Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 15.
Published in final edited form as: J Mol Biol. 2024 Feb 14;436(6):168492. doi: 10.1016/j.jmb.2024.168492

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability

Sarah C Miller 1, Elena B Tikhonova 1, Sarah M Hernandez 1, Jannette M Dufour 1, Andrey L Karamyshev 1,*
PMCID: PMC11675392  NIHMSID: NIHMS2041206  PMID: 38360088

Abstract

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

Graphical Abstract

graphic file with name nihms-2041206-f0001.jpg

Introduction

The protein hormone insulin is synthesized and secreted by pancreatic β-cells in response to rising blood glucose levels [1]. Insufficient insulin production and/or signaling underlies the development of diabetes [2]. There are more than a dozen heterozygous variants in the insulin (INS) gene which alter the protein sequence and result in monogenic diabetes [3]. INS mutations vary in their severity, resulting in either a predisposition to diabetes, the development of mature-onset diabetes of the young, or the presence of diabetes in utero and at birth [3]. While INS gene mutations are rare, their study has provided important insights into insulin biosynthesis and the pathogenesis of diabetes.

As for many other secreted proteins, insulin is synthesized as a precursor (preproinsulin) containing a signal peptide (SP) and processed to proinsulin after cleavage of the signal peptide (Figure 1). N-terminal signal peptides (SPs) serve as tags that are recognized by protein targeting factors. There are two major routes for protein secretion in mammals, the signal recognition particle (SRP)-dependent and SRP-independent protein targeting pathways. It is still disputed if preproinsulin uses SRP-dependent or independent pathway [2, 4]. SRP is a cytosolic ribonucleoprotein containing six protein subunits arranged on non-coding 7SL RNA [5]. Defects in SRP are linked to human diseases [6]. SRP cotranslationally recognizes SPs of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor, which is associated with the SEC61 translocon in endoplasmic reticulum (ER) [5, 7]. In case of SRP-independent targeting, SPs are recognized by other factors. SPs are cleaved by a signal peptidase on the luminal side of the ER membrane, and the proteins are released to the ER lumen for further transport, processing, modification and folding. SPs of different human proteins vary in their length with a median length of 22 amino acid residues [8]. There is no consensus sequence for SPs; however, they have three distinct physicochemical regions: a positively charged n-terminus (n-region), a hydrophobic core (h-region), and a polar c-region (Figure 1A) [9, 10]. It is proposed that the n-region facilitates the correct orientation of a nascent peptide into the ER membrane through the SEC61 translocon [11, 12]. In bacteria, positively charged residues in the n-region promote favorable electrostatic interactions with the negatively charged phosphate group of a lipid molecule [13]. The h-region represents a central stretch of hydrophobic residues that interact with the methionine-lined pocket of the SRP54 subunit and are critically important for SRP recognition [7, 14]. The c-region contains the cleavage site for signal peptidase, and it is critical for processing [15, 16]. In addition to SP cleavage (preproinsulin to proinsulin), insulin synthesis involves two more cleavage steps by prohormone convertases inside secretory granules that result in the excision of the connecting peptide (proinsulin to insulin) [17] (Figure 1B).

Figure 1. Diabetes-causing mutations in the preproinsulin signal peptide.

Figure 1

Open in a new tab

(A) The three physicochemical regions of a typical signal peptide. The n-region contains one or two positive residues near the n-terminus that help determine the orientation of a signal peptide in the SEC61 translocon; the h-region represents a sequence of hydrophobic residues that bind to the signal recognition particle (SRP); and the c-region contains the signal peptide cleavage site recognized by signal peptidase at the ER membrane.

(B) Processing of precursor insulin. Insulin is synthesized as precursor protein containing an n-terminal signal peptide (preproinsulin). Once targeted to the endoplasmic reticulum (ER), the signal peptide is cleaved from the mature protein (proinsulin). Targeting to the ER is essential for proper proinsulin folding and the formation of its three disulfide bonds. Prohormone convertases in secretory granules excise the C-peptide to form insulin.

(C) Autosomal dominant heterozygous mutations are found in each region of the preproinsulin signal peptide, and all of them result in monogenic diabetes as either Mature Onset Diabetes of the Young (MODY) or Neonatal Diabetes (ND) due to insulin deficiency.

Earlier, we discovered a protein quality control termed Regulation of Aberrant Protein Production (RAPP) that modulates secretory proteins [18–23]. It senses aberrant secretory proteins during translation on the ribosome and specifically degrades their mRNAs. Aberrant secretory proteins sensed by RAPP contain signal peptide mutations that result in the loss of SRP54 interactions with the signal peptide [18, 22]. The loss of other components in the SRP-dependent pathway, including SRP receptor subunit α or β or the SEC61α translocase, did not affect secretory protein mRNA, indicating that RAPP specifically senses SRP54 interactions [18]. We hypothesized that disease-causing mutations in the preproinsulin signal peptide pathologically activate the RAPP pathway leading to insulin insufficiency and disease. Five clinical preproinsulin SP mutations (R6C, R6H, P9R, L13R, and A24D) are distributed throughout the preproinsulin SP in the n-, h-, and c-regions (Figure 1C). Notably, all of them result in monogenic diabetes [3, 24]. The n-region mutations (R6C, R6H) lead to diabetes onset in adolescence or adulthood that is typically mild (maturity-onset diabetes of the young (MODY)), while mutations in the hydrophobic region (P9R, L13R) and in the c-region (A24D) result in the severe onset of diabetes that presents at birth and can be mistaken for the autoimmune disorder, type I diabetes [3]. Thus, the presence of the disease-causing mutations in all three SP-specific regions provides a rare opportunity to study their role in triggering the RAPP pathway as a molecular mechanism of diabetes. Here, we demonstrate for the first time that insulin is a RAPP substrate and that mutations which decrease the SP hydrophobicity inhibit the interaction between nascent preproinsulin and SRP, activating RAPP and leading to decreased insulin mRNA and protein.

Results

SRP interacts with signal peptides of short preproinsulin nascent chains at ribosomes

SRP interacts only transiently with nascent peptide chains, making it difficult to capture SRP interactions with standard immunoprecipitation methods. To detect SRP interactions with preproinsulin peptides, we used a unique site-specific photocrosslinking technique [18]. During in vitro translation, a modified lysine with the photoreactive probe, Nε-(5-azido-2-nitrobenzoyl), attached to the tRNAamb (εANB-Lys-tRNAamb) is incorporated into the preproinsulin signal peptide by tRNA-mediated amber suppression via recognition of the amber stop codon, UAG, in preproinsulin mRNA (Figure 2A, B). The positioning of the photocrosslinking probe in the preproinsulin SP allows for the capture of interacting molecules upon exposure to UV light. Crosslinked complexes are then isolated, separated by SDS-PAGE, and photo adducts are visualized with a phosphoimager.

Figure 2. SRP54 directly interacts with signal peptide of preproinsulin nascent polypeptide.

Figure 2

Open in a new tab

(A) Schematic for site-specific photocrosslinking (photo XL). In a cell-free translation system, a chemical probe is incorporated into the preproinsulin signal peptide via tRNA-mediated amber suppression. With UV light, the probe forms covalent bonds with nearby proteins that can be visualized by SDS-PAGE and phosphor plate radiography with the addition of [35S] methionine to translation reactions.

(B) The amber (UAG) stop codon was placed in positions of insulin mRNA corresponding to the residues A15 or P19 in the preproinsulin signal peptide. Amino acid sequence of preproinsulin signal peptide is shown, positions of A15 or P19 are marked.

(C) Representative image of SRP54 photocrosslinking to preproinsulin signal peptide from 2 replicate experiments. Preproinsulin nascent polypeptides of 66 amino acids (aa) and 86 aa containing photocrosslinking probes were synthesized in vitro. The samples were UV irradiated, analyzed on a 4–15% gradient SDS-PAGE and visualized by a phosphoimager. The positions of SRP54-preproinsulin photo adducts and preproinsulin translated products on the gel are marked. The addition of purified SRP increases the specific photocrsslinking products demonstrating that these photo adducts are indeed SRP54-preproinsulin complexes.

(D) Quantification of SRP54-preproinsulin photo adducts from two replicate experiments. A numerical value based on pixel density was determined with ImageJ for the SRP54 photo adduct and translated product in each lane. The graph shows the intensity value of the adduct relative to the same reaction with no additional SRP (+SRP/−SRP). A value greater than 1 indicates more SRP54 interactions with additional SRP than without additional SRP.

To find the optimum length of the polypeptide nascent chain for interaction with SRP, we completed photocrosslinking experiment with preproinsulin translational intermediates containing the first 66, 86, or all 110 amino acids (aa). For all mRNA constructs, we observed photo adducts at the expected size for the SRP54-preproinsulin nascent chain complex, but SRP interactions were the most prominent for the 66 aa nascent chain (Figure 2C, D; Supplemental Figure 1A). Considering that the ribosomal exit tunnel is ~100 angstroms, which can fit ~30–65 amino acids, these data demonstrate that SRP54 interacts immediately when the preproinsulin SP (24 amino acid residues) is exposed from the ribosome [25]. We also tested two different positions of the photo probe in the SP corresponding to original codons for Ala15 or Pro19 (Figure 2B; Supplemental Figures 1B, C) and found that the amber codon in position 15 is the most optimal for detection of the SRP54-preproinsulin interaction (Supplemental Figure 1B, C). The addition of purified SRP validates that the indicated photo adducts are indeed SRP54-preproinsulin (Figure 2C). Using the artificial L10R mutation, which has a large effect on the hydrophobicity of the preproinsulin SP (Supplemental Figure 2D), we observed lost SRP54 interactions across all nascent chains and for both probe positions (Supplemental Figure 1B, C).

Thus, our data demonstrate that SRP54 interacts with preproinsulin SP. This interaction is the strongest when a very short nascent chain is synthesized, and the SP is just exposed from the ribosomal tunnel. The hydrophobicity of the SP plays a crucial role in this interaction. These experiments laid the foundation to study disease-causing mutations in the preproinsulin SP.

Disease-causing mutations in the preproinsulin signal peptide affect interaction with SRP54

To evaluate the interaction efficiencies of preproinsulin SP mutants with SRP, we used 66 aa preproinsulin mRNAs with the crosslinking probe at position 15 (Figure 2B, Figure 3A). L13R and L10R mutations show the strongest effects - their photocrosslinking intensities respectively decrease by 65% and 73% relative to wildtype (Figure 3B). R6H and P9R also affect crosslinking, but show smaller decreases by 37% and 22%, respectively, while A24D does not have a significant impact. The photocrosslinking efficiency of R6C mutant is notably higher, suggesting stronger interaction with SRP54. Overall, the photocrosslinking data parallel the differences in preproinsulin SP hydrophobicity across mutants, such that decreased hydrophobicity in the h-region inhibits SRP interactions, while increased h-region hydrophobicity promotes SRP interactions (Supplemental Figure 2). The P9R mutant is the exception; however, in a predicted structure of the preproinsulin SP by Google’s Alpha Fold (AF-P01308-F1), Leu10 and Leu13 face the same side of the alpha helix, while Pro9 faces the opposite side, suggesting that SRP interacts with one side of the alpha helix, making critical contacts with Leu10 and Leu13 but not Pro9. Additionally, a Leu to Arg mutation has a greater overall effect on signal peptide hydrophobicity than a Pro to Arg mutation [8]. In summary, disease-causing mutations in preproinsulin SP affect interaction with SRP complex through its SRP54 subunit.

Figure 3. Preproinsulin signal peptide mutations affect SRP interactions.

Figure 3

Open in a new tab

(A) Representative image of the effect of signal peptide mutations on SRP54 interactions with 66-residue preproinsulin nascent chains. Additional SRP was added to all reactions. The samples were analyzed as in Figure 2C.

(B) Quantification of mutant preproinsulin-SRP54 photo adducts relative to wildtype preproinsulin-SRP photo adduct (mutant/wildtype) from three to five independent reactions. N = 3 (R6C, R6H, L13R, and A24D), n=4 (L10R), and n=5 (P9R). The graph shows the mean ± the standard deviation (error bars). One sample t- and Wilcoxon tests were used to show the difference of each normalized mutant interaction from a theoretical mean of 1. *p<0.05, **p<0.01, ***p<0.001, and ns = not significant.

SRP54 depletion decreases expression of wildtype insulin mRNA and protein

RAPP is a protein quality control that regulates secretory proteins. To test the hypothesis that the loss of SRP54, the subunit of SRP which binds to SP, triggers the RAPP pathway and leads to decrease in insulin mRNA and protein expression, as was shown for other RAPP substrates [18, 22, 26, 27], we used RNAi technology to knock it down in cultured HeLa cells transiently expressing wildtype preproinsulin. We achieved an efficient knockdown of SRP54 in cultured human cells (Figure 4A, B). Upon SRP54 depletion, insulin mRNA expression decreased by 55%, and total proinsulin protein expression (intracellular + secreted) decreased by 90% (Figures 4C, D). Our data clearly demonstrate that preproinsulin is a substrate for RAPP, and that RAPP tightly controls insulin biogenesis by monitoring preproinsulin SP interactions on the ribosome during translation.

Figure 4. SRP54 depletion reduces wildtype insulin mRNA and proinsulin protein levels.

Figure 4

Open in a new tab

(A) Confirmation of SRP54 knockdown by qPCR from HeLa cells transfected with wildtype preproinsulin from five independent experiments. Graph shows the mean ± standard deviation (error bars). Statistical significance was determined using a t test. ****p<0.0001.

(B) Representative SRP54 western blot from siSRP54 experiments. Actin was used as a loading control.

(C) Insulin mRNA levels during SRP54 depletion relative to control from five independent experiments. Graph shows the mean ± standard deviation (error bars). Statistical significance was determined using a t test. ****p<0.0001.

(D) Proinsulin protein levels from a proinsulin ELISA assay in cell lysate (intracellular) and media (secreted) from three independent experiments. Protein levels during SRP54 depletion are shown relative to control (+siSRP54/−siSRP54). Graph shows mean ± standard deviation (error bars). Statistical significance was determined using a t test. **p<0.01.

Mutations in the signal peptide h-region decrease insulin mRNA and protein levels, suggesting pathological activation of RAPP as a molecular mechanism of diabetes

To show the effect of preproinsulin SP mutants on insulin expression in cells, we transfected plasmids bearing preproinsulin wildtype (WT) or mutant preproinsulin into HeLa cells and compared WT and mutant insulin mRNA and proinsulin protein levels by qPCR and ELISA, respectively. At the mRNA level, only the clinical L13R mutant and the artificial L10R mutant significantly decreased relative to WT (31% and 51%) (Figure 5A). During in vitro translation, the L10R and L13R mutants were also the only mutants whose interaction with SRP54 decreased by more than 50% relative to WT (Figure 3B). This substantial loss of interaction with SRP together with low insulin mRNA level corroborate activation of the RAPP pathway. Although the SRP interactions with the clinical mutants R6H and P9R are also somewhat affected in vitro, the impact of these mutations on the mRNA level in cells was not detected (Figure 5A).

Figure 5. Effects of signal peptide mutations on insulin mRNA and proinsulin protein levels.

Figure 5

Open in a new tab

(A) qPCR of RNA extracted from HeLa cell lysates transiently transfected with preproinsulin. HeLa cells were transfected with plasmids bearing preproinsulin WT, R6C, R6H, P9R, L10R, L13R, or A24D signal peptide mutations. Graph shows mRNA levels of mutants relative to WT from three to six independent experiments. (mutant/WT; n=6 for WT, P9R, and L10R; n=5 for R6C, and L13R; n=3 for R6H; n=4 for A24D). Graph shows the mean ± the standard deviation (error bars). Statistical significance was determined using a one-way ANOVA test with multiple comparisons (Dunnett’s test). *p<0.05, ***p<0.001, and ns = not significant.

(B) Total proinsulin level (cells + media) from transiently transfected HeLa cells from three independent experiments. Proinsulin was measured by an ELISA assay and mutated proinsulin levels are shown relative to WT (mutant/WT; n=3). All graphs show the mean ± the standard deviation (error bars). Statistical significance was determined using a one-way ANOVA test with multiple comparisons (Dunnett’s test). ****p<0.0001, and ns = not significant.

The effect of mutant SPs on the proinsulin protein was more robust - all mutants show decreased proinsulin levels (Figure 5B). Mutations which result in severe neonatal diabetes (P9R, L13R, A24D) and the artificial L10R mutation, cause the greatest reduction in total proinsulin protein levels with depletion at 75%, 86%, 93%, and 99%, respectively; while R6C and R6H, which are associated with a relatively mild form of the disease, decreased by 47% and 25% relative to WT. Our results suggest that the molecular mechanisms of the mutation pathology are diverse and depends on the position in the SP and their severity, or greater change in the physicochemical properties of the altered amino acid residue.

Insulin mRNA is less susceptible to the effect of decreased SP hydrophobicity than prolactin mRNA

Earlier we demonstrated that decrease of hydrophobicity of preprolactin SP due to deletions of leucines dramatically reduced the protein mRNA level [18]. However, we have found that mutations in the proinsulin SP have a smaller effect on its mRNA (Figure 5A). To find out the maximized effect of an aberrant SP on the insulin mRNA expression we constructed preproinsulin chimeras containing WT or mutated (deletion of four leucines, Δ4L) preprolactin SPs and measured their mRNA levels in HeLa cells in comparison with WT and Δ4L preprolactin (Figure 6A). Remarkably, effect of the Δ4L mutation in the preprolactin SP-insulin chimera was notably less than in preprolactin (compare relative mRNA levels in Figure 6B and C). These data are consistent with effect of SRP54 depletion on insulin mRNA (Figure 4C). Our results demonstrate that the maximum effect of the lost interaction of preproinsulin SP and SRP on insulin mRNA level is a half decrease relative to control.

Figure 6. The effect of decreased signal peptide hydrophobicity on the mRNA level of a Prl-Ins chimera.

Figure 6

Open in a new tab

(A) The preprolactin (PRL) signal peptide (30 residues) replaced the preproinsulin (INS) signal peptide (24 residues) to create a wildtype (WT) and mutant (△4L) PRL-INS chimera.

(B) The mRNA level of mutant PRL in which four leucines were deleted from the h-region (△4L). Data are shown relative to PRL WT from four independent experiments. In all experiments, cells were collected 44–46 h after plasmid transfection and mRNA level was measured by qPCR. Graph shows the mean ± standard deviation (error bars). Statistical significance was determined using a t test. ***p<0.001.

(C) The mRNA level of mutant PRL-INS, shown relative to WT PRL-INS for four independent experiments. Graph shows the mean ± the standard deviation (error bars). Statistical significance was determined using a t test. *p<0.05.

Discussion

Insulin, as many secreted proteins, possesses a signal peptide that is required for its targeting to the ER membrane. Signal peptides are recognized by SRP or some other targeting factor in SRP-dependent or SRP-independent pathways, respectively. The question of what pathway is used for insulin secretion is still disputable [2, 4]. Moreover, if preproinsulin is subject to the RAPP protein quality control was also unknown. RAPP, a recently discovered pathway, controls many secretory proteins by monitoring their interaction with SRP during translation, and it degrades their mRNAs if this interaction is disrupted [18, 21]. By site-specific photocrosslinking, we show for the first time that the SRP54 subunit of SRP directly interacts with the preproinsulin SP. Experiments with SRP54 depletion also show that RAPP monitors this interaction - both insulin mRNA and proinsulin protein were notably decreased upon SRP54 knockdown. Although these results do not completely exclude other targeting pathways in insulin biogenesis that may also be involved, our data demonstrate that SRP recognizes preproinsulin SP, and RAPP is involved in control of this process.

Intensive studies of patients with different forms of diabetes or related diseases led to identification of multiple mutations in preproinsulin. Although these mutations are rare, understanding their effect on the insulin biogenesis will help to elucidate the mechanism of diabetes and find novel conceptual treatments of the disease. Five disease-causing mutations are analyzed in this study. They were selected on their presence in preproinsulin signal peptide. For the first time, using site-specific photocrosslinking we studied how these mutations affect interaction with SRP54. The biggest inhibitory effect was observed for mutations in h-domain (L13R, L10R) and one of the mutations in n-domain (R6H). Disease-causing L13R and artificial L10R were the only mutations significantly decreasing the insulin mRNA level. These results corroborate photocrosslinking data demonstrating that these residues are important for SRP54 interaction, and that the molecular mechanism of the disease is caused by pathological activation of the RAPP pathway. Interestingly, P9R mutation in the h-domain has only mild inhibitory effect on interaction with SRP and does not significantly affect insulin mRNA level, but notably decreased the total proinsulin protein levels. These results suggest that effect of P9R on the proinsulin protein is not caused by interaction with SRP but is likely associated with protein translocation at ER. It has been proposed that this mutation affects preproinsulin’s interaction with TRAP (the translocon-associated protein complex), which is required for preproinsulin translocation through the ER membrane [25]. The P9R and L13R SP mutations were discovered in infants in 2013 and 2019 who presented with extremely low birth weight and hyperglycemia within the first 7 weeks of age [28, 29]. So far, no studies have uncovered potential molecular mechanisms behind β-cell failure in these cases of permanent neonatal diabetes mellitus (PNDM). Analysis of the mutated protein expressions suggest that while decrease of L13R and L10R mutants is associated with the RAPP activation (consistent with photocrosslinking data), the mutations in the SP n-domain (R6C, R6H) are not. These mutations have been shown to affect the signal peptide interaction with translocation machinery at ER (SEC61 complex) [11, 12, 30]. Guo et al. found that a large proportion of R6C molecules and a smaller proportion of R6H molecules are not efficiently translocated into the ER or cleaved and are degraded by the proteosome [11, 12]. Patients with autosomal dominant R6C or R6H SP mutations develop non-obese diabetes between 15–65 years of age [3, 30, 31].

We also found that the effects of A24D in the SP c-region on interaction with SRP and on mRNA level were not significant. These data support a hypothesis that c-region of the preproinsulin SP is not important for SRP binding. Mutations in this region may inhibit SP cleavage and lead to retention of the protein inside the cell. Aspartate in the −1 position is extremely rare in human signal peptides [8]. Several studies have shown that while A24D is translocated into the ER lumen, its SP is not cleaved, and as a result it remains stuck in the ER membrane and is not secreted [11, 32, 33]. Thus, the mechanism of this mutant pathology is associated with disruption of the protein processing, but not with inhibition of SRP recognition and RAPP activation.

Constructing a hybrid preproinsulin with WT or mutated preprolactin SPs allowed us to determine a maximum depletion level of the corresponding mRNAs. While the mRNA of the mutated preprolactin with deletions of four leucines in the SP is decreased, the same mutated SP in the preprolactin (PRL)-insulin (INS) hybrid had notably smaller effect (Figure 6). These data are consistent with mRNA level decrease by disease-causing mutations (Figure 5A). However, the proinsulin protein level was dramatically affected (Figure 5B) similarly to the mutated preprolactin level observed earlier [18]. These data suggest that induction of RAPP may be sufficient to cause translational repression of insulin on the ribosome preventing its synthesis while degrading only portion of insulin mRNA. It is possible that the mature part of the protein affects signal peptide interaction with SRP, probably inducing engagement of other factors that may consequently influence mRNA stability. For instance, the loss of interaction with SRP may shift to SRP-independent targeting involving SEC62 [4]. While our photocrosslinking experiments clearly demonstrate that SRP interacts with preproinsulin signal peptide, a limitation of this in vitro approach is that ribosome stalling due to transcripts without a stop codon may provide more time for SRP recognition, and thus facilitate interactions that may not completely reflect targeting in cells. Preproinsulin is notably small (110 residues). By the time the signal peptide emerges from the ribosome exit tunnel, ~60 residues of preproinsulin have already been synthesized. SRP has a very limited time to be engaged with preproinsulin SP before completion of protein synthesis. Therefore, SRP may target insulin less efficiently than longer polypeptides. It was proposed that SRP can efficiently target proteins larger than 160 amino acid residues, while proteins smaller than 100 amino acids are mostly SRP-independent [4]. Preproinsulin is between these two categories of proteins and may switch between SRP-dependent and - independent pathways and engage other factors in addition to SRP for its targeting. This could explain why mutant preprolactin (225 residues) activates RAPP more efficiently than the mutant preprolactin SP-insulin hybrid (112 residues). It is also possible that insulin mRNA by itself is more resistant to degradation.

Although some details of the fine mechanisms of insulin biogenesis and effects of mutations need to be addressed in the future, our study demonstrates that SRP interacts with the preproinsulin SP immediately once it emerges from the exit tunnel; RAPP controls the quality of preproinsulin nascent chains; and molecular mechanisms of disease-causing mutations in the SP are diverse and include pathological activation of RAPP, and defects in processing and protein translocation.

Materials and Methods

In Vitro Translation and Site-Specific photocrosslinking

In vitro translation and site-specific photocrosslinking experiments were performed as previously described [18, 22]. To make constructs for in vitro transcription, polymerase chain reactions (PCRs) were performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific). PCR-specific primers are listed in Supplemental Table 1. Two methionine codons were added at the c-terminus of each construct. PCR reactions were purified using the NucleoSpin Gel and PCR Clean-Up Kit (Macherey-Nagel). PCR products were transcribed in vitro into mRNAs using SP6 RNA polymerase (New England Biolabs). mRNAs were purified by the NucleoSpin RNA Isolation Kit (Macherey-Nagel) and used for in vitro translation. These mRNAs did not have natural stop codons to prevent disassociation of the ribosome subunits. In vitro translation reactions (15 μl) contained rabbit reticulocyte lysate (Green Hectares, LLC), 2.6 mM magnesium acetate, 70mM potassium acetate, 0.8 U/μl RNasin (Promega), 0.8 μCi/μl [35S] methionine, 1μg of mRNA, and 15 pmol of εANB-Lys-tRNAamb (tRNA Probes, LLC). Rabbit reticulocyte lysate was pretreated with ribonucleases to remove endogenous mRNAs. Purified canine SRP (40 nM; tRNA Probes, LLC) was added where indicated. Translation reactions (40 min at 26°C) were performed in the dark and then exposed to UV light for 15 min on ice (Newport Oriel, 500-W mercury arc lamp). Ribosome-nascent chain complexes were then pelleted by centrifugation (532,140g), treated with RNase A and analyzed on a 4–15% gradient SDS-PAGE. A [Methyl-14C] methylated protein molecular weight marker (PerkinElmer) was used to estimate molecular weight. Photo adducts were visualized with a Typhoon FLA 9000 Biomolecular Imager.

DNA Techniques

The plasmids used in the study are presented in Supplemental Table 2. The human INS open reading frame (NM_000207.3) was cloned into the pCS2 vector using the restriction sites FseI and AscI. T4 DNA ligase (New England Biolabs) was used for ligation reactions. Signal peptide mutations were introduced using the QuikChange II Site-Directed Mutagenesis Kit (Agilent). DH5-alpha competent cells were transformed with plasmids containing preproinsulin, grown overnight, and DNA was purified using the PureYield Plasmid Midiprep System (Promega). The wildtype and mutated preprolactin-insulin chimeras were generated using PCR. PCR primers to make the preprolactin-insulin chimeras are listed in Supplemental Table 1. PCR products were cleaned using the NucleoSpin Gel and PCR Clean-up kit (Clontech). Restriction sites for chimeras were introduced by PCR primers. The chimeric constructs were cloned into the pBI-CMV2 vector (Clontech) using the restriction sites, HindIII and EcoRV, which is the same vector used for the expression of preproprolactin. All constructs were confirmed by DNA sequencing from Genewiz.

Cell Culture and Transfection

HeLa Tet-On cells (Clontech) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 units/mL penicillin, and 100 μg/ml streptomycin (Sigma Aldrich) in a 37°C incubator with 5% CO2. Cells were seeded into 6-well plates one day before transfection. Plasmids (0.5 μg) were mixed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol and added to each well. Cells were collected 44–46 h after plasmid transfection for analysis. For SRP54 knockdown experiments, 13.75 nM siSRP54 was added to cells with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The siSRP54 sequence was synthesized by Dharmacon and reads 5’-GAAAUGAACAGGAGUCAAUdTdT-3’ [18]. 24h after siRNA transfection, cells were transfected with 0.5 μg of plasmid DNA. Cells were collected 72h after siRNA transfection.

Quantitative PCR

Total RNA was purified from cell lysates using the NucleoSpin RNA purification kit (Takara), and cDNA samples were prepared using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative polymerase chain reactions (qPCR) were made using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol. Reactions were performed on a 384-well plate and cycled using the Quant Studio 12K Flex Real-Time PCR System. qPCR primers are listed in Supplemental Table 1. Actin mRNA was used for normalization. The comparative (ΔΔCT) method was used to analyze the qPCR data [34].

Western Blotting

Cells were directly lysed on 6-well plates using sample buffer with 1% β-mercaptoethanol and protease inhibitor cocktail (Roche). Total protein lysates were boiled for 5 min at 95°C, separated by 12% SDS-PAGE, and electro-transferred to a PVDF membrane (0.45 μm pore size, BioRad) for probing with primary antibody (diluted in Tris-buffered saline with 1% Tween with 5% milk) overnight at 4°C. Mouse anti-human SRP54 antibody (1:4000 dilution) was from BD Bioscience (#610940). Mouse anti-human β-actin (1:30000 dilution) was from ProteinTech (#66009–1). PVDF membranes were incubated in horseradish peroxidase-conjugated secondary antibody (goat anti-mouse, 1:30000 dilution, Jackson Labs #115-035-003) at room temperature for 1h. Protein signals were detected using peroxidase reactions with Pierce ECL or SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific).

Enzyme-Linked Immunosorbent Assay (ELISA)

Cells were directly lysed and scraped on 6-well plates using RIPA buffer (20mM Tris pH 7.6, 150mM NaCl, 0.1% SDS, 1% NP-40) and frozen overnight at −80°C. Cell lysates were then sonicated and centrifuged to pellet cell debris. Cell culture media was collected at the same time and centrifuged at 3000 rpm for 5 min. Media samples were incubated with 10% (v/v) TCA (trichloroacetic acid) overnight to precipitate proteins and washed twice using cold acetone and high-speed centrifugation. Precipitates were dried and resuspended in RIPA buffer. Protein concentrations of cell lysates and media were determined using the Pierce BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific). Total protein (0.6 to 2.4 ug) from cell lysate or media was used to determine proinsulin concentration using Human Total Proinsulin ELISA (Millipore, # EZHPI-15K). Proinsulin was not detected in negative control (no transfection of plasmids with preproinsulin), either intracellularly or in media. Absorbance values of proinsulin standards were used to generate a standard curve.

Statistical Analyses

A Student t test was used to compare the differences between control and knockdown from SRP54 depletion experiments. A one-way ANOVA test with multiple comparisons (Dunnett’s test) was used to compare the differences between each mutant and wildtype on mRNA and protein levels. Quantifications of photocrosslinking scans were done using ImageJ software. The intensities of the band for each mutant preproinsulin-SRP interaction were normalized to the intensity of the band for the wildtype preproinsulin-SRP interaction for each experiment. One sample t- and Wilcoxon tests were used to show the difference of each normalized mutant interaction from a theoretical mean of 1. All graphs (GraphPad Prism 9 software) show the mean ± S.D. P < 0.05 was considered statistically significant.

Supplementary Material

1

HIGHLIGHTS.

  • Molecular mechanism of many disease-causing mutations in insulin is unknown

  • SRP interaction with insulin signal peptide is required for its mRNA stability

  • Mutations decreasing signal peptide hydrophobicity affect insulin mRNA and protein expression

  • RAPP is implicated in the development of diabetes caused by the Leu13Arg mutation

Acknowledgements

The authors thank Gurvinder Kaur for providing plasmid containing cloned insulin. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM135167, by start-up fund from the Texas Tech University Health Sciences Center (TTUHSC), and by the South Plains Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the TTUHSC, or the South Plains Foundation.

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • [1].Cottet-Dumoulin D, Lavallard V, Lebreton F, Wassmer CH, Bellofatto K, Parnaud G, et al. Biosynthetic Activity Differs Between Islet Cell Types and in Beta Cells Is Modulated by Glucose and Not by Secretion. Endocrinology. 2020;162:bqaa239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Liu M, Huang Y, Xu X, Li X, Alam M, Arunagiri A, et al. Normal and defective pathways in biogenesis and maintenance of the insulin storage pool. J Clin Invest. 2021;131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Støy J, Franco ED, Ye H, Park S-Y, Bell GI, Hattersley AT. In celebration of a century with insulin – Update of insulin gene mutations in diabetes. Mol Metab. 2021:101280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Lakkaraju AKK, Thankappan R, Mary C, Garrison JL, Taunton J, Strub K. Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation. Mol Biol Cell. 2012;23:2712–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Kellogg MK, Miller SC, Tikhonova EB, Karamyshev AL. SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection. Int J Mol Sci. 2021;22:6284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Kellogg MK, Tikhonova EB, Karamyshev AL. Signal Recognition Particle in Human Diseases. Front Genet. 2022;13:898083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Nilsson I, Lara P, Hessa T, Johnson AE, Heijne Gv, Karamyshev AL. The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence. J Mol Biol. 2015;427:1191–201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Gutierrez Guarnizo SA, Kellogg MK, Miller SC, Tikhonova EB, Karamysheva ZN, Karamyshev AL. Pathogenic signal peptide variants in the human genome. Nar Genom Bioinform. 2023;5:lqad093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Gv Heijne. Signal sequences The limits of variation. J Mol Biol. 1985;184:99–105. [DOI] [PubMed] [Google Scholar]
  • [10].von Heijne G The signal peptide. J Membr Biol. 1990;115:195–201. [DOI] [PubMed] [Google Scholar]
  • [11].Guo H, Xiong Y, Witkowski P, Cui J, Wang L-j, Sun J, et al. Inefficient Translocation of Preproinsulin Contributes to Pancreatic β Cell Failure and Late-onset Diabetes*. J Biol Chem. 2014;289:16290–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Guo H, Sun J, Li X, Xiong Y, Wang H, Shu H, et al. Positive charge in the n-region of the signal peptide contributes to efficient post-translational translocation of small secretory preproteins. J Biol Chem. 2018;293:1899–907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Nesmeyanova MA, Karamyshev AL, Karamysheva ZN, Kalinin AE, Ksenzenko VN, Kajava AV. Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids. Febs Lett. 1997;403:203–7. [DOI] [PubMed] [Google Scholar]
  • [14].Tirincsi A, Sicking M, Hadzibeganovic D, Haßdenteufel S, Lang S. The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum. Int J Mol Sci. 2021;23:143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Liaci AM, Steigenberger B, Telles De Souza PC, Tamara S, Gröllers-Mulderij M, Ogrissek P, et al. Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage. Mol Cell. 2021;81:3934–48.e11. [DOI] [PubMed] [Google Scholar]
  • [16].Karamyshev AL, Karamysheva ZN, Kajava AV, Ksenzenko VN, Nesmeyanova MA. Processing of Escherichia coli alkaline phosphatase: role of the primary structure of the signal peptide cleavage region. J Mol Biol. 1998;277:859–70. [DOI] [PubMed] [Google Scholar]
  • [17].Liu M, Weiss MA, Arunagiri A, Yong J, Rege N, Sun J, et al. Biosynthesis, structure, and folding of the insulin precursor protein. Diabetes Obes Metabolism. 2018;20:28–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Karamyshev AL, Patrick AE, Karamysheva ZN, Griesemer DS, Hudson H, Tjon-Kon-Sang S, et al. Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway. Cell. 2014;156:146–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Karamyshev AL, Karamysheva ZN. Lost in Translation: Ribosome-Associated mRNA and Protein Quality Controls. Front Genet. 2018;9:431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Karamyshev AL, Tikhonova EB, Karamysheva ZN. Translational Control of Secretory Proteins in Health and Disease. Int J Mol Sci. 2020;21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Karamysheva ZN, Karamyshev AL. Aberrant protein targeting activates quality control on the ribosome. Front Cell Dev Biol. 2023;11:1198184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Tikhonova EB, Karamysheva ZN, Heijne Gv, Karamyshev AL. Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations. J Mol Biol. 2019;431:2567–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Karamysheva ZN, Tikhonova EB, Karamyshev AL. Granulin in Frontotemporal Lobar Degeneration: Molecular Mechanisms of the Disease. Front Neurosci. 2019;13:395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Liu M, Sun J, Cui J, Chen W, Guo H, Barbetti F, Arvan P. INS-gene mutations: From genetics and beta cell biology to clinical disease. Mol Aspects Med. 2015;42:3–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Kriegler T, Kiburg G, Hessa T. Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin. J Mol Biol. 2020;432:166694. [DOI] [PubMed] [Google Scholar]
  • [26].Pinarbasi ES, Karamyshev AL, Tikhonova EB, Wu IH, Hudson H, Thomas PJ. Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability. Cell Reports. 2018;23:2844–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Tikhonova EB, Gutierrez Guarnizo SA, Kellogg MK, Karamyshev A, Dozmorov IM, Karamysheva ZN, Karamyshev AL. Defective Human SRP Induces Protein Quality Control and Triggers Stress Response. J Mol Biol. 2022;434:167832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Hussain S, Ali JM, Jalaludin MY, Harun F. Permanent neonatal diabetes due to a novel insulin signal peptide mutation. Pediatr Diabetes. 2013;14:299–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Laurenzano SE, McFall C, Nguyen L, Savla D, Coufal NG, Wright MS, et al. Neonatal diabetes mellitus due to a novel variant in the INS gene. Mol Case Stud. 2019;5:a004085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Meur G, Simon A, Harun N, Virally M, Dechaume A, Bonnefond A, et al. Insulin Gene Mutations Resulting in Early-Onset Diabetes: Marked Differences in Clinical Presentation, Metabolic Status, and Pathogenic Effect Through Endoplasmic Reticulum Retention. Diabetes. 2010;59:653–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Edghill EL, Flanagan SE, Patch AM, Boustred C, Parrish A, Shields B, et al. Insulin Mutation Screening in 1,044 Patients With Diabetes: Mutations in the INS Gene Are a Common Cause of Neonatal Diabetes but a Rare Cause of Diabetes Diagnosed in Childhood or Adulthood. Diabetes. 2007;57:1034–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Park S-Y, Ye H, Steiner DF, Bell GI. Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted. Biochem Bioph Res Co. 2010;391:1449–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Cui J, Chen W, Sun J, Guo H, Madley R, Xiong Y, et al. Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos*. J Biol Chem. 2015;290:28131–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–8. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2041206-supplement-1.pdf (1.4MB, pdf)

RESOURCES