Structure-Function Studies of PANDER, an Islet Specific Cytokine Inducing Cell Death of Insulin-Secreting Cells
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- 作者：Jichun Yang ; Zhiyong Gao ; Claudia E. Robert ; Brant R. Burkhardt ; Helena Gaweska ; Amary Wagner ; Jianmei Wu ; Scott R. Greene ; Robert A. Young ; and Bryan A. Wolf
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 30, 2005
- 年：2005
- 卷：44
- 期：34
- 页码：11342 - 11352
- 全文大小：675K
- 年卷期：v.44,no.34(August 30, 2005)
- ISSN：1520-4995
文摘
PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretorygranules, that induces apoptosis of 
and 
cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of4 -helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutatedPANDERs were constructed and expressed in -TC3 cells to identify the essential region of PANDERinvolved in -cell death. -Cell function was assessed by assays of cell viability and insulin secretion.Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized anddetected in -TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncationof helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices Cand D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A,the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced -celldeath. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfidebond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the activepart of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices Band C and the second disulfide bond of PANDER are essential for PANDER-induced -cell death.
and cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of4 -helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutatedPANDERs were constructed and expressed in -TC3 cells to identify the essential region of PANDERinvolved in -cell death. -Cell function was assessed by assays of cell viability and insulin secretion.Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized anddetected in -TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncationof helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices Cand D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A,the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced -celldeath. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfidebond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the activepart of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices Band C and the second disulfide bond of PANDER are essential for PANDER-induced -cell death.