptica BI and Synechocystis sp. PCC 6803, respectively. The catalytic triad and serine-protease residues (GXSXG) were identified in the CphEStrept. sequence. Dipeptides and tetrapeptides were identified as hydrolysis products. Biotechnological exploitation of S. pratensis strain YSM for CGPase production might have an advantage due to the reduction of separation costs and its ability to degrade CGP in phosphate buffer saline using actively growing or resting cells.

Light Pollution Changes the Toxicological Effects of Cadmium on Microbial Community Structure and Function Associated with Leaf Litter Decomposition.

Artificial light at night (ALAN/A) can not only alter the behavior and communication of biological organisms, it can also interact with other stressors. Despite its widespread use and the numerous potential ecological effects, little is known about the impact of ALAN on plant litter decomposition under cadmium (Cd) pollution in aquatic ecosystems. In an indoor microcosm experiment, we tested single and combined effects of ALAN and Cd on the activities and community structure of fungi associated with plant litter. The results showed that ALAN and/or Cd can change both water and leaf litter characteristics. ALAN exposure not only altered fungal community structure and their correlations, but also increased the activities of alkaline phosphatase, ß-glucosidase, and cellobiohydrolase. The leaf litter decomposition rate was 71% higher in the A-Cd treatment than that in the N-Cd treatment, indicating that the presence of ALAN weakened the negative impact of Cd on leaf litter decomposition. These results suggested that ALAN exposure mitigated the negative effect of Cd on leaf litter decomposition, contributing to the duel effect of ALAN on leaf litter decomposition. Overall, the results expand our understanding of ALAN on the environment and highlight the contribution of ALAN to Cd toxicity in aquatic ecosystems.

Design, Synthesis, and Characterization of a Paclitaxel Formulation Activated by Extracellular MMP9.

The concept of triggered drug release offers a possibility to overcome the toxic side effects of chemotherapeutics in cancer treatment by reducing systemic exposure to the active drug. In the present work, the concept foresees the use of the extracellular enzyme MMP9 as an enzymatic trigger for drug release in the proximity of tumor cells. METHODS: A paclitaxel-hemisuccinate-peptide conjugate as a building block for self-assembling nanoparticles was synthesized using standard conjugation approaches. The building block was purified via preparative HPLC and analyzed by LC-MS. Nanoparticles were formed using the nanoprecipitation method and characterized. For selection of a suitable in vitro model system, common bioanalytical methods were used to determine mRNA expression, enzyme amount, and activity of MMP9. RESULTS: The MMP9-labile prodrug was synthesized and characterized. Nanoparticles were formed out of MMP9-labile conjugate-building blocks. The nanoparticle's diameter averaged at around 120 nm and presented a spherical shape. LN-18 cells, a glioblastoma multiforme derived cell line, were chosen as an in vitro model based on findings in cancer tissue and cell line characterization. The prodrug showed cytotoxicity in LN-18 cells, which was reduced by addition of an MMP9 inhibitor. CONCLUSION: taken together, we confirmed increased MMP9 in several cancer tissues (cervical, esophageal, lung, and brain) compared to healthy tissue and showed the effectiveness of MMP9-labile prodrug in in vitro tests.