Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Moreno, Ramón; Gil, Juana; Rodríguez-Sáinz, Carmen; Cela, Elena; LaFay, Victor; Oloizia, Brian; Herr, Andrew B.; Sümegi, János; Jordan, Michael B.; Risma, Kimberly A.

doi:10.1182/blood-2008-08-172924

Cited by 25 publications

(16 citation statements)

References 34 publications

(72 reference statements)

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“…These remarkable studies establish that pro-PFN as expressed in baculovirus is as active as the mature form and lends support to the observation that PFN may be secreted constitutively under certain conditions in vivo (Makedonas et al, 2009; Urrea Moreno et al, 2009). Instead, the C-terminus appears to regulate trafficking of this highly toxic protein.…”

supporting

confidence: 65%

Intracellular Trafficking of Perforin: To Thwart a Killer

Froelich

Metkar

2011

Immunity

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supporting

confidence: 65%

Intracellular Trafficking of Perforin: To Thwart a Killer

Froelich

Metkar

2011

Immunity

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“…(26) EL4 target cells were pulsed with 100ng/mL of LCMV gp33–41 for one hour prior to 51 Cr labeling. Effector and EL4 target cells (5,000 cells/well) were co-incubated for 4 hours at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Real-Time Detection of CTL Function Reveals Distinct Patterns of Caspase Activation Mediated by Fas versus Granzyme B

Figueira

Vrazo

et al. 2014

The Journal of Immunology

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Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas- mediated pathways of killing by cytotoxic T lymphocytes (CTL); however, the kinetics of caspase activation remain undefined due to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease specific activity in target cells. Biosensors were engineered from a circularly-permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Co-incubation of murine CTL with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor utilized. Additionally, the luciferase signal at 30 minutes correlated with target cell death, as measured by 51Cr release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B compared to Fas-mediated signal induction in murine CTL; the latter appeared gradually after a 90 minute delay in perforin or granzyme B deficient CTL. Remarkably, the Fas dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTL was also detectable after a 90 minute delay when measured by re-directed killing. Thus we have utilized a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTL.

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“…These three domains are further organized into eight subdomains (Figure a,c). We have determined using the ExAC Browser database and primary literature sources that there are 460 SNVs present in the coding region of human PRF1 (Figure b) (An et al., ; Clementi et al., ; Feldmann et al., ; Goransdotter Ericson et al., ; Horne et al., ; Husami, ; Lee et al., ; Lek et al., ; Stepp et al., ; Urrea Moreno et al., ; Voskoboinik et al., ; Zhang et al., ; Zur Stadt et al., ). Of the 555 amino acids in perforin, over half are associated with a mutation.…”

Section: Resultsmentioning

confidence: 99%

Human perforin gene variation is geographically distributed

Willenbring

Ikeda

Pease

et al. 2017

Molec Gen & Gen Med

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BackgroundDeleterious mutations in PRF1 result in lethal, childhood disease, familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). However, not all mutations in PRF1 are deleterious and result in FHL 2. Currently, these nondeleterious mutations are being investigated in the onset of numerous disorders, such as lymphomas and diabetes. Yet, there is still an overwhelmingly large amount of PRF1 mutations that are not associated with disease.MethodsWe conducted a post hoc analysis of the PRF1 mutations in the coding region using the recently published Exome Aggregation Consortium genomes, Leiden Open Variation Database, NCBI SNP database, and primary literature to better understand PRF1 variation in the human population.ResultsThis study catalogs 460 PRF1 mutations in the coding region, and demonstrates PRF1 is more variant then previously predicted. We identify key PRF1 mutations with high allelic frequency and are only found in certain populations. Additionally, we define PRF1 SNVs are geographically distributed.ConclusionsThis study concludes with a novel hypothesis that nondeleterious mutation in PRF1, which decreases perforin expression and/or activity, may be an example of selective advantage in the context of environmental stressors prevalent near the equator. Our studies illustrate how perforin deficiency can be protective from injuries resulting in blood–brain barrier (BBB) disruption.

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Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Cited by 25 publications

References 34 publications

Intracellular Trafficking of Perforin: To Thwart a Killer

Intracellular Trafficking of Perforin: To Thwart a Killer

Real-Time Detection of CTL Function Reveals Distinct Patterns of Caspase Activation Mediated by Fas versus Granzyme B

Human perforin gene variation is geographically distributed

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