Cell binding, internalization and cytotoxic activity of human granzyme B expressed in the yeast Pichia pastoris.
ABSTRACT: Granzyme B (GrB) is an apoptosis-inducing protease of cytotoxic lymphocytes. We have investigated intracellular and extracellular effects of human GrB using recombinant protein expressed in the yeast Pichia pastoris. GrB was rapidly taken up by HeLa cells, and accumulated in vesicular structures in the cytoplasm. There it remained inactive and could not be liberated by the endosomolytic reagent chloroquine, indicating that the vesicular structures are distinct from late endosomes and lysosomes. Direct cytosolic delivery of GrB with a cationic lipid-based transduction reagent, however, resulted in the induction of apoptotic cell death. After prolonged incubation at or above 125 nM, GrB on its own induced pronounced morphological changes in human tumour cells, leading to partial loss of contact to the culture support. This extracellular effect was dependent on enzymatic activity and could be reversed by removal of the protein, suggesting GrB-dependent cleavage of extracellular matrix components as the underlying mechanism.
Project description:Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.
Project description:Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumour cells. A major player in this process is GrB (granzyme B), which triggers apoptosis in both caspase-dependent and caspase-independent pathways. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. In the present study, we confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least twice at two distinct aspartic acids, Asp1860 and Asp1961. GrB cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also displayed perforin-independent functions by cleaving the extracellular domain of Notch1. Overall, cleavage of Notch1 by GrB resulted in a loss of transcriptional activity, independent of Notch1 activation. We conclude that GrB disables Notch1 function, probably resulting in anti-cellular proliferation and cell death signals.
Project description:Human plasmacytoid dendritic cells (pDCs) are crucially involved in the modulation of adaptive T-cell responses in the course of neoplastic, viral, and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDCs can be an abundant source of GrB and that such GrB(+) pDCs potently suppress T-cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving Janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and STAT5 and that interleukin-3 (IL-3), a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while Toll-like receptor agonists and CD40 ligand strongly inhibit, GrB secretion by pDCs. GrB-secreting pDCs may play a regulatory role for immune evasion of tumors, antiviral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T-cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.
Project description:Granzyme B (GrB) is a serine protease produced by immune and non-immune cells, able to promote multiple processes, like apoptosis, inflammation, extracellular matrix remodeling and fibrosis. GrB expression in visceral adipose tissue (VAT) was associated with tissue damage, local inflammation and insulin resistance in obesity murine model, but there is no data in humans. Aim of this study was to explore the expression of GrB in VAT from obese subjects in relation to adipose tissue injury, inflammation, metabolic alterations and GrB circulating levels. For this purpose, 85 obese individuals undergoing bariatric surgery and 35 healthy subjects (as control) were recruited at Sapienza University, Rome, Italy. Study participants underwent clinical work-up and routine biochemistry. mRNA expression of GrB in VAT and of a panel of VAT inflammatory markers was analyzed by real-time PCR. Serum GrB levels were measured by Elisa Affymetrix EBIO. We observed that 80% of obese patients expressed GrB mRNA in VAT, and GrB VAT expression was associated with the presence of local inflammation and glucose homeostasis alterations. Moreover, GrB serum levels, which were higher in obese subjects compared to non-obese healthy individuals, were associated with GrB expression in VAT and glyco-metabolic impairment. Our data show, for the first time in humans, that obese subjects with "sick" fat and altered glucose tolerance exhibit GrB expression in VAT, and suggest that GrB might contribute to obesity-related VAT in?ammatory remodeling and glucose homeostasis dysregulation. Moreover, increased circulating GrB levels might represent a possible peripheral marker of VAT dysfunction in metabolic diseases.
Project description:Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.
Project description:The systemic autoimmune diseases are a complex group of disorders characterized by elaboration of high titer autoantibodies and immune-mediated damage of tissues. Two striking features of autoimmune rheumatic diseases are their self-sustaining nature and capacity for autoamplification, exemplified by disease flares. These features suggest the presence of a feed-forward cycle in disease propagation, in which immune effector pathways drive the generation/release of autoantigens, which in turn fuel the immune response. There is a growing awareness that structural modification during cytotoxic granule-induced cell death is a frequent and striking feature of autoantigens, and may be an important principle driving disease. This review focuses on granzyme B (GrB)-mediated cleavage of autoantigens including (i) features of GrB cleavage sites within autoantigens, (ii) co-location of cleavage sites with autoimmune epitopes, and (iii) GrB sensitivity of autoantigens in disease-relevant target tissue. The mechanisms whereby GrB-induced changes in autoantigen structure may contribute to the initiation and propagation of autoimmunity are reviewed and reveal that GrB has the potential to create or destroy autoimmune epitopes. As there remains no direct evidence showing a causal function for GrB cleavage of antigens in the generation of autoimmunity, this review highlights important outstanding questions about the function of GrB in autoantigen selection.
Project description:Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
Project description:<h4>Purpose</h4>Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG).<h4>Methods</h4>GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis.<h4>Results</h4>GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3.<h4>Conclusions</h4>These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.
Project description:Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
Project description:The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF??? binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF??? fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF??? that correlated closely to total VEGFR-2 expression. GrB/VEGF??? localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF??? induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF??? localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF??? to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF??? showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF??? at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.