Serine Protease Family Research Articles

Pro-tumoral role of Granzyme B to aid in invasion and metastasis.

Abstract The granule exocytosis pathway has long been known to be involved in potent responses to pathogens and cancers. This is executed by the action of Perforin and a family of granule-associated serine proteases known as Granzymes (Gzm). Many studies have shown that these proteases exert their effects by killing affected cells, executing the major function by Cytotoxic T cells (Tc) and Natural Killer cells expressing GzmB. Studies have found that the tumor cells ability to create a suppressive environment is dependent on Gzm expression by Regulatory T lymphocytes (Treg). Bird et al published data showing GzmB aids in the transmigration of Tcs, however the ability of GzmB to aid in the migration of non-T cells has not been investigated. We hypothesize that Granzyme B secretion may cause extracellular matrix remodeling in the tumor microenvironment, aiding in the outgrowth of the tumor cells via promoting invasion or metastasis. We test our hypothesis by measuring tumor invasion in vitro utilizing the xCelligence transmigration system. We found that culturing B16 cells with recombinant GzmB from Biolegend or Peprotech on Matrigel resulted in a 31% and 47% increase in cellular invasion, respectively. Also, the rate of invasion resulted in a 35% and 61% increase, respectively. We also tested our hypothesis in vivo with a preliminary study using Treg-specific GzmB KO mice (Foxp3creGzmBfl/fl). We depleted CD8+ T cells, injected B16 cells intravenously, and activated Tregs. We found a 48% decrease in metastatic nests in mice deficient of GzmB in Treg cells after 8 days and a 37% decrease after 16 days compared to WT. These findings shed light on the pro-migratory ability of secretory GzmB and helps us understand an unassuming role of GzmB as a pro-tumoral factor. Supported by funds through NIH (RO1 HL135325, T32 AI095190, P30 CA134274) and Maryland Department of Health's Cigarette Restitution Fund Program.

Evolutionary history of type II transmembrane serine proteases involved in viral priming.

Type II transmembrane serine proteases (TTSPs) are a family of trypsin-like membrane-anchored serine proteases that play key roles in the regulation of some crucial processes in physiological conditions, including cardiac function, digestion, cellular iron homeostasis, epidermal differentiation, and immune responses. However, some of them, in particular TTSPs expressed in the human airways, were identified as host factors that promote the proteolytic activation and spread of respiratory viruses such as influenza virus, human metapneumovirus, and coronaviruses, including SARS-CoV-2. Given their involvement in viral priming, we hypothesized that members of the TTSP family may represent targets of positive selection, possibly as the result of virus-driven pressure. Thus, we investigated the evolutionary history of sixteen TTSP genes in mammals. Evolutionary analyses indicate that most of the TTSP genes that have a verified role in viral proteolytic activation present signals of pervasive positive selection, suggesting that viral infections represent a selective pressure driving the evolution of these proteases. We also evaluated genetic diversity in human populations and we identified targets of balancing selection in TMPRSS2 and TMPRSS4. This scenario may be the result of an ancestral and still ongoing host–pathogen arms race. Overall, our results provide evolutionary information about candidate functional sites and polymorphic positions in TTSP genes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00439-022-02435-y.

Isolation of Bacillus sp. A5.3 Strain with Keratinolytic Activity.

Simple SummaryIn this study, we described keratinolytic properties of a strain of Bacillus (sp. A5.3) isolated from sites of feather waste accumulation. The proteolytic enzymes secreted by Bacillus sp. A5.3 are serine proteases, are alkaline enzymes, have a wide substrate specificity, and have high thermal stability. Bacillus sp. A5.3 effectively hydrolyzes feathers and can be used in feather-processing technologies and as a source of alkaline and thermostable proteases and keratinases.Environmental safety and economic factors necessitate a search for new ways of processing poultry farm feathers, which are 90% β-keratin and can be used as a cheap source of amino acids and peptones. In this study, feather-decomposing bacteria were isolated from a site of accumulation of rotten feathers and identified as Bacillus. Among them, the Bacillus sp. A5.3 isolate showed the best keratinolytic properties. Scanning electron microscopy indicated that Bacillus sp. A5.3 cells closely adhere to the feather surface while degrading the feather. It was found that Bacillus sp. A5.3 secretes thermostable alkaline proteolytic and keratinolytic enzymes. Zymographic analysis of the enzymatic extract toward bovine serum albumin, casein, gelatin, and β-keratin revealed the presence of proteases and keratinases with molecular weights 20–250 kDa. The proteolytic and keratinolytic enzymes predominantly belong to the serine protease family. Proteome analysis of the secreted proteins by nano-HPLC coupled with Q-TOF mass spectrometry identified 154 proteins, 13 of which are proteases and peptidases. Thus, strain Bacillus sp. A5.3 holds great promise for use in feather-processing technologies and as a source of proteases and keratinases.

Human Proteinase 3, an important autoantigen of c-ANCA associated vasculitis, shares cross-reactive epitopes with serine protease allergens from mites: an in silico analysis.

Background: In autoimmune vasculitis, autoantibodies to Human Proteinase 3 (PR3), a human serine protease, seems to have a role on the inception of c-ANCA associated vasculitis. The origin of this autoreactive response remains unclear. However, for several autoreactive responses, molecular mimicry between environmental antigens and human proteins is key to trigger autoantibodies and finally autoimmunity manifestations. Considering that PR3 is a serine protease and house dust mite (HDM) group 3 allergens share this biochemical activity, the aim of this study was to identify cross-reactive epitopes between serine proteases from human and mites using an in silico approach. Methods: Multi alignment among amino acid sequences of PR3 and HDM group 3 allergens was performed to explore identity and structural homology. ElliProandBepiPred in silicotoolswereusedtopredictBand T cell epitopes. Consurf tool was used to conduct identification of conserved regions in serine proteases family. Results: PR3 and HDM group 3 allergens shared moderate identity and structural homology (rootmeansquaredeviation<1). One B cell cross reactive epitope among serine proteases was identified (29I, 30V, 31G, 32G, 34E, 36K, 37A, 38L, 39A and 54C) and two T cell epitopes. Conclusions: PR3 have structural homology and share cross reactive epitopes with HDM group 3 allergens.

Structure-based discovery of small molecules improving stability of human broadly-neutralizing anti-HIV antibody 2F5 in plant suspension cells.

The production of biopharmaceuticals in engineered plant-based systems is a promising technology that has proven its suitability for the production of various recombinant glyco-proteins that are currently undergoing clinical trials. However, compared to mammalian cell lines, the productivity of plant-based systems still requires further improvement. A major obstacle is the proteolytic degradation of recombinant target proteins by endogenous plant proteases mainly from the subtilisin family of serine proteases. In the present study, the authors screened for putative small molecule inhibitors for subtilases that are secreted from tobacco BY-2 suspension cells using an in silico approach. The effectiveness of the substances identified in this screen was subsequently tested in degradation assays using the human broadly-neutralizing anti-HIV monoclonal antibody 2F5 (mAb2F5) and spent BY-2 culture medium as a model system. Among 16 putative inhibitors identified by in silico studies, three naphthalene sulfonic acid derivatives showed inhibitory activity in in vitro degradation assays and are similar to or even more effective than phenylmethylsulfonyl fluoride (PMSF), a classical inhibitor of serine proteases, which served as positive control.

Unraveling the Dichotomy of Enigmatic Serine Protease HtrA2.

Mitochondrial high-temperature requirement protease A2 (HtrA2) is an integral member of the HtrA family of serine proteases that are evolutionarily conserved from prokaryotes to humans. Involvement in manifold intricate cellular networks and diverse pathophysiological functions make HtrA2 the most enigmatic moonlighting protease amongst the human HtrAs. Despite perpetuating the oligomeric architecture and overall structural fold of its homologs that comprises serine protease and regulatory PDZ domains, subtle conformational alterations and dynamic enzymatic regulation through the distinct allosteric mode of action lead to its functional diversity. This mitochondrial protease upon maturation, exposes its one-of-a-kind N-terminal tetrapeptide (AVPS) motif that binds and subsequently cleaves Inhibitor of Apoptosis Proteins (IAPs) thus promoting cell death, and posing as an important molecule for therapeutic intervention. Interestingly, unlike its other human counterparts, HtrA2 has also been implicated in maintaining the mitochondrial integrity through a bi-functional chaperone-protease activity, the on-off switch of which is yet to be identified. Furthermore, its ability to activate a wide repertoire of substrates through both its N- and C-terminal regions presumably has calibrated its association with several cellular pathways and hence diseases including neurodegenerative disorders and cancer. Therefore, the exclusive structural attributes of HtrA2 that involve multimodal activation, intermolecular PDZ-protease crosstalk, and an allosterically-modulated trimeric active-site ensemble have enabled the protease to evolve across species and partake functions that are fine-tuned for maintaining cellular homeostasis and mitochondrial proteome quality control in humans. These unique features along with its multitasking potential make HtrA2 a promising therapeutic target both in cancer and neurodegeneration.

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Biosynthesis of Gut-Microbiota-Derived Lantibiotics Reveals a Subgroup of S8 Family Proteases for Class III Leader Removal.

Lanthipeptides are a group of ribosomally synthesized and post-translationally modified peptides with diverse structural features and bioactivities. Gut-microbiota-derived lanthipeptides play important roles in gut homeostasis of the host. Herein, we report the discovery and biosynthesis of class III lantibiotics named amylopeptins, which are derived from the gut microbiota of Sprague-Dawley rats and display a narrow antimicrobial spectrum. In contrast to known class III lanthipeptides, the biosynthesis of amylopeptins employs AmyP, which belongs to a subgroup of S8 family serine proteases, to remove the leader of corresponding precursor peptides in a site-specific manner during the last step of their maturation. Overall, this study shows for the first time that S8 family proteases participate in the biosynthesis of class III lanthipeptides.

Biosynthesis of Gut‐Microbiota‐Derived Lantibiotics Reveals a Subgroup of S8 Family Proteases for Class III Leader Removal

AbstractLanthipeptides are a group of ribosomally synthesized and post‐translationally modified peptides with diverse structural features and bioactivities. Gut‐microbiota‐derived lanthipeptides play important roles in gut homeostasis of the host. Herein, we report the discovery and biosynthesis of class III lantibiotics named amylopeptins, which are derived from the gut microbiota of Sprague‐Dawley rats and display a narrow antimicrobial spectrum. In contrast to known class III lanthipeptides, the biosynthesis of amylopeptins employs AmyP, which belongs to a subgroup of S8 family serine proteases, to remove the leader of corresponding precursor peptides in a site‐specific manner during the last step of their maturation. Overall, this study shows for the first time that S8 family proteases participate in the biosynthesis of class III lanthipeptides.

Human Proteinase 3, an important autoantigen of c-ANCA associated vasculitis, shares cross-reactive epitopes with serine protease allergens from mites: an in silico analysis

Background: In autoimmune vasculitis, autoantibodies to Human Proteinase 3 (PR3), a human serine protease, seems to have a role on the inception of c-ANCA associated vasculitis. The origin of this autoreactive response remains unclear. However, for several autoreactive responses, molecular mimicry between environmental antigens and human proteins is key to trigger autoantibodies and finally autoimmunity manifestations. Considering that PR3 is a serine protease and house dust mite (HDM) group 3 allergens share this biochemical activity, the aim of this study was to identify cross-reactive epitopes between serine proteases from human and mites using an in silico approach. Methods: Multi alignment among amino acid sequences of PR3 and HDM group 3 allergens was performed to explore identity and structural homology. ElliPro and BepiPred in silico tools were used to predict B and T cell epitopes. Consurf tool was used to conduct identification of conserved regions in serine proteases family. Results: PR3 and HDM group 3 allergens shared moderate identity and structural homology (root mean square deviation < 1). One B cell cross reactive epitope among serine proteases was identified (29I, 30V, 31G, 32G, 34E, 36K, 37A, 38L, 39A and 54C) and two T cell epitopes. Conclusions: PR3 have structural homology and share cross reactive epitopes with HDM group 3 allergens.

Oral immunization with attenuated Salmonella encoding an elastase elicits protective immunity against Trichinella spiralis infection

Elastase belongs to the serine protease family. Previous studies showed that Trichinella spiralis elastase (TsE) was highly expressed in intestinal infective larvae (IIL). Recombinant TsE (rTsE) promoted the larval intrusion of enteral epithelium cells (IECs), whereas anti-rTsE antibodies and siRNA impeded larval intrusion. Subcutaneous vaccination of mice with rTsE showed a partial protective immunity, suggesting that TsE might be a promising vaccine target against Trichinella infection. In this study, complete TsE cDNA sequence was cloned into pcDNA3.1, and the rTsE DNA was transformed into attenuated S. typhimurium strain ΔcyaSL1344. Oral vaccination of mice with TsE DNA elicited a systemic Th1/Th2/Treg mixed immune response and gut local mucosal sIgA response. Immunized mice exhibited a significant immune protection against T. spiralis larval challenge, as demonstrated by a 52.48% reduction of enteral adult worms and a 69.43% reduction of muscle larvae. The protection might be related to the TsE-induced production of intestinal mucus, specific anti-TsE sIgA and IgG, and secretion of IFN-γ, IL-2, IL-4 and IL-10, which protected gut mucosa from larval intrusion, suppressed worm development and impeded female reproduction. The results demonstrated that attenuated Salmonella-delivered TsE DNA vaccine provided a prospective strategy for the control of Trichinella infection in food animals.

Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach.

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.

A Novel Mutation of the KLK6 Gene in a Family With Knee Osteoarthritis.

To investigate the correlation between gene mutation and knee osteoarthritis (KOA), a whole-exome sequencing (WES) was applied to analyze blood samples of four KOA patients and two normal subjects in a family. Gene mutations were identified by gene-trapping and high-throughput sequencing analysis across the differences between the patients and normal subjects. The interactive gene network analysis on the retrieval of interacting genes (STRING) database and the KOA-related genes expression data sets was performed. A possibly detrimental and nonsynonymous mutation at the kallikrein-related peptidase 6 (KLK6) gene (rs201586262, c. C80A, P27H) was identified and attracted our attention. KLK6 belongs to the kallikrein family of serine proteases and its serum level is known as a prevalent biomarker in inflammatory and malignant diseases. KLK6 expresses in the extracellular compartment for matrix degradation, highlighting that KLK6 plays a role in the pathogenesis of KOA. By using the gene databases, the KOA-related genes were mined after de-duplication and IL6 was selected as the most relevant gene through interactive analysis of protein-protein interaction (PPI) network. The data suggested that KLK6 gene mutation and the related expression alteration of IL6 gene might determine the occurrence of hereditary KOA. The is the first study discovering the gene mutation of KLK6 as a factor of pathogenesis of KOA, especially the hereditary KOA.

The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases.

Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.

Downregulation of matriptase suppresses the PAR‑2/PLCγ2/PKC‑mediated invasion and migration abilities of MCF‑7 breast cancer cells

Matriptases, members of the type II transmembrane serine protease family, are cell surface proteolytic enzymes that mediate tumor invasion and metastasis. Matriptase is highly expressed in breast cancer and is associated with poor patient outcome. However, the cellular mechanism by which matriptase mediates breast cancer invasion remains unknown. The present study aimed to determine the role of matriptase in the protein kinase C (PKC)-mediated metastasis of MCF-7 human breast cancer cells. Matriptase small interfering RNA-mediated knockdown significantly attenuated the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasiveness and migration of MCF-7 cells, and inhibited the activation of phospholipase C γ2 (PLCγ2)/PKC/MAPK signaling pathways. Matriptase-knockdown also suppressed the expression of MMP-9 and inhibited the activation of NF-κB/activator protein-1 in MCF-7 cells. Additionally, GB83 [an inhibitor of protease-activated receptor-2 (PAR-2)] inhibited PKC-mediated MMP-9 expression and metastatic ability in MCF-7 cells. Furthermore, downregulation of matriptase suppressed TPA-induced MMP-9 expression and invasiveness via PAR-2/PLCγ2/PKC/MAPK activation. These findings shed light on the mechanism underlying the role of matriptase in MCF-7 cell invasion and migration ability, and suggest that matriptase modulation could be a promising therapeutic strategy for preventing breast cancer metastasis.

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation, proteolytic activity, and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.

Feasibility and Therapeutic Potential of 177Lu-Fibroblast Activation Protein Inhibitor-46 for Patients With Relapsed or Refractory Cancers: A Preliminary Study.

Fibroblast activation protein (FAP) is a member of the serine protease family and has a high expression in the stroma of approximately 90% of epithelial malignancies. The present investigation aimed to assess the feasibility, safety, and dosimetry data of 177Lu-FAPI-46 in diverse malignancies. Patients with advanced cancers with nonoperable tumors, or tumors refractory to conventional therapies, were enrolled. Treatment included escalating doses of 177Lu-FAPI-46 (1.85-4.44 GBq) per cycle using a combination of clinical and statistical expertise design, and intervals of 4 to 6 weeks were considered between the cycles. Biodistribution and dosimetry were examined by whole-body scans. We applied the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 to measure peptide-targeted radionuclide therapy (PTRT)-associated toxicity. A total of 21 patients (11 females and 10 males) with a median age of 50 years (range, 6-79 years) were investigated. Of 21 participants, 18 cases were selected for PTRT. Overall, 36 PTRT cycles were performed. The median number of PTRT cycles and the median injected amount of activity in each cycle were 2 and 3.7 GBq, respectively. The dosimetric analysis revealed median absorbed doses of 0.026, 0.136, 0.886, and 0.02 with ranges of 0.023-0.034, 0.001-0.2, 0.076-1.39, and 0.002-0.2 mGy/MBq for the whole body, liver, kidneys, and spleen, respectively. The therapy was well tolerated in almost all patients. The findings of this preliminary investigation might indicate the potential feasibility and safety of PTRT using 177Lu-FAPI-46 for different aggressive tumors. Moreover, the current study could be beneficial in determining the suitable amount of activity for a phase 2 study.

NOVEL IMMUNOTHERAPEUTIC TARGETED GRANZYME DELIVERY SYSTEMS IN TREATMENT OF MALIGNANT TUMORS

Cytotoxicity is the main human killer cell property. The cytotoxicity reaction of human killer cells is achieved through a complex of molecules, including perforins, granzyme, cathepsin and others. However, only one molecule is enough for target cell death: granzyme. Other molecules are intended for granzyme activation and its delivery to the target cell cytoplasm. Granzymes are a whole family of serine proteases that perform their function in the human body as integral cytolytic effectors during programmed cell death of cancer and pathogen-infected cells. Secreted mainly by cytotoxic T-lymphocytes and NK-cells, granzymes initiate apoptosis via caspase-dependent and caspase-independent pathways. These natural properties make granzymes one of the most promising human enzymes for use in the development of targeted therapeutic strategies in the treatment of various types of cancer.The most promising is granzyme B, because it has the most powerful effector properties. Due to the initiation of cascade reactions that activate apoptosis, granzyme is attractive as a basis for the development of medicines applicable in clinical oncology. At this time, several approaches have been developed for delivering granzyme molecules to tumor cells and facilitating its penetration through the cell membrane. Moreover, some solutions are proposed to overcome the resistance of target cells to granzyme-mediated apoptosis. These approaches are discussed in this review.The purpose of this review was to systematize information on the use of granzyme B as a nanostructured drug delivery system in the treatment of solid and hematological malignancies. In addition, this review discusses ways to overcome the resistance of granzyme penetration into target cells.

Abstract 3158: Characterization and targeting of TMPRSS13 in breast cancer

Abstract Breast cancer (BCa) remains a significant public health burden in the United States (US) and globally, despite advances in screening, detection, and therapeutics. In the US alone, BCa is the most commonly diagnosed cancer and the second leading cause of cancer deaths in women. The discovery of novel, targetable biomarkers is therefore paramount to reduce the morbidity and mortality associated with BCa. Cancer progression is often accompanied by increased expression of pericellular proteases capable of degrading the extracellular matrix and cleaving pro-oncogenic signaling molecules. BCa cells use pericellular proteolysis in order to enhance their proliferation, survival, and invasiveness. TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, has emerged as a potential biomarker and therapeutic target in BCa. Prior data indicates that TMPRSS13 is significantly upregulated in invasive ductal carcinoma samples at the mRNA and protein levels compared to normal breast tissue. Furthermore, TMPRSS13-deficient mice have few phenotypic defects but in a genetic model of mammary cancer, TMPRSS13-deficient mice have a significantly reduced tumor burden as well as a reduction in lung metastases. This data indicates that TMPRSS13 may be a worthwhile target to treat BCa patients. However, selective targeting of TMPRSS13 above other TTSPs is difficult due to the homology of their catalytic domains. Therefore, any information that we can glean regarding the unique biochemical and oncogenic properties of TMPRSS13 will aid in the future development of inhibitors. Our group has found that TMPRSS13 is the only TTSP family member confirmed to be phosphorylated in its intracellular domain. This phosphorylation is critical for the trafficking and cell surface expression of the protease. Mutagenesis of individual amino acids as well as deletion constructs of the phosphorylated residues in the intracellular domain have indicated that the inability of TMPRSS13 to be phosphorylated does not affect its catalytic activity, but inhibits proper cell membrane localization. Thus, phosphorylation may be imperative for TMPRSS13 to interact with pro-oncogenic substrates in the tumor microenvironment. Furthermore, preliminary data has indicated that TMPRSS13 may be a substrate for phosphorylation by CDK5, a neuronal kinase that has recently emerged as pro-oncogenic in various cancer types. CDK5, when bound to myristoylated p35, likely phosphorylates TMPRSS13 at the cell membrane, allowing TMPRSS13 to stably localize to the cell surface of both luminal and triple negative BCa cells. The overarching goal of our project is to fully understand the manner in which TMPRSS13 promotes the development and progression of BCa, which will allow for the design of selective targeted inhibitors in the future. Citation Format: Carly E. Martin, Andrew S. Murray, Evan Harrison, Karin List. Characterization and targeting of TMPRSS13 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3158.