A Novel Nonsense INS Mutation Causes Inefficient Preproinsulin Translocation Into the Endoplasmic Reticulum

Preproinsulin (PPI) translocation across the membrane of the endoplasmic reticulum (ER) is the first and critical step of insulin biosynthesis. Inefficient PPI translocation caused by signal peptide (SP) mutations can lead to β-cell failure and diabetes. However, the effect of proinsulin domain on the efficiency of PPI translocation remains unknown. With whole exome sequencing, we identified a novel INS nonsense mutation resulting in an early termination at the 46th residue of PPI (PPI-R46X) in two unrelated patients with early-onset diabetes. We examined biological behaviors of the mutant and compared them to that of an established neonatal diabetes causing mutant PPI-C96Y. Although both mutants were retained in the cells, unlike C96Y, R46X did not induce ER stress or form abnormal disulfide-linked proinsulin complexes. More importantly, R46X did not interact with co-expressed wild-type (WT) proinsulin in the ER, and did not impair proinsulin-WT folding, trafficking, and insulin production. Metabolic labeling experiments established that, despite with an intact SP, R46X failed to be efficiently translocated into the ER, suggesting that proinsulin domain downstream of SP plays an important unrecognized role in PPI translocation across the ER membrane. The study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.


INTRODUCTION
Insulin is an essential hormone for maintaining glucose homeostasis of the body. In pancreatic bcells, the insulin biosynthesis starts from its precursor, preproinsulin (PPI), which is composed of the N-terminal signal peptide (SP) followed by the C-terminal proinsulin domain. Newly synthesized PPI driven by its SP translocates across the membrane of the endoplasmic reticulum (ER) through the Sec61 translocon. Upon delivery into the ER, SP of PPI is cleaved by signal peptidase at the luminal side of the ER membrane, forming another insulin precursor, proinsulin (PI) (1). In the oxidized ER lumen, PI undergoes rapid oxidative folding, forming three highly conserved disulfide bonds (B7-A7, B19-A20, and A6-A11). Well-folded PI exits from the ER and traffics through the Golgi to the secretory granules where it is processed by prohormone convertase (PC1/3 and PC2) and carboxypeptidase E (CPE), forming mature insulin and Cpeptide (2)(3)(4). It takes approximately 30-150 min to finish these intracellular processes. Among these events, the earliest step during which PPI translocation into the ER had been long thought to be very efficient and exclusively through the signal recognition particle (SRP)-dependent co-translational translocation (5). However, recent evidence indicates that, as a small secretory protein with a suboptimal signal sequence, the fully synthesized PPI may not be efficiently recognized by SRP and cotranslationally translocated into the ER. SRP independent posttranslational translocation then functions as an important backup to enhance PPI translocation (4,6,7). The pathophysiological significance of inefficient PPI translocation is highlighted by recent evidence showing that deficiency of TRAPa [translocon associated protein alpha, a type 2 diabetes associated gene (8)], TRAPb, and/ or TRAPd impairs PPI translocation and insulin production (9,10), and also by the discoveries of PPI SP mutations that impair PPI translocation causing b-cell failure and diabetes in humans (2,(11)(12)(13)(14).
In addition to the PPI SP mutations, at least 70 INS gene mutations located in the proinsulin domain have been reported to be associated with diabetes in humans (15)(16)(17)(18)(19)(20). The majority of these mutations impair proinsulin oxidative folding in the ER, causing proinsulin misfolding, and decreasing insulin production (21)(22)(23)(24). However, no INS mutation located in the proinsulin domain has been reported to affect translocation of PPI. Herein, we identified a novel nonsense INS mutation causing an early termination at the 46th residue of PPI (PPI-R46X) in two unrelated patients with early-onset diabetes. We found that, unlike other proinsulin domain mutations, R46X did not appear to induce strong ER stress or form abnormal disulfide-linked proinsulin complexes (DLPC) with coexpressed wild-type (WT) proinsulin in the ER. Metabolic labeling experiments established that, despite with an intact SP, R46X failed to be efficiently translocated into the ER, suggesting an important unrecognized role of proinsulin domain in determining the efficiency of PPI translocation. Genetic testing, pedigree analysis, and clinical presentations revealed a broad spectrum of diabetes phenotypes among the members carrying R46X in the two families, suggesting that other genetic and environmental factors may contribute to actual clinical presentations associated with R46X. This study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.

Patients
Two unrelated patients with early-onset diabetes were referred for genetic testing of the monogenic diabetes. The clinical information was collected and recorded by the Tianjin Medical University General Hospital and the second Part of Jilin University First Hospital. The clinical information of these two probands and their family members is described in Tables 1-3.

Genetic Testing and Analyses
Targeted gene capture followed by whole exome sequencing with massively parallel next-generation sequencing (NGS) was performed with the genomic DNA extracted from white blood cells. The identified INS gene mutation was confirmed by Sanger sequencing with the primer: Forward 5'-TCAGCCCTG CCTGTCTCC-3', Reverse 5'-AAAAGTGCACCTGAC CCCCTG-3'. The members of the two families of two probands were recruited and tested for the mutation by Sanger sequencing.
Cell Culture and Transfection, 35 35 S-(Met+Cys) and chased for the times indicated. The cells were harvested and lysed. Trichloroacetic acid (TCA)-precipitable counts were performed to normalize the amount of the total proteins among samples. The cell lysates were immunoprecipitated with anti-insulin antibody overnight at 4°C, and analyzed using 4%-12% NuPage gel under reducing condition as previously described (25). For co-immunoprecipitation (Co-IP), the cells were lysed with co-IP buffer (100 mM NaCl, 25 mM Tris, pH 7.0, 0.1% Triton X-100, 5 mM EDTA, and protease inhibitors mixture). Ninety percent of the total lysates were immunoprecipitated with anti-GFP at 4°C for 3 h. The remaining 10% of the total lysates were used to determine the expression levels of Myc-tagged and GFP-tagged PPI WT and Myc-tagged mutants. The immunoprecipitates and the total lysates were resolved using 4%-12% NuPage gel under reducing conditions followed by immunoblotted with anti-Myc and anti-proinsulin antibodies as indicated.

Immunofluorescence Assay
An immunofluorescence assay was employed in INS832/13 cells transfected with plasmids encoding Myc-tagged PPI WT or mutants (C96Y and R46X). Briefly, transfected INS832/13 cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.2% Triton for an additional 15 min and then blocking with 5% BSA. The cell samples were incubated with primary antibodies (Anti-Myc 1:3000 dilution, anti-insulin 1:5,000 dilution) followed by appropriate secondary antibodies conjugated with different fluor as indicated. Immunofluorescence images were acquired by using Axio Imager M2 (ZEISS, Baden-Württemberg, Germany).

Statistical Analysis
All data were processed with GraphPad Prism 8 and presented as means ± SEM. One-way ANOVA test was used to determine significance between the groups. A p-value < 0.05 was considered as statistically significant.

Identification of a Novel Nonsense INS Mutation R46X Associated With Diabetes in Two Unrelated Patients
We identified an INS gene mutation (NM_001042376.2: p. Arg46*/c.136C>T) in two unrelated patients with early-onset  ) were all negative. She was treated with insulin (0.71 U/kg/day) at the time of diagnosis. Self-monitoring of blood glucose (SMBG) showed that her fasting blood glucose (FBG) was 4-5 mmol/L and 2 h postprandial blood glucose (2hPBG) was 6-9 mmol/L, suggesting that her diabetes was well controlled. Subsequently 1 year later, she withdrew insulin without doctor's advice and only took metformin 1.5 g/day. Her HbA1c was 7.1% at the last visit. Notably, the proband's mother and older brother were found to carry the same mutation but only had impaired glucose regulation ( Figure 1A and Table 2). Her maternal uncle was diagnosed with diabetes at the age of 40. Unfortunately, his blood sample was unavailable for genetic testing because he has passed away. The second proband was diagnosed with diabetes when she was 8 years old. She was accidentally found to have hyperglycemia when she had an upper respiratory tract infection. At the time of diabetes diagnosis, her BMI was 20.9 kg/m 2 and HbA1c was 13% without typical symptoms of diabetes. She was initiated insulin therapy (0.73 U/kg/day). However, the patient adherence was low and her blood glucose was poorly controlled even with a high dose of insulin therapy (1.87 U/kg/day). Except for her maternal grandmother whose fasting blood glucose were 6-7 mmol/L, her family members carrying this mutation were all diagnosed with diabetes at various onset ages ( Figure 1B and Table 3).  (2). We therefore firstly examined the secretion of R46X in transfected 293T cells and INS1 cells. An established proinsulin mutant PPI-C96Y (also called Akita proinsulin), which causes proinsulin misfolding and neonatal diabetes (19,23), was used as a positive control. Although the total amount of proinsulin was comparable (Figure 2A, left panel), both R46X and C96Y failed to be secreted from the cells to the media in transfected cells (Figures 2A, B). This secretion defect was further confirmed in INS1 cells transfected with PPI-WT and mutants ( Figures 2C, D).

R46X Mutant Impaired
Theoretically, the amount of insulin expressed by a single allele is enough for control blood glucose in vivo (2). However, most diabetes causing INS mutants are heterozygous and present as autosomal dominant fashion, indicating trans-dominant negative effect of proinsulin mutants on bystander proinsulin-WT. We have previously reported that C96Y could interact with coexpressed WT proinsulin and prevent its ER export (26). We therefore asked whether this mechanism also applies for R46X mutant. We co-expressed WT-untagged with WT-Myc or mutants (C96Y-Myc and R46X-Myc) in 293T cells. Consistent with our previous observation, C96Y blocked secretion of coexpressed proinsulin-WT. In contrast, although R46X could not be secreted from the cells, it did not impair the secretion of bystander proinsulin-WT ( Figures 2E, F). Next, we asked whether R46X could generate a dominant negative effect on insulin production from endogenous proinsulin in b cells. We transfected WT-Myc or mutants (C96Y-Myc and R46X-Myc) into INS832/13 cells. In the cells expressing C96Y-Myc (red), endogenous insulin (green) was significantly decreased compared with the neighboring control cells. However, although R46X itself displayed a diffused pattern within the cells, it did not appear to affect the insulin content of b cells ( Figure 2G). Together, these results indicated that although the secretion efficiency of R46X was reduced, R46X did not affect secretion or production of bystander proinsulin-WT, nor did it impair insulin production from endogenous proinsulin.

R46X Did Not Form Disulfide-Linked Proinsulin Complexes, nor Did It Interact With Proinsulin-WT
In order to examine the effect of R46X on the folding of the mutant, we expressed Myc-tagged PPI-WT mutants in 293T cells and INS1 cells, and analyzed their oxidative folding under nonreducing conditions. Consistent with our previous reports (21,27), we found that C96Y formed more misfolded disulfide-linked proinsulin complexes (DLPC, including dimer, trimer, tetramer, and high-molecular-weight proinsulin complexes) than that of WT. Surprisingly, however, unlike C96Y, R46X did not form DLPC itself in 293T cells ( Figures 3A, B). Similar results were confirmed in INS1 cells transfected with Myc-tagged PPI-WT, C96Y, or R46X ( Figures 3C, D). To examine whether R46X interacts with co-expressed PPI-WT, we co-expressed untagged PPI-WT with empty vector or Myc-tagged PPI-WT, or mutants (C96Y-MYC and R46X-Myc) in 293T cells. We found that, under non-reducing conditions, untagged PPI-WT and Myctagged PPI-WT could form disulfide-linked homodimers (marked as D and D', respectively) as well as heterodimers (arrow, and also marked as "H"). Importantly, C96Y-Myc not only formed more disulfide-linked homodimers, but also formed heterodimers with co-expressed untagged PPI-WT ( Figure 3E). On the contrary, R46X-Myc did not form homodimers by itself or form heterodimers with untagged PPI-WT ( Figure 3E). To further confirm whether R46X interacted with proinsulin-WT in the ER, we performed co-immunoprecipitation experiments, in which GFP-tagged PPI-WT with Myc-tagged PPI-WT or mutants (C96Y-MYC and R46X-Myc) were expressed in 293T cells. We found that C96Y-Myc and WT-Myc could be coimmunoprecipitated by GFP-tagged PI-WT (WT-GFP). However, R46X could not be pulled down by PI-WT-GFP ( Figures 3F, G), indicating that R46X did not interact with PI-WT in the ER. Furthermore, we explored the effect of R46X mutation on ER stress and b-cell survival. We used an established ER stress reporter (21,25,28,29) Bip-promoter driven luciferase assay to evaluate ER stress response in both INS1 and Min6 cells expressing Myc-tagged PPI-WT or mutants. We found that unlike other INS mutations, R46X mutation did not induce significant increases in ER stress response (Supplemental Figures 1A, B). In order to examine whether R46X could affect b-cell survival, we have examined cleaved caspase 3 in Min6 cells expressing PPI-WT and mutants. Min6 cells treated with ER stress inducer thapsigargin was used as a positive control. We found that R46X did not increase cleaved caspase 3, a critical executioner of apoptosis, suggesting that it did not cause b-cell apoptosis (Supplemental Figures 1C, D). Together, those data indicate that R46X does not interact and affect co-expressed PI-WT and does not induce strong enough ER stress as other tested mutations do, which does not appear to lead to apoptosis at least based on the short-term in vitro assays in cell lines. The future in vivo experiments using transgenic/ knock-in mice expressing R46X may bring more certainty regarding the effect of the mutant on b-cell stress responses and survival.

R46X Causes an Inefficient Translocation Into the ER and Is Less Stable in the Cells
To further investigate biological behavior of R46X, we performed metabolic labeling experiments. Since the two methionine residues present only in the signal peptide of PPI and six cysteine residues locate in the proinsulin domain ( Figure 4A, upper panel), 35 S-methionine ( 35 S-Met only) can only label signal peptide attached PPI, but not proinsulin, whereas 35 S-methionine/cysteine ( 35 S-Met/Cys) mixture can label both PPI and proinsulin. As shown in Figure 4A, the newly synthesized PPI-WT and R46X were successfully detected in the cells labeled with 35 S-Met/Cys. However, only R46X was detected in the cells  6 and 7). Forty-eight hours post-transfection, the cell lysates were treated with or without PNGaseF before analyzing using Western blotting under reducing conditions. The non-specific bands at~17 Kd were marked with a black triangle.
labeled with 35 S-Met only, suggesting that the signal peptide was still attached to the proinsulin domain of the R46X mutant ( Figure 4A, the first five lanes of the bottom panel). Upon 10 min chase, a majority of the newly synthesized R46X was degraded ( Figure 4A, the last two lanes of the bottom panel), suggesting that R46X was not stable in transfected 293T cells. Indeed, when we compared the stability of C96Y and R46X by Western blotting, we found that up to 60% of R46X was degraded in 20 min (Figures 4B, C). At least two possible reasons can cause failure of signal peptide removal of PPI-R46X ( Figure 4A). First, R46X caused inefficient translocation; therefore, the signal peptide cleavage site was not exposed to signal peptidase at the luminal side of the ER membrane. Second, R46X is successfully translocated into the ER; however, it cannot be efficiently cleaved by signal peptidase. To distinguish these possible causes, we mutated histidine to threonine at the 5th residue of proinsulin domain, generating an "N-X-T" N-linked glycosylation site (named as H29T). Consistent with our previous report (28), proinsulin WT was glycosylated as demonstrated by glycosylated proinsulin-WT that was deglycosylated upon the digestion with PNGase F ( Figure 4D, lanes 4 vs. 5). However, R46X/H29T generated two bands ( Figure 4D, lane 6). The upper band shifted down upon digestion of PNGase F, establishing that it entered the ER and acquired an N-linked glycan ( Figure 4D, lane 7), whereas the lower band in the lane 6 did not shift upon treatment of PNGaseF, suggesting that it did not enter the ER lumen and was not glycosylated ( Figure 4D. lanes 6 vs. 7). It is worth noting that, compared with R46X, more than 50% of the double mutant R46X/H29T did translocate into the ER and acquire an N-linked glycan ( Figure 4D, lanes 3 vs. 6), suggesting that mutating histidine to threonine appeared to improve translocation efficiency of R46X. We have previously shown that increased PPI N-terminal charge gradient was favorable for translocation of PPI (6,11). Therefore, the improvement of translocation efficiency of R46X/H29T was likely caused by mutation of histidine that increased charge gradient of PPI N-terminus. Altogether, these results established that R46X caused inefficient translocation and revealed an important role of proinsulin domain in determining translocation efficiency of PPI.

DISCUSSION
To date, at least 70 INS mutations have been reported to cause autosomal dominant diabetes in humans (2,20,30). Some additional mutations that disrupt transcription and translation of insulin gene due to INS deletion, promoter inactivation, and loss of translation initiation cause neonatal diabetes only when both INS alleles were affected (31), suggesting that one insulin gene allele may be sufficient to maintain normoglycemia under normal physiological conditions, although these recessive INS mutations are linked to an increased risk of diabetes (32,33). Most dominant INS mutations are located in the proinsulin domain, impairing proinsulin oxidative folding and causing proinsulin misfolding in the ER (2,16,24,34). Two main mechanisms underlie b-cell failure and diabetes caused by misfolded proinsulin mutants. First, misfolded proinsulin may accumulate in the ER, disturbing ER protein homeostasis, inducing chronic ER stress that may activate apoptotic pathway, leading to a decrease of b-cell mass (24,(35)(36)(37). Second, misfolded proinsulin can abnormally interact with coexpressed proinsulin-WT via proinsulin dimerization interface, impairing folding and the ER export of proinsulin-WT, decreasing insulin production, and leading to insulin-deficient diabetes (16,21,23,28). More recently, a study shows that misfolded proinsulin may impair intracellular trafficking and processing of the precursor of insulin receptor, causing an impairment of insulin signaling in b cells, which may also contribute to b-cell failure caused by INS mutations (38). Interestingly, the clinical phenotypes associated with different INS mutations or even with same mutations range from severe neonatal diabetes to mild adult-onset diabetes, or some even without overt diabetes as the time of studies (2,13,16,22,(39)(40)(41)(42). In this report, we identified a novel nonsense INS mutation PPI-R46X associated with early-onset diabetes in two unrelated patients. Pedigree analysis showed a broad spectrum of clinical presentations among the members carrying R46X in the two families ( Figure 1 and Tables 1, 3), suggesting that other factors that need to be further determined may contribute to actual clinical presentations associated with R46X. Functional studies showed that, although R46X failed to be secreted from the cells, unlike the mutations causing neonatal diabetes, R46X did not form DLPC, did not affect PPI-WT secretion and insulin production, and did not appear to induce strong ER stress or cause b-cell apoptosis (Figures 2, 3 and Supplemental Figure 1). In addition, R46X is less stable in the cells compared with C96Y ( Figures 4B, C). All these data suggest that R46X may not be exposed to the oxidative ER environment. Indeed, two independent approaches demonstrated that, despite with an intact SP, R46X failed to be efficiently translocated into the ER ( Figures 4A, D), highlighting a critically unrecognized role of proinsulin domain in efficient PPI translocation. Together, pedigree and functional analyses suggest that additional contributors may be required for developing diabetes in the patients carrying R46X. Further whole exome sequencing with linkage analysis among family members with or without R46X mutation, combined with functional studies, may shed light on better understanding what and how multiple genetic and/or environmental risk factors in concert with R46X lead to the development and progression of diabetes.
Preproinsulin is a small secretory protein with a suboptimal signal sequence that may not be efficiently recognized by the SRP and undergo SRP-dependent co-translational translocation across the ER membrane (7,11). SRP-independent posttranslational translocation is an important backup for efficient PPI translocation. However, the factors that determine the route and efficiency of PPI translocation remain to be further elucidated. PPI signal peptide mutations provide excellent molecular models and great opportunities to address these fundamental biological questions. To date, PPI signal peptide mutations that have been experimentally established to affect PPI translocation are PPI-R6C/H. These two mutations locate in the n-region of the PPI signal peptide, resulting in a decrease of charge gradient from PPI N-terminus to proinsulin domain. This charge gradient has been shown to play an important role in orientating PPI signal peptide in the Sec61 translocon during translocation (11), and it is more important for small secretory proteins including PPI for the post-translational translocation, because of the large secretory proteins that do not depend on the N-terminal positive charge for their translocation (6,43), suggesting that the length of secretory proteins may also play a role in selecting the translocation pathway and efficiency. Indeed, R46X results in translating a very small truncated PPI, causing inefficient translocation. However, increasing a charge gradient in the N-terminus of R46X by mutating histidine to threonine at the 5th residue of proinsulin domain improves efficiency of R46X translocation by more than 50%, supporting the notion that both N-terminal positive charge and the length of PPI are important for efficient PPI translocation.
In summary, this study reveals that PPI translocation efficiency is affected not only by the natural property of its signal sequence, but also by its downstream of proinsulin domain. To the best of our knowledge, this is the first report to experimentally demonstrate that a mutation in the proinsulin domain causes PPI translocation inefficiency, which may underlie increased risk of diabetes-associated R46X. This study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.

DATA AVAILABILITY STATEMENT
The data sets generated during and analyzed during the current study are available from the corresponding author upon reasonable request. The data presented in this study are deposited in the GenBank repository, accession number PRJNA787369. The information will be accessible with the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA787369.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Tianjin Medical University General Hospital Ethics Committee (IRB2017-047-01 of 4 April 2017). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.