Abstract
The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.
| Original language | English |
|---|---|
| Pages (from-to) | 1517-1529 |
| Number of pages | 13 |
| Journal | Cell Death and Differentiation |
| Volume | 25 |
| DOIs | |
| Publication status | Published - 7 Feb 2018 |
Keywords
- blood proteins
- immune cell death
Cite this
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Perforin proteostasis is regulated through its C2 domain : supra-physiological cell death mediated by T431D-perforin. / Brennan, Amelia J.; Law, Ruby H. P.; Conroy, Paul J.; Noori, Tahereh; Lukoyanova, Natalya; Saibil, Helen; Yagita, Hideo; Ciccone, Annette; Verschoor, Sandra; Whisstock, James C.; Trapani, Joseph A.; Voskoboinik, Ilia.
In: Cell Death and Differentiation, Vol. 25, 07.02.2018, p. 1517-1529.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Perforin proteostasis is regulated through its C2 domain
T2 - supra-physiological cell death mediated by T431D-perforin
AU - Brennan, Amelia J.
AU - Law, Ruby H. P.
AU - Conroy, Paul J.
AU - Noori, Tahereh
AU - Lukoyanova, Natalya
AU - Saibil, Helen
AU - Yagita, Hideo
AU - Ciccone, Annette
AU - Verschoor, Sandra
AU - Whisstock, James C.
AU - Trapani, Joseph A.
AU - Voskoboinik, Ilia
PY - 2018/2/7
Y1 - 2018/2/7
N2 - The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.
AB - The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.
KW - blood proteins
KW - immune cell death
UR - http://www.scopus.com/inward/record.url?scp=85041547104&partnerID=8YFLogxK
U2 - 10.1038/s41418-018-0057-z
DO - 10.1038/s41418-018-0057-z
M3 - Article
VL - 25
SP - 1517
EP - 1529
JO - Cell Death and Differentiation
JF - Cell Death and Differentiation
SN - 1350-9047
ER -