Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.

ABSTRACT: Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.

SUBMITTER: Sutton VR

PROVIDER: S-EPMC2063978 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema.

Project description:BackgroundIt is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8+ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8+ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.MethodsWe assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8+ T lymphocytes, CD4+ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8+ T lymphocytes by real-time PCR.ResultsEmphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8+ T lymphocytes, CD4+ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.ConclusionDespite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.

| S-EPMC2018693 | biostudies-literature

Cytotoxic Lymphocytes Target HIV-1 Gag Through Granzyme M-Mediated Cleavage.

Project description:Untreated HIV-1 infection leads to a slow decrease in CD4+ T cell lymphocytes over time resulting in increased susceptibility to opportunistic infections (acquired immunodeficiency syndrome, AIDS) and ultimately death of the infected individual. Initially, the host's immune response controls the infection, but cannot eliminate the HIV-1 from the host. Cytotoxic lymphocytes are the key effector cells in this response and can mediate crucial antiviral responses through the release of a set of proteases called granzymes towards HIV-1-infected cells. However, little is known about the immunological molecular mechanisms by which granzymes could control HIV-1. Since we noted that HIV-1 subtype C (HIV-1C) Gag with the tetrapeptide insertion PYKE contains a putative granzyme M (GrM) cleavage site (KEPL) that overlaps with the PYKE insertion, we analyzed the proteolytic activity of GrM towards Gag. Immunoblot analysis showed that GrM could cleave Gag proteins from HIV-1B and variants from HIV-1C of which the Gag-PYKE variant was cleaved with extremely high efficiency. The main cleavage site was directly after the insertion after leucine residue 483. GrM-mediated cleavage of Gag was also observed in co-cultures using cytotoxic lymphocytes as effector cells and this cleavage could be inhibited by a GrM inhibitor peptide. Altogether, our data indicate towards a noncytotoxic immunological mechanism by which GrM-positive cytotoxic lymphocytes target the HIV-1 Gag protein within infected cells to potentially control HIV-1 infection. This mechanism could be exploited in new therapeutic strategies to treat HIV-1-infected patients to improve immunological control of the infection.

| S-EPMC8089382 | biostudies-literature

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.

Project description:Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

| S-EPMC2830763 | biostudies-literature

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

Project description:How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

| S-EPMC3140544 | biostudies-literature

Adenosine leakage from perforin-burst extracellular vesicles inhibits perforin secretion by cytotoxic T-lymphocytes.

Project description:Extracellular vesicles (EVs) in the tumor microenvironment facilitate intercellular communication. Cancer cell-derived EVs act as an immunosuppressor by transporting cargos and presenting transmembrane proteins. By contrast, CD8+ cytotoxic T-lymphocytes (CTLs) exert anti-cancer cytotoxicity via the pore-forming protein perforin. Here, we hypothesize that although EVs are destroyed by perforin, cancer cell-derived EVs might possess mechanisms that enable them to avoid this destruction. We used a breast cancer cell line, MDA-MB-231-luc-D3H2LN (D3H2LN), to generate EVs. Destruction of the EVs by perforin was demonstrated visually using atomic force microscopy. To investigate immunosuppressive metabolites within cancer cell-derived EVs, we performed metabolomic profiling of EVs from D3H2LN cells cultured for 48 h with or without IFN-γ, which induces metabolic changes in the cells. We found that both types of EV from IFN-γ treated D3H2LN cells and non-treated D3H2LN cells contained adenosine, which has immunosuppressive effects. When we exposed cancer cell-derived EVs to CTLs, perforin secretion by CTLs fell significantly. In addition, the decreases in perforin secretion were ameliorated by treatment with adenosine deaminase, which degrades extracellular adenosine. Taken together, these results suggest that after perforin secreted by CTLs disrupts the membrane of EVs, adenosine released from the EVs acts as an immunosuppressive metabolite by binding to the adenosine receptor on the CTL membrane. This mechanism provides a novel survival strategy using cancer cell-derived EVs.

| S-EPMC7147783 | biostudies-literature

Cytotoxicity of CD19-CAR-NK92 cells is primarily mediated via perforin/granzyme pathway.

Project description:Chimeric antigen receptors (CARs) have improved cancer immunotherapy in recent years. Immune cells, such as Natural killer cells (NK-cells) or T cells, are used as effector cells in CAR-therapy. NK92-cells, a cell line with known cytotoxic activity, are of particular interest in CAR-therapy since culturing conditions are simple and anti-tumor efficacy combined with a manageable safety profile was proven in clinical trials. The major pathways of immune effector cells, including NK92-cells, to mediate cytotoxicity, are the perforin/granzyme and the death-receptor pathway. Detailed knowledge of CAR-effector cells' cytotoxic mechanisms is essential to unravel resistance mechanisms, which potentially arise by resistance against apoptosis-inducing signaling. Since mutations in apoptosis pathways are frequent in lymphoma, the impact on CAR-mediated cytotoxicity is of clinical interest. In this study, knockout models of CD19-CAR-NK92 cells were designed, to investigate cytotoxic pathways in vitro. Knockout of perforin 1 (Prf1) and subsequent abrogation of the perforin/granzyme pathway dramatically reduced the cytotoxicity of CD19-CAR-NK92 cells. In contrast, knockout of FasL and inhibition of TRAIL (tumor necrosis factor-related apoptosis-inducing ligands) did not impair cytotoxicity in most conditions. In conclusion, these results indicate the perforin/granzyme pathway as the major pathway to mediate cytotoxicity in CD19-CAR-NK92 cells.

| S-EPMC10361870 | biostudies-literature

Targeted Cell-to-Cell Delivery of Protein Payloads via the Granzyme-Perforin Pathway.

Project description:There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the co-localization of the chaperones with lytic granules and the chaperone-mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full-length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

| S-EPMC5700818 | biostudies-literature

The combination of the prodrugs perforin-CEBPD and perforin-granzyme B efficiently enhances the activation of caspase signaling and kills prostate cancer.

Project description:The survival of prostate cancer (PrCa) patients is associated with the transition to hormone-independent tumor growth and metastasis. Clinically, the dysregulation of androgen action has been associated with the formation of PrCa and the outcome of androgen deprivation therapy in PrCa. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor that has been reported to act as an oncogene or tumor suppressor, depending on the extra- and intracellular environments following tumorigenesis. We found that androgen can activate CEBPD transcription by direct binding of the androgen receptor (AR) to the CEBPD promoter region. Increases of suppressor of zeste 12 (SUZ12) and enhancer of zeste homolog 2 (EZH2) attenuated the androgen-induced transcription of CEBPD. Importantly, the increases in E2F1, SUZ12 and EZH2 as well as the inactivation of CEBPD were associated with the clinicopathological variables and survival of PrCa patients. We revealed that caspase 8 (CASP8), an apoptotic initiator, is responsive to CEBPD induction. Reporter and in vivo DNA-binding assays revealed that CEBPD directly binds to and activates CASP8 reporter activity. A prodrug system was developed for therapeutic application in AR-independent or androgen-insensitive PrCa to avoid the epigenetic effects on the suppression of CEBPD expression. Our results showed that the combination of a perforin (PF)-CEBPD prodrug (which increases the level of procaspase-8) and a PF-granzyme B prodrug (which activates CASP8 and caspase 3 (CASP3)) showed an additive effect in triggering the apoptotic pathway and enhancing apoptosis in PrCa cells.

| S-EPMC4047860 | biostudies-literature

Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosis.

Project description:How granzymes gain entry into the cytosol of target cells during killer cell attack has been the subject of several studies in the past, but the effective delivery mechanism during target cell encounter has not been clarified. Here we show that granzyme B (GzmB) mutants lacking binding to negatively charged, essentially heparan-sulfate-containing membrane receptors are poorly endocytosed yet are delivered to the cytosol with efficacy similar to that of WT GzmB. In a cell-based system GzmB-deficient natural killer cells provided perforin (pfn) by natural polarized secretion and synergized with externally added GzmB. Whereas receptor (heparan sulfate)-dependent endocytosis was dispensable, delivery of larger cargo like that of GzmB fusion proteins and GzmB-antibody complexes was restricted by their size. Our data support the model in which granzymes are primarily translocated through repairable membrane pores of finite size and not by the disruption of endocytosed vesicles. We conclude that structurally related translocators, i.e., perforin and cholesterol-dependent cytolysins, deliver deathly cargo across host cell membranes in a similar manner.

| S-EPMC2544534 | biostudies-literature

Temporal Dynamics of Serum Perforin and Granzyme during the Acute Phase of SARS-CoV-2 Infection.

Project description:BackgroundAs many SARS-CoV-2 infections are asymptomatic, it could be useful to be able to determine how much time has passed since infection. We explored the changes in the temporal levels of T cell-related proteins (including perforin and granzymes) in the sera of patients with SARS-CoV-2 infection using a commercially available assay.MethodsThis study enrolled 36 patients infected with SARS-CoV-2 and 20 healthy control participants. Blood samples were collected at three different times based on the number of days since symptom onset (early phase: 1-5 days, mid-phase: 6-10 days, late phase: 11-18 days). We assessed the temporal changes in the serum levels of perforin and granzymes in patients with SARS-CoV-2 infection by comparing the results with those obtained in the healthy control group.ResultsWe identified a significantly low level of perforin in the early phase of SARS-CoV-2 infection (p < 0.01), which was restored to normal during the mid- and late phases of the infection. However, there was no difference in the temporal change in the level of granzymes in SARS-CoV-2-infected patients compared to the healthy control group.ConclusionsThis finding suggests that SARS-CoV-2 infection paralyzed the perforin expression in the early period immediately after infection. Thus, serum perforin is a potential marker for identifying the acute phase of SARS-CoV-2 infection.

| S-EPMC10459539 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data