Supplemental Materials [+] pdf here - DOI: 10.19277/bbr.19.2.301_Supplemental
Ablation of Kex2 activity enhances proIAPP proteotoxicity in yeast
Sofia Ferreira 1,2, Ana F. Raimundo 3,4,5, Inês Farrim 1,2, Regina Menezes 1,3,4*
1CBIOS - Center for Biosciences & Health Technologies, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal; 2Universidad de Alcalá, Escuela de Doctorado, Departamento de Ciencias Biomédicas, Madrid, Spain; 3iBET - Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; 4NOVA Medical School | Faculdade de Ciências Médicas, NMS|FCM, Universidade Nova de Lisboa, Lisboa, Portugal; 5ITQB-NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA Lisboa, Oeiras, Portugal
Pancreatic deposition of Islet Amyloid PolyPeptide (IAPP) is a histopathological hallmark of type 2 diabetes. Inadequate processing of immature IAPP by proprotein convertases (PCs) leads to the accumulation of unprocessed forms, which in turn favors increased aggregate formation in pancreatic β-cells. Kexin 2 (Kex2) of Saccharomyces cerevisiae is the prototype of eukaryotic PCs family, but to date no direct correlations between Kex2 activity and preproIAPP processing in yeast have been reported. In this study we aimed to address the possible role of Kex2 on IAPP maturation as a tool to investigate the contribution of impaired processing towards IAPP proteototoxicity. Genetically modified S. cerevisiae models lacking the KEX2 gene and carrying different chimeric fusions of human IAPP linked to GFP were used. The cytotoxic effects of Kex2 ablation were assessed by means of growth curve analysis and cell viability assays using flow cytometry. IAPP protein profile was evaluated by immunoblotting assays using an anti-IAPP antibody. Intracellular IAPP aggregates were monitored by confocal fluorescence microscopy. Data showed that kex2 mutants exhibit growth defects, potentiated by preproIAPP-GFP, proIAPP-GFP and mature IAPP-GFP expression with an increased cytotoxicity for proIAPP-GFP. Notwithstanding, Kex2 absence does not seem to affect IAPP protein pattern nor the frequency/distribution of intracellular IAPP aggregates in yeast. Our findings suggest that Kex2 is not essential for IAPP processing in yeast, at least under the conditions tested.
Keywords: Amylin, Islet Amyloid Polypeptide (IAPP), Kexin 2, Prohormone processing, Saccharomyces cerevisiae
Received: 13/11/2022; Accepted: 31/12/2022
Protein misfolding diseases cover a heterogeneous group of pathologies in which an amyloid-forming peptide acquires an alternative folding state, starts to oligomerize and to accumulate in tissues where the disease-specific damage occurs. This process can occur systemically or at the site of protein production, as it is the case of type 2 diabetes (T2D) (1). The uncontrolled deposition of Islet Amyloid PolyPeptide (IAPP) is a hallmark of T2D, being present in nearly 90% of the individuals at post-mortem (2) and in 50% of insulinomas (3). IAPP, or amylin, is a 37-amino acid peptide which has regulatory functions in metabolism and glucose homeostasis. It acts on muscle cells and fat tissue by stimulating the uptake of glucose and by slowing gastric emptying through signaling in the Central Nervous System (4). Concerning its synthesis, IAPP is expressed and processed concomitantly with insulin in pancreatic β-cells. The initial and immature form of IAPP (preproIAPP, ppIAPP) is comprised of a sequence of 89 amino acid residues with a signal peptide. The signal peptide of ppIAPP is removed throughout its transfer to the endoplasmic reticulum (ER), originating a proIAPP (pIAPP) molecule that matures in the late Golgi complex into IAPP through the cleavage of two flanking peptides by protein convertase (PC)1/3, PC2 and carboxypeptidase E. Amidation of the C-terminal end and the formation of a disulfide bridge also occur, contributing to the yield of an active IAPP hormone (5). Mature IAPP is then stored in tight ratios with insulin in secretory granules, being released upon glucose stimulation (6,7). Yet, when there is a high demand for insulin, β-cells are required to perform with high turnover and may lose the capacity to properly process all the insulin and IAPP. Consequently, immature IAPP forms arise in the islets of individuals with diabetes, which are seen as even more amyloidogenic than its mature form (8). In human IAPP transgenic mice, immunoelectron microscopy revealed the presence of fibrillar aggregates immunoreactive for pIAPP-specific sera in the secretory granules of β-cells (8). In addition, amyloid composed of proIAPP was observed in cells lacking both PCs. Such evidence was not documented in cell models carrying functional PC2 and PC1/3 where efficient processing occurred (9). Corroborating the relevance of aberrant processing in the accumulation of immature IAPP intermediates and disease progress, the ratio of serum pIAPP/IAPP levels was found to be altered in individuals with T2D displaying impaired glucose regulation (10). Accumulation of IAPP intermediate species favors to the formation of oligomers that can act as trigger agents for β-cell depletion (4). These IAPP oligomers are thought to be involved in multiple toxic mechanisms. They have been described to disrupt cellular membrane dynamics (11-12), inhibit cell proliferation, impair autophagy (13), dysregulate calcium homeostasis (14) and induce overproduction of reactive oxygen species (4,15-16). Augmented concentrations of pIAPP in the ER have been reported to activate the unfolded protein response (UPR) and other ER stress pathways which, if not checked, can culminate in cell apoptosis (17-18). Despite that, the role of these incompletely processed forms within the plethora of cytotoxic effects attributed to IAPP is not yet fully understood.
Recently, we described a Saccharomyces cerevisiae model expressing differently processed forms of IAPP fused to Green Fluorescent Protein (GFP) (19). These models recapitulated the major molecular pathways related to IAPP aggregation, thus representing an unprecedented tool to study the molecular mechanisms affected by IAPP proteotoxicity. The same study showed that ppIAPP-GFP is at least partially processed towards its mature form, which implies the action of a protein convertase equivalent to PC1/3 and PC2 in β-cells. In that view, Kexin 2 (Kex2), appears as the most probable candidate to perform such processing. Kex2 has been described as a prohormone-processing protease that catalyzes the cleavage of peptides both in Lys-Arg and Arg-Arg motifs, in a Ca2+-dependent manner (20). Selectively located to the late compartment of the Golgi complex in yeast, it contains a single transmembrane domain and a retention signal on C-terminal cytosolic tail sequence. Consistent with this compartmentalization, Kex2 has emerged as a protease that acts on a strict spectrum of target proteins in the late compartments of secretory pathway (e.g. precursors of prohormones, neuropeptides and integral membrane proteins) (21). Further supporting a possible role of Kex2 in IAPP processing, this convertase was shown to cleave the insulin precursor protein in the late secretory pathway of yeast (22). This is particularly interesting when considering that IAPP and insulin are co-processed by the same protein convertases in pancreatic β-cells (23).
Although some proteins have been predicted as potential targets of Kex2, the number of substrates for which there is experimental evidence of cleavage by Kex2 remains scarce (24). In the scope of IAPP processing events, the substrate specificity of Saccharomyces cerevisiae Kex2 protease has great importance. Kex2 is considered the prototype for the family of eukaryotic subtilisin-like serine proprotein convertases that comprises the human PC1/3, PC2 and furin (25). Specifically, in regard to IAPP, no direct correlations between Kex2 activity and the processing of such amyloidogenic peptide have been reported to date. To fill this gap of knowledge, we used genetically modified S. cerevisiae models expressing different forms of human IAPP to investigate the possible involvement of Kex2 on IAPP enzymatic processing. By sharing conserved cellular and molecular mechanisms with human cells, yeast represents a versatile and powerful experimental model to study fundamental biological processes involved in human diseases (26). Thus, the use of yeast models to dissect Kex2 proteolytic action on IAPP may represent an important tool to elucidate the mechanisms underlying aberrant IAPP processing and consequent accumulation of immature species observed in disease scenarios.
Materials and methods
Strains and plasmids
The strain BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 and the isogenic kex2 mutant strain (EUROSCARF) were used. Both strains were transformed with different chimeric fusions of IAPP linked to GFP. Yeast transformation protocols were performed following the lithium acetate standard technique (27). The constructs used are indicated in Table 1.
Yeast growth conditions
Cells were maintained in synthetic dropout (SD)-glucose medium. Prior to experiments, an inoculum was made in SD-raffinose medium at 30ºC for 24 h and under agitation. Cell cultures were diluted in new medium and incubated as indicated above until reaching an optical density of 0.5 ± 0.05 at 600 nm (OD600). Measurements were acquired using a Biotek Power Wave XS plate spectrophotometer (Winooski, VT, USA). Constructs expression was modulated by incubating the cells in SD medium with glucose (repression) or galactose (induction). Detailed protocols were previously described by our research group (19).
Cultures were diluted in SD-galactose to OD600 0.1 ± 0.01 and maintained for 12 h at 30°C under orbital shaking. Flow cytometry was carried out using propidium iodide (PI) as previously described (19).
Protein extraction and Immunoblotting
Cultures were diluted in SD-galactose medium to OD600 0.1 ± 0.01 and maintained at 30°C for 12 h under orbital shaking. Protein extraction and immunoblotting were performed as previously described (19) using the antibodies indicated in Supplementary Table S1.
For growth assays, yeast cells were diluted in SD-glucose or SD-galactose medium to OD600 0.05 ± 0.005 and maintained at 30°C with agitation during 24 h. Yeast growth was checked hourly by reading OD600 using a Biotek Power Wave XS Microplate Spectrophotometer.
Cell cultures were diluted to OD600 0.1 ± 0.01 in SD-galactose medium and incubated at 30°C for 12 h under orbital shaking. The culture was centrifuged at 3000 g for 3 min and resuspended in PBS. Slides were prepared using a mixture of 4 μl of low melting point agarose and 4 μL of cell suspension. GFP signal was monitored using a Carl Zeiss LSM 710 (Jena, Germany) for confocal microscopy or Leica Z2 (Wetzlar, Germany) for fluorescence microscopy. The number of aggregates-containing cells was counted by visualizing at least 800 cells for each condition. Photos were counted using Fiji-ImageJ1.51j8 software (USA).
GraphPad Prism 6 software was used to carry out the statistical analysis. Two-way ANOVA with the Tukey’s multiple comparison test was used to access differences between conditions in the viability test. Two-way ANOVA with Sidak’s multiple comparison analysis was applied to test for differences between conditions in immunoblotting and microscopy analysis.
Results And Discussion
Kex2 as a possible prohormone convertase involved in ppIAPP processing
IAPP is the main element of amyloid inclusions in pancreatic islets of diabetic patients. Like insulin, IAPP is produced as a prepropeptide in the ER and processed to the mature and active form in secretory granules before its extracellular release. IAPP is formed by exclusion of both C- and N-terminal flanking peptides at basic amino acids pairs, through the action of prohormone convertases (PC2 and PC1/3) that recognize specific and highly conserved sites of cleavage in the peptide sequence (5) (Figure 1A). In S. cerevisiae, Kex2 (or Kexin) appears as the yeast homolog of such processing enzymes. Kex2 cleaves target peptides at paired dibasic sites, being essential to generate the final and active forms of excreted proteins such as killer toxin, α-factor, insulin and, eventually, IAPP (28). Kex2 is a membrane-attached protein that is originally targeted to the ER. Ultimately, it acts at the trans-Golgi network (TGN), circulating between trans-Golgi vesicles and the late endosomal compartments (29,30). Structurally, it contains an ER signal peptide in the N-terminus, followed by a catalytic domain that bears most of the similarities between PC2, PC3 and Kex2 residue sequences. Within the first 600 amino acids, 34-45% of the residues are identical between any pair of these proteins. Beyond the first 600 residues, near the end of PC2, Kex2 contains a hyperglycosylated serine/threonine rich region and a single transmembrane domain that is important for its function and localization as an integral membrane protein (Figure 1B) (30,31).
Absence of Kex2 protease increases cytotoxicity of proIAPP-GFP in yeast
In diabetes, the increased need for insulin production caused by increased blood glucose levels is accompanied by an overproduction of IAPP which completely overloads and impairs the β-cell processing machinery. Abnormal post-translational processing of IAPP precursor peptide has been associated to the buildup of intermediate immature forms of IAPP and consequent deposition of cytotoxic intracellular oligomers (1,4,5).
To address the impact of Kex2 lack on IAPP processing, we generated genetically modified yeast models that do not harbor the KEX2 gene (kex2 strain) and simultaneously express different chimeric constructs of human IAPP (p426-ppIAPP-GFP (ppIAPP-GFP), p426-pIAPP-GFP (pIAPP-GFP) and p426-matIAPP-GFP (matIAPP-GFP)) (19). In these constructs, each cDNA was cloned in fusion with GFP and under the regulation of a promoter inducible by galactose (GAL1). For comparison, the isogenic strain BY4741 was used as a control encoding a functional processing machinery. These models were previously described and characterized, showing that expression and aggregation of immature ppIAPP species causes a marked cellular toxicity (19). The expression of IAPP constructs in the null-mutant strain was confirmed by the presence of GFP positive cells as checked by flow cytometry analysis (Supplementary Figure S1). Cell viability was next evaluated upon 12 h galactose induction of IAPP expression, through PI staining by flow cytometry. PI is membrane-impermeant and excluded from viable cells. It can only cross compromised cellular membranes, being therefore considered an indicator of membrane integrity (33). According to our previous data, at this timepoint only ppIAPP-GFP expression significantly reduced the cell viability in BY4741 cells, with other constructs showing lower levels of toxicity (19). Interestingly, genetic ablation of KEX2 gene significantly increased pIAPP-GFP cytotoxicity, while no effects were observed in strains expressing the other constructs. In mutant ppIAPP-expressing cells, PI levels remained unchanged suggesting that the absence of Kex2 protease per se is not sufficient to further exacerbate the toxicity of the most immature IAPP form (Figure 2). A plausible explanation is that signal peptide-mediated ppIAPP-GFP addressing to ER may exacerbate accumulation of immature forms and clog the secretory pathway creating a problem upstream to the cleavage by protein convertases.
The cellular membrane of yeast is known to be involved in major biological processes, serving as a barrier to solute(s) diffusion, stores energy as transmembrane ions and solute gradients, offers sites of enzyme pathways engaged in the synthesis of cell components, among others. These functions are crucial to maintain the intracellular homeostasis and gather the elements required for cell growth and division (34). Since PI analysis showed alterations in membrane integrity in certain conditions (Figure 2), we decided to address how the expression of different IAPP constructs impacts the growth of Kex2-lacking yeast. Growth curve analyses were performed for wild type and mutant strains (Figure 3A). Cultures of BY4741 expressing the differently processed IAPP species showed an initial lag phase that is well-matched with the change in the carbon source from raffinose to galactose. Subsequently, these strains resumed growth and behave similarly, with ppIAPP-GFP-expressing cells growing slower than the control cells after 12 h of galactose induction. Regarding the comparison between the growth curves of wild type and mutant strains, a marked difference was observed, with kex2 cultures showing lower levels of final biomass. Under equal conditions, control BY4741 cultures reached an optical density of 0.43 ± 0.013 after 24 h induction with galactose, whereas control kex2 culture attained values of 0.35 ± 0.008 at the same time point (Figure 3A). Monitoring of kex2 culture growth also showed a reduced Area Under the Curve (AUC), when compared to BY4741 cells, supporting the occurrence of growth defects in the mutant strain (Figure 3B). Such results are in accordance with systematic studies showing that kex2 mutant grows at slower rates in comparison to the isogenic wild type strain (35). Analysis of the AUC also revealed that the absence of Kex2 potentiates growth impairment mediated by ppIAPP-GFP, pIAPP-GFP and matIAPP-GFP expression as concluded by the increase of ΔAUC calculated as the difference between the AUC of the control and the respective IAPP construct (Figure 3C). These results were corroborated by the spot assays showing that cytotoxicity in all strains was enhanced in the kex2 mutant, in particular in the strains expressing ppIAPP-GFP and pIAPP-GFP (Figure 4).
IAPP processing and intracellular distribution are not affected in kex2 mutants
Transmission electron microscopy studies of intracellular amyloid in human IAPP transgenic mice pancreas identified intragranular fibrils containing pIAPP molecules. Fibrils at this location were associated to prohormone processing impairment and consequent pIAPP accumulation (8). In β-cells containing functional processing machinery, expression of pIAPP did not lead to amyloid deposition. On the contrary, pIAPP expression in GHC4 cells lacking PC2 and PC1/3 resulted in amyloid composed of pIAPP (36), indicating that abnormal processing is a key factor for the accumulation of amyloidogenic pIAPP intermediates. To verify if increased pIAPP cytotoxicity in kex2 mutants was linked to the accumulation of IAPP unprocessed forms, we performed immunoblotting assays. As shown in Figure 5, no visible differences were observed in IAPP profile between the two strains. In both cases, total protein samples from cells expressing the most immature form of IAPP showed the presence of a 38 kDa signal which is well-matched with the molecular weight of ppIAPP-GFP. An extra signal of ~32 kDa was also noticed in these samples (Figure 5). In a similar way, total protein samples from cells expressing pIAPP-GFP revealed the existence of a 36 kDa signal, corresponding to the molecular weight of the pIAPP-GFP. Two other signals of ~32 and ~70 kDa were also identified. In the light of these results, we hypothesize that such signals may be associated to the generation of processing intermediates (~32 kDa signals) and dimers (~70 kDa signal) of IAPP species. matIAPP-GFP total protein samples disclosed the existence of a 31 kDa-single signal that should match the polypeptide sequence of the mature construct. Comparable signals were detected in membranes probed with anti-GFP antibody (Supplementary Figure S2). Also, no significant differences were found in total levels of IAPP between wild type and mutant strains. In both strains, pIAPP-GFP-expressing cells showed higher levels of total IAPP (Figure 5), however with no significant effect on the number of GFP positive cells, as indicated by flow cytometry studies (Supplementary Figure S1). More robust techniques such as size exclusion chromatography should be useful to elucidate this question.
Confocal microscopy was then used to evaluate the impact of the absence of Kex2 on the subcellular distribution of chimeric proteins. At a first glance, it can be observed that kex2 cells are larger than the wild type cells, a phenomenon previously reported in the literature. In addition to S. cerevisiae, a wide-ranging of phenotypes have been reported for kex2 deletion mutants in other yeasts. The stated phenotypic descriptions of the respective mutants comprise morphological alterations that seems to be associated with the deficient activity of cell-wall modifying enzymes (24).
Regarding protein aggregation, as observed in BY4741 cells, ppIAPP-expressing kex2 cells exhibited a heterogeneous distribution of well-defined intracellular inclusions (Figure 6, A and B), whereas a more diffuse GFP signal was observed in kex2 cells expressing pIAPP-GFP or matIAPP-GFP. Remarkably, in these cells, less defined aggregates were detected in a non-fluorescent compartment, likely the vacuole, suggesting the accumulation of intravacuolar inclusions. This could represent a cellular defense mechanism through the compartmentalization of toxic protein assemblies. Further experiments are required to elucidate this issue. Although visual inspection suggests that aggregates in ppIAPP-GFP expressing cells seem to be larger and more frequent in the kex2 mutant (Figure 6,A and B), systematic counting of the number of cells containing proteinaceous IAPP inclusions using ImageJ software indicated no differences between the two strains (Figure 6C).
In view of these data, our results do not support the idea of IAPP as a target for Kex2-mediated processing at least in conditions tested. The phenotypic features of kex2 mutants can be attributed to compromised processing of substrate proteins rendering them dysfunctional and thereby affecting cell growth. An example is the case of the α-pheromone where the deficiency of Kex2-mediated processing disables mutant mating (37). Notwithstanding, the marked increase in cell permeability observed in the kex2 mutant expressing pIAPP-GFP reveals some degree of response specificity that needs further investigation.
In our study, the absence of Kex2 does not seem to result in cleavage impairment and accumulation of unprocessed forms of pIAPP-GFP. The restricted Kex2 localization in the late trans-Golgi network and prevacuolar compartments explain its limited target spectrum to those proteins anchored to the cell surface or involved in the secretory pathway (24). Accordingly, kex2 mutants phenotypes have been characterized by the release of incompletely processed protein precursors into the extracellular environment, as the case of the secretory xylanase of T. reesei (38). However, evidence has demonstrated that these effects are blurred since the phenotypes described in kex2 strains may simply be secondary effects themselves. Moreover, failure of Kex2-mediated processing mechanisms may be masked by the function of other proteinases, as the case of yapsins, a family of glycosylphosphatidylinositol (GPI)-linked aspartyl proteases (24,39). Further studies are mandatory to better comprehend the role of players involved in the processing of immature forms of IAPP in yeast.
The kex2 mutant phenotypes have been characterized by the extracellular release of unprocessed protein precursors. Although few proteins have been indicated as possible substrates for Kex2, there is not experimental proof of Kex2 cleavage for the majority of the proteins. There is no reported evidence regarding the effects of Kex2 on the IAPP peptide. Through the generation of a S. cerevisiae model lacking KEX2 gene and expressing different chimeric constructs, our study provided novel information about the potential enzymatic activity of this protease on IAPP. The lack of Kex2 reduced cell viability of cells expressing pIAPP, without affecting IAPP processing and intracellular distribution. These data call into question the initially established hypothesis that IAPP would be a potential substrate for Kex2 activity. Additional studies are necessary to better comprehend the mechanisms behind increased pIAPP toxicity in the kex2 mutant. Moreover, determining the substrates of Kex2 protease would contribute to clarify the phenotypes described in deletion mutants and afford insights into crucial regulatory mechanisms of the cells.
Authors contributions statement
RM conceived and designed the study experiments. SF, AFR and IF executed the experiments. SF, AFR and RM analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.
This study was funded by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência e do Ensino Superior, grant numbers PTDC/BIA-MOL/31104/2017 (RM) and UIDB/04567/2020 and UIDP/ 04567/2020 (CBIOS). Authors would like to acknowledge FCT for the financial support of AFR (PD/BD/135504/2018); SF (UI/BD/151421/2021), and RM (CEEC/04567/CBIOS/2020).
Conflict of interests
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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