Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells.
Defective Proinsulin Handling Modulates the MHC I Bound Peptidome and Activates the Inflammasome in β-Cells.
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DOI：
10.3390/biomedicines10040814
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发表时间：
2022-03-30
期刊：
影响因子：
4.7
通讯作者：
中科院分区：
文献类型：
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引发自身免疫性1型糖尿病的胰腺β细胞肽是如何失去免疫耐受性的，这是一个谜。我们已经证明，内质网(ER)中胰岛素原分子伴侣葡萄糖调节蛋白(GRP)94的缺失会导致胰岛素原的错误处理、内质网应激以及免疫蛋白酶体的激活。我们推测，相对于生物合成需求而言，内质网胰岛素原折叠能力不足可能导致β细胞主要组织相容性复合体(MHC)I类结合的多肽和炎性小体激活改变，从而使β细胞对免疫攻击敏感。我们使用有或没有GRP94基因敲除(KO)的INS-1E细胞，或有或不有GRP94抑制剂PU-WS13(GRP94I，20µM)的INS-1E细胞，或暴露于促炎症细胞因子IL-1β或干扰素γ(分别为15 pg/m L和10 ng/m L)24 h后，用流式细胞仪检测大鼠MHC I的表达。用反相高效液相色谱(RP-HPLC)分离细胞裂解产物，然后用液相色谱-串联质谱仪(LC-MS/MS)分析总RT1.A结合的多肽。用Western blotting检测核苷酸结合的寡聚区、富含亮氨酸重复序列和吡咯区的蛋白(NLRP1)、B细胞抑制因子α中kappa轻多肽基因增强子的核因子(IκBα)和(Pro)IL-1β的表达和分泌。与相关对照组相比，GRP94KO增加了β-细胞中RT1.A的表达，细胞因子暴露也增加了RT1.A的表达。免疫组学分析显示，GRP94 KO/I细胞和细胞因子处理后的细胞中，RT1.A结合的多肽库均增加。GRP94KO/细胞因子暴露组在他们的肽库中显示出部分重叠。值得注意的是，随着细胞因子的暴露，GRP94KO/I中的胰岛素原衍生多肽多样性增加。在GRP94缺陷的细胞中，随着IκBα含量的降低，NLRP1的表达上调，而ProIL 1β的细胞水平下降，并伴随着成熟IL 1β的分泌增加。我们的结果表明，限制β细胞胰岛素原伴侣作用增强了β。A表达改变了包括胰岛素原多肽在内的MHC-I型多肽，并激活了炎症途径，提示与阻碍胰岛素原处理相关的应激可能使RT1细胞对免疫攻击敏感。
How immune tolerance is lost to pancreatic β-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered β-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing β-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1β or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in β-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1β cellular levels declined, coupled with increased secretion of mature IL-1β. Our results suggest that limiting β-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize β-cells to immune-attack.
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DOI：
10.1126/science.aad2791
发表时间：
2016-02-12
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Delong T;Wiles TA;Baker RL;Bradley B;Barbour G;Reisdorph R;Armstrong M;Powell RL;Reisdorph N;Kumar N;Elso CM;DeNicola M;Bottino R;Powers AC;Harlan DM;Kent SC;Mannering SI;Haskins K
通讯作者：
Haskins K
影响因子：
29.7
作者：
Blum JS;Wearsch PA;Cresswell P
通讯作者：
Cresswell P
影响因子：
6
作者：
Ghemrawi R;Battaglia-Hsu SF;Arnold C
通讯作者：
Arnold C
影响因子：
32.4
作者：
Basu, S;Binder, RJ;Srivastava, PK
通讯作者：
Srivastava, PK
影响因子：
4.4
作者：
Berwin, B;Hart, JP;Nicchitta, CV
通讯作者：
Nicchitta, CV