Inefficient Translocation of Preproinsulin Contributes to Pancreatic β Cell Failure and Late-onset Diabetes

Guo, Huan; Xiong, Yi; Witkowski, Piotr; Cui, Jingqing; Wang, Ling Jia; Sun, Jin; Lara-Lemus, Roberto; Haataja, Leena; Hutchison, Kathryn; Shan, Shu‐ou; Arvan, Peter; Liu, Ming

doi:10.1074/jbc.m114.562355

Cited by 61 publications

(87 citation statements)

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“…We have reported recently that a positive charge in the N region and charge gradient flanking the H region of SP plays a critical role in orienting the SP of preproinsulin during translocation. Indeed, two SP mutations, pPI-R6C and pPI-R6C/D20R, cause ϳ50% and ϳ90%, respectively, of preproinsulin to become misoriented in the ER membrane during translocation (28). We therefore introduced F25P into pPI-R6C or pPI-R6C/D20R, generating double and triple mutants, respectively (Fig.…”

Section: Uncleaved Ppi-f25p Is Integrated Into the Er Membrane And Imentioning

confidence: 99%

“…For quantification of the mRNA levels of HCV structural proteins in transfected HEK293T cells, 48 h after transfection, the total RNA was isolated and used for synthesizing cDNA, followed by quantitative real-time PCR using the forward primer 5Ј GCG-CCTCACCAGCTTATTTG and the reverse primer 5Ј ATAA-AGCCGGTGTGCAAGGA. Sodium carbonate extraction was performed as described previously (28). Briefly, 48 h post-transfection, HEK293T cells were suspended in 0.1 M sodium carbonate (pH 12), homogenized, and incubated on ice for 1 h, followed by sedimentation at 50,000 rpm at 4°C for 1 h. The supernatants and pellets were boiled for 5 min in SDS sample buffer containing 100 mM DTT and analyzed by Western blotting as described above.…”

Section: Cell Culture and Transfection Metabolic Labeling Immunoprementioning

confidence: 99%

“…A loss of positive charge in the N region (R6C/F25P) causes a defect in orientation and translocation in 50% of pPI molecules. Reversing the charge gradient flanking the H region (R6C/D20R/F25P) leads to a defect in orientation and translocation of ϳ90% of pPI molecules (28). C, 293T cells were co-transfected with untagged pPI-WT plus either 2Myc-tagged pPI-F25P or pPI-R6C/F25P at a 1:2 DNA ratio.…”

Section: Uncleaved Ppi-f25p Is Integrated Into the Er Membrane And Imentioning

confidence: 99%

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Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Cui

Chen

Sun

et al. 2015

Journal of Biological Chemistry

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Background: Signal peptidase (SPase) excises the signal peptide of secretory preproteins. Results: A variant preproinsulin with a proline following the signal peptide cleavage site binds to and inhibits SPase. Conclusion: Inhibition of SPase impairs, in trans, the intracellular processing, trafficking, and maturation of secretory proteins and viral polypeptides. Significance: Our findings suggest eukaryotic SPase as a potential antiviral target.

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Section: Uncleaved Ppi-f25p Is Integrated Into the Er Membrane And Imentioning

confidence: 99%

Section: Cell Culture and Transfection Metabolic Labeling Immunoprementioning

confidence: 99%

Section: Uncleaved Ppi-f25p Is Integrated Into the Er Membrane And Imentioning

confidence: 99%

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Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Cui

Chen

Sun

et al. 2015

Journal of Biological Chemistry

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“…Pancreatic b-cells do not secrete whole PPI as an extracellular source for uptake by DC. However, a small proportion of whole PPI (including the signal sequence) is present in the cytosol of human islets that may become accessible to DC under pathological conditions in T1D, such as b-cell stress, thus ending up in HLA-DQ for presentation to the immune system (37,38). We previously reported PPI 54-69 as preferentially presented by the highest-risk HLA-DQ8trans molecule (30).…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Selective Islet Peptidome Presented by the Highest-Risk HLA-DQ8trans Molecule

Lummel

Veelen

et al. 2015

Diabetes

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HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLADQ8trans-binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLADQ8trans. The selective processing and presentation of HLA-DQ8trans-binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T-cell-mediated destruction of the insulin-producing pancreatic b-cells (1-4). The search for naturally processed and presented epitopes (NPPE) for high-risk HLA class II as a target for autoreactive CD4 T cells in T1D has been focus of attention over the years. Most attention was given to HLA-DR-binding epitopes (5,6), whereas HLA-DQ-binding epitopes deserve investigation, too. Indeed, subjects heterozygous for HLA-DQ2 and -DQ8 are endorsed with by far the highest risk for development of T1D, but what functional consequences explain this synergistically increased risk compared with a double dose of HLA-DQ2 or -DQ8 remain unclear. We previously revealed the unique peptide binding properties of HLA-DQ molecules composed of the products of DQA1*0201 (coding for the a-chain of DQ2) and DQB1*0302 (coding for the b-chain of DQ8), the so-called HLA-DQ8trans molecule (7-9). The islet epitopes presented by HLA-DQ8trans are largely unknown. Importantly, HLA-DQ8cis/trans-restricted CD4 T-cell clones have been isolated from human insulitis lesions, and T-cell autoreactivity was confirmed for several proinsulin peptides, underscoring the potential relevance of preproinsulin (PPI) peptides presented by HLA-DQ in diabetogenesis (10-13). We contend that knowledge of the HLA-DQ8trans islet peptidome provides insight in the mechanism by which HLA-DQ2/8 hete...

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“…However, binding of this short signal peptide to HLA-DR4 was confirmed. PPI as whole protein is only present in low quantities in the cytosol of pancreatic b cells, but the amount increases under pathological conditions (45,46). It is conceivable that b cells suffering from hyperglycemia, inflammation, or viral assaults may develop endoplasmic reticulum stress, which in turn upregulates PPI quantities (47).…”

Section: Discussionmentioning

confidence: 99%

Dendritic Cells Guide Islet Autoimmunity through a Restricted and Uniquely Processed Peptidome Presented by High-Risk HLA-DR

Lummel

Veelen

et al. 2016

The Journal of Immunology

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Identifying T cell epitopes of islet autoantigens is important for understanding type 1 diabetes (T1D) immunopathogenesis and to design immune monitoring and intervention strategies in relationship to disease progression. Naturally processed T cell epitopes have been discovered by elution from HLA-DR4 of pulsed B lymphocytes. The designated professional APC directing immune responses is the dendritic cell (DC). To identify naturally processed epitopes, monocyte-derived DC were pulsed with preproinsulin (PPI), glutamic acid decarboxylase (65-kDa isoform; GAD65), and insulinoma-associated Ag-2 (IA-2), and peptides were eluted of HLA-DR3 and -DR4, which are associated with highest risk for T1D development. Proteome analysis confirmed uptake and processing of islet Ags by DC. PPI peptides generated by DC differed from those processed by B lymphocytes; PPI signal-sequence peptides were eluted from HLA-DR4 and -DR3/4 that proved completely identical to a primary target epitope of diabetogenic HLA-A2–restricted CD8 T cells. HLA-DR4 binding was confirmed. GAD65 peptides, eluted from HLA-DR3 and -DR4, encompassed two core regions overlapping the two most immunodominant and frequently studied CD4 T cell targets. GAD65 peptides bound to HLA-DR3. Strikingly, the IA-2 ligandome of HLA-DR was exclusively generated from the extracellular part of IA-2, whereas most previous immune studies have focused on intracellular IA-2 epitopes. The newly identified IA-2 peptides bound to HLA-DR3 and -DR4. Differential T cell responses were detected against the newly identified IA-2 epitopes in blood from T1D patients. The core regions to which DC may draw attention from autoreactive T cells are largely distinct and more restricted than are those of B cells. GAD65 peptides presented by DC focus on highly immunogenic T cell targets, whereas HLA-DR–binding peptides derived from IA-2 are distinct from the target regions of IA-2 autoantibodies.

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Inefficient Translocation of Preproinsulin Contributes to Pancreatic β Cell Failure and Late-onset Diabetes

Cited by 61 publications

References 63 publications

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Discovery of a Selective Islet Peptidome Presented by the Highest-Risk HLA-DQ8trans Molecule

Dendritic Cells Guide Islet Autoimmunity through a Restricted and Uniquely Processed Peptidome Presented by High-Risk HLA-DR

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