Editor's evaluation: Evolutionary rescue of phosphomannomutase deficiency in yeast models of human disease
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N-linked glycosylation. The yeast gene SEC53 encodes a homolog of human PMM2. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53-V238M and sec53-F126L, or wild-type SEC53. We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles. Editor's evaluation This valuable paper shows that experimental evolution can shed new and unbiased light on mutations involved in human diseases by showing how growth defects can be compensated. The evidence is convincing, benefiting from not only genetics but also well-established biochemical assays. This paper will be of interest to a broad group of evolutionary biologists and biologists interested in human diseases. https://doi.org/10.7554/eLife.79346.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Protein glycosylation is an important cotranslational and posttranslational modification involving the attachment of glycans to polypeptides. These glycans play vital roles in protein folding, stability, activity, and transport (Varki, 2017). Glycosylation is one of the most abundant protein modifications, with evidence indicating that over 50% of human proteins are glycosylated (Roth et al., 2012; Wong, 2005). Despite this, we lack a global understanding of the complex pathologies involving protein glycosylation. Congenital disorders of glycosylation (CDG) are a group of inherited metabolic disorders arising from defects in the protein glycosylation pathway, including N-linked glycosylation, O-linked glycosylation, and lipid/glycosylphosphatidylinositol anchor biosynthesis (Chang et al., 2018). N-linked CDG are further categorized into two groups based on the affected process: synthesis and transfer of glycans (Type 1) or processing of protein-bound glycans (Type 2) (Aebi et al., 1999). Mutations in the human phosphomannomutase 2 gene (PMM2) cause Type 1 CDG and are the most common cause of CDG (Ferreira et al., 2018). PMM2 forms a homodimer and catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate (M1P) (EC 5.4.2.8). M1P is then converted to GDP-mannose, a required substrate for N-linked glycosylation glycosyltransferases. Two of the most common pathogenic PMM2 variants found in humans are p.Val231Met and p.Phe119Leu. The V231M protein is less stable and exhibits defects in protein folding (Citro et al., 2018; Silvaggi et al., 2006), whereas the F119L protein is stable but exhibits dimerization defects (Andreotti et al., 2015; Kjaergaard et al., 1999; Pirard et al., 1999). The budding yeast Saccharomyces cerevisiae contains a homologous phosphomannomutase enzyme encoded by the gene SEC53. SEC53 is essential in yeast and expression of human PMM2 rescues lethality (Hansen et al., 1997; Lao et al., 2019). Previously, Lao et al., 2019 constructed strains harboring the yeast-equivalent mutations of the most common human-disease-associated PMM2 alleles including V231M (sec53-V238M) and F119L (sec53-F126L). Using experimental evolution, we sought to identify compensatory mutations able to overcome glycosylation deficiency. Previous studies have used experimental evolution of compromised organisms, to study evolutionary outcomes and identify compensatory mutations (Harcombe et al., 2009; Helsen et al., 2020; Laan et al., 2015; Michel et al., 2017; Moser et al., 2017; Szamecz et al., 2014). Experimental evolution allows for a more robust approach than traditional suppressor screens, enabling the identification of weak suppressors and compensatory interactions between multiple mutations (Cooper, 2018; LaBar et al., 2020). Here, we present a 1000-generation evolution experiment of yeast models of PMM2-CDG. We evolved 96 populations of yeast with wild-type SEC53, 192 populations with the sec53-V238M allele, and 96 populations with the sec53-F126L allele for 1000 generations. We sequenced 188 evolved clones to identify mutations that arose specifically in the populations harboring the sec53 human-disease-associated alleles. We find an overrepresentation of mutations in genes whose human homologs are other Type 1 CDG genes, including PGM1 (the minor isoform of phosphoglucomutase), the most commonly mutated gene in our experiment. We show that evolved mutations in PGM1 restore protein N-linked glycosylation and alleviate the slow-growth phenotype caused by the sec53-V238M disease-associated allele. We performed genetic and biochemical characterization of the pgm1 mutations and show that these mutations are dominant and allele-specific SEC53 genetic interactors. Results Experimental evolution improves growth of yeast models of PMM2-CDG Mutations in the phosphomannomutase 2 gene, PMM2, are the most common cause of CDG. Lao et al., 2019 constructed yeast models of PMM2-CDG by making the yeast-equivalent mutations for five human-disease-associated alleles in SEC53, the yeast ortholog of PMM2. These strains have growth rate defects that correlate with enzymatic activity and promoter strength. Increasing expression levels of the mutant sec53 alleles twofold, by replacing the endogenous promoter (pSEC53) with the ACT1 promoter (pACT1), modestly improves the growth rate of sec53 disease-associated alleles (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset Experimental evolution of yeast models of congenital disorders of glycosylation. (A) Table of the various sec53 alleles used in this study. In vitro enzymatic activities of Pmm2 are relative to wild-type Pmm2 and were previously reported (Pirard et al., 1999). Relative growth rates of yeast carrying various SEC53 alleles were previously reported (Lao et al., 2019). (B) Diagram of the evolution experiment. Yeast carrying sec53 mutations implicated in human disease were used to initiate replicate populations in 96-well plates: 96 populations of each mutant genotype and 48 populations of each wild-type genotype. Populations were propagated in rich glucose media, unshaken, for 1000 generations. (C) Single time-point OD600 readings of populations were taken every 50 generations during the evolution experiment as a measure of growth rate. Each line represents one population. We chose to focus on sec53-V238M and sec53-F126L because they are two of the most common disease alleles in humans and because the mutations have distinct and well-characterized effects on protein structure and function (Figure 1A; Andreotti et al., 2015; Briso-Montiano et al., 2022; Citro et al., 2018; Kjaergaard et al., 1999; Pirard et al., 1999; Silvaggi et al., 2006). We evolved 96 haploid populations of each mutant sec53 genotype (pSEC53-sec53-V238M, pACT1-sec53-V238M, and pACT1-sec53-F126L) and 48 haploid populations of each wild-type SEC53 genotype (pSEC53-SEC53-WT and pACT1-SEC53-WT) for 1000 generations in rich glucose medium (Figure 1B). Strains with the pSEC53-sec53-F126L allele grew too slowly to keep up with a 1:210 dilution every 24 hr. Every 50 generations we measured culture density (single time-point OD600) for each population as a proxy for growth rate (Figure 1C; Figure 1—figure supplement 1). Populations with the pACT1-sec53-V238M allele show a range of dynamics, with some populations reaching maximum saturation (OD600 ≈ 1.0) early in the experiment, while some do not even by Generation 1000 (Figure 1C). Three pACT1-sec53-V238M populations went extinct before Generation 200. While saturation levels of pACT1-sec53-F126L populations also increased over the course of the evolution experiment, none reached an OD600 close to 1.0, indicating that these populations are likely less-fit than the evolved pACT1-sec53-V238M populations. Each pSEC53-sec53-V238M population started the evolution experiment with growth rates comparable to SEC53-WT populations, indicating compensatory mutation(s) likely arose in the starting inocula (Figure 1—figure supplement 1). We therefore exclude these populations from subsequent analyses. Together, our data show that the PMM2-CDG yeast models acquired compensatory mutations throughout the evolution experiment and that the extent of compensation depends on the specific disease-associated allele. Putative compensatory mutations are enriched for other Type 1 CDG-associated homologs To identify the compensatory mutations in our evolved populations, we sequenced single clones from 188 populations isolated at Generation 1000 to an average sequencing depth of ~50×. These included 91 pACT1-sec53-V238M clones, 36 pACT1-sec53-F126L clones, 32 pSEC53-SEC53-WT clones, 11 pACT1-SEC53-WT clones, and 18 of the initially suppressed pSEC53-sec53-V238M clones (Supplementary file 1). Autodiploids are a common occurrence in our experimental system (Fisher et al., 2018; Johnson et al., 2021). We attempted to avoid sequencing autodiploids by screening our evolved populations for sensitivity to benomyl, an antifungal agent that inhibits growth of S. cerevisiae diploids more severely than haploids (Venkataram et al., 2016). We find that the average number of de novo mutations (single nucleotide polymorphisms [SNPs] and small indels) varied between SEC53 genotypes, with the pACT1-sec53-F126L clones accruing more mutations per clone (8.14 ± 1.00, 95% confidence interval [CI]) compared to pACT1-sec53-V238M (5.76 ± 0.54) and SEC53-WT clones (combined pSEC53-SEC53-WT and pACT1-SEC53-WT) (6.49 ± 0.90) (Figure 2—figure supplement 1). Despite screening for benomyl sensitivity, most of the sequenced clones (131/188) appear to be autodiploids, with evidence of multiple heterozygous loci. In addition, we detect several copy number variants (CNVs) and aneuploidies shared between independent populations (Figure 2—source data 1, Figure 2—source data 2). We identified putative adaptive targets of selection as genes with more mutations than expected by chance across replicate clones. Some of these common targets of selection are not specific to sec53. For example, mutations in negative regulators of Ras (IRA1 and IRA2) are found in clones from each experimental group and are also observed in other laboratory evolution experiments across a wide range of conditions (Figure 2A; Fisher et al., 2018; Gresham and Hong, 2014; Johnson et al., 2021; Kvitek and Sherlock, 2013; Lang et al., 2013; Venkataram et al., 2016). Figure 2 with 1 supplement see all Download asset Open asset Mutations in Type 1 congenital disorders of glycosylation (CDG) homologs are enriched in sec53-V238M populations. (A) Heatmap showing the number of nonsynonymous mutations per gene that arose in the evolution experiment. Genes with two or more unique nonsynonymous mutations (and each Type 1 CDG homolog with at least one mutation) are shown. pSEC53-SEC53-WT and pACT1-SEC53-WT are grouped as 'SEC53-WT'. For comparison, we show data from previously reported evolution experiments where the experimental conditions were identical to the conditions used here, aside from strain background and experiment duration (bottom three rows). Commonly mutated pathways are grouped for clarity. Asterisks (*) indicate previously known SEC53 genetic interactors (Costanzo et al., 2016; Kuzmin et al., 2018). (B) Binomial test for enrichment or depletion of mutations in Type 1 CDG homologs based on the number of nonsynonymous mutations observed in each experiment, the total number of yeast genes (5906), and the number of genes that are Type 1 CDG homologs (24). 'Combined WT' includes the SEC53-WT populations as well as three additional datasets: Lang et al., 2013, Fisher et al., 2018, and Johnson et al., 2021. Figure 2—source data 1 Table of recurrent copy number variants (CNVs) and aneuploidies in the evolution experiment. https://cdn.elifesciences.org/articles/79346/elife-79346-fig2-data1-v2.zip Download elife-79346-fig2-data1-v2.zip Figure 2—source data 2 Coverage plots of evolved clones. https://cdn.elifesciences.org/articles/79346/elife-79346-fig2-data2-v2.zip Download elife-79346-fig2-data2-v2.zip Figure 2—source data 3 Table of mutations in Type 1 congenital disorders of glycosylation (CDG) homologs. https://cdn.elifesciences.org/articles/79346/elife-79346-fig2-data3-v2.zip Download elife-79346-fig2-data3-v2.zip We identified putative sec53-compensatory targets as genes mutated exclusively in pACT1-sec53-V238M and/or pACT1-sec53-F126L populations (Figure 2A). The most frequently mutated genes among pACT1-sec53-F126L clones are common targets in other evolution experiments such as IRA1, IRA2, and KRE6, suggesting that mutations capable of compensating for Sec53-F126L dimerization defects are rare or not easily accessible. In contrast, while recurrently mutated genes in pACT1-sec53-V238M populations include common targets, we also identified a number of unique targets of selection, most notably in homologs of other Type 1 CDG-associated genes. We find an enrichment of mutations in CDG homologs in pACT1-sec53-V238M clones (Figure 2B; Figure 2—source data 3). Across all sequenced populations we identified ~620 nonsynonymous mutations. As there are nearly 6000 yeast genes, the probability of any given gene receiving even a single nonsynonymous mutation is low. We are therefore well powered to detect enrichment of mutations, but underpowered to detect depletion of mutations. To gain statistical power we aggregated our SEC53-WT data with three other large datasets using similar strains and propagation regimes (Fisher et al., 2018; Johnson et al., 2021; Lang et al., 2013). In this aggregate dataset, we observe a statistically significant depletion of mutations in Type 1 CDG homologs, suggesting that they are typically under purifying selection in experimental evolution (Figure 2B). One of the CDG homologs, PGM1, is the most frequently mutated gene among pACT1-sec53-V238M clones. PGM1 encodes the minor isoform of phosphoglucomutase in yeast (Bevan and Douglas, 1969). We only find PGM1 mutations in pACT1-sec53-V238M populations which suggests their compensatory effects are unique to the sec53-V238M mutation and not glycosylation deficiency in general. Each of the five mutations in PGM1 are missense mutations, rather than frameshift or nonsense. This is consistent with selection acting on alteration-of-function rather than loss-of-function (LOF; posterior probability of non-LOF = 0.96, see Methods). To identify the compensatory mutation(s) that arose prior to the start of the evolution experiment in the initially suppressed pSEC53-sec53-V238M populations, we analyzed the 18 sequenced clones. For each clone, we find both wild-type SEC53 and sec53-V238M alleles among sequencing reads. This could result from integration or maintenance of the covering plasmid. No sequencing reads align to the URA3 locus of our reference genome, suggesting that the plasmid marker was lost, as expected following counterselection. Compensatory mutations restore growth In order to validate putative compensatory mutations we reconstructed evolved mutations in PGM1 and ALG9, two genes whose human homologs are implicated in Type 1 CDG (Frank et al., 2004; Timal et al., 2012). These heterozygous evolved mutations were constructed in a homozygous pACT1-sec53-V238M diploid background (note that although each pgm1 and alg9 mutation assayed here arose in haploid-founded populations, they arose as heterozygous mutations in autodiploids). We quantified the fitness effect of each mutation using a flow cytometry-based fitness assay, competing the reconstructed strains against a fluorescently labeled, diploid version of the pACT1-SEC53-WT ancestor. The pACT1-sec53-V238M ancestor has a fitness deficit of −25.86 ± 2.3% (95% CI) relative to wild-type. We find that each of the five evolved pgm1 mutations are compensatory in the pACT1-sec53-V238M background. The fitness effects of the sec53/pgm1 double mutants range between −19.10 ± 1.07% and −9.84 ± 0.77% (i.e., the pgm1 mutations confer a fitness benefit between 6.76% and 16.02% in the pACT1-sec53-V238M background) (Figure 3A). We also find that the evolved alg9-S230R mutation is compensatory, as the sec53/alg9 double mutant has a fitness effect of −17.89 ± 0.59% (Figure 3—figure supplement 1). We compared the fitness of these reconstructed double mutants to the fitness of the evolved clones which carry three to nine additional SNPs. In each case, the evolved clones were more fit than the reconstructed strain, except for the evolved clone containing the pgm1-D295N mutation (Figure 3—figure supplement 1). Thus, while the alg9 and pgm1 mutations compensate for the sec53-V238M defect, other mutations contribute to the overall fitness of these evolved clones. Figure 3 with 6 supplements see all Download asset Open asset Evolved mutations rescue fitness and protein glycosylation defects of pACT1-sec53-V238M. (A) Average fitness effects and standard deviations of reconstructed heterozygous pgm1 mutations. Fitness effects were determined by competitive fitness assays against a fluorescently labeled version of the diploid pACT1-SEC53-WT ancestor. Replicate measurements are plotted as gray circles. Pairs of pgm1 fitness effects with non-statistically significant differences: R64C-D295N, R64C-pgm1Δ, R64C-S120A, D295N-pgm1Δ, G514C-T521K, G514C-S120A, and pgm1Δ-S120A (df = 194, F = 208.1, each p > 0.05, one-way analysis of variance [ANOVA] with Tukey post hoc test). (B) Western blots of invertase (left) and carboxypeptidase Y (right) from ancestral and reconstructed strains. In panels A and B, plus signs (+) indicate wild-type alleles. Genotypes are either homozygous wild-type (+/+), homozygous mutant (mutation/mutation), or heterozygous (mutation/+). Figure 3—source data 1 Raw images of blots and Ponceau stains. https://cdn.elifesciences.org/articles/79346/elife-79346-fig3-data1-v2.zip Download elife-79346-fig3-data1-v2.zip We constructed and assayed the fitness effects of the pgm1 mutations in diploid pACT1-sec53-F126L and pACT1-SEC53-WT backgrounds. Each pACT1-sec53-F126L strain was too quickly outcompeted by the reference strain to measure fitness, suggesting that the compensatory effects of the evolved pgm1 mutations are specific for the sec53-V238M mutation or any fitness improvements in the pACT1-sec53-F126L background are too minimal to detect. Each pgm1 mutation is nearly neutral in the pACT1-SEC53-WT background (Figure 3—figure supplement 2). To determine if LOF of PGM1 would phenocopy evolved mutations, we deleted one copy of PGM1 (pgm1Δ) in the diploid pACT1-sec53-V238M background and again measured fitness. We find that the heterozygous pgm1Δ mutation improves fitness (−15.28 ± 0.76%), indicating that LOF of PGM1 is compensatory in the conditions of the evolution experiment (Figure 3A). It is not immediately clear, then, why we only identify missense mutations in PGM1. Each of the mutated Pgm1 residues is located around the active site of the enzyme, based on predicted protein structure (Jumper et al., 2021; Stiers and Beamer, 2018). It could be that the evolved pgm1 mutations are LOF given this clustering but maintaining protein expression provides an ancillary benefit. To account for this possibility, we constructed a catalytically dead allele of PGM1 by mutating the enzyme's catalytic serine (pgm1-S120A) (Stiers et al., 2017a). We find that pgm1-S120A improves pACT1-sec53-V238M fitness (−13.32 ± 0.88%) and this does not significantly differ from pgm1Δ (Figure 3A), indicating that PGM1 LOF is compensatory regardless of if that arises from loss of coding sequence or enzyme activity. We also measured fitness of homozygous pgm1 mutations (Figure 3—figure supplement 3). In all cases, the fitness effect of heterozygous and homozygous pgm1 mutants is not substantially different from each other, indicating that, by this assay, the evolved pgm1 mutations are dominant in the pACT1-sec53-V238M background, as are the pgm1Δ and pgm1-S120A mutations. Compensatory mutations restore protein glycosylation We have established that pgm1 mutations compensate for the growth rate defect of the sec53-V238M allele. To determine whether the evolved pgm1 mutants also compensate for the molecular defects in N-linked glycosylation we examined two representative yeast glycoproteins, invertase and carboxypeptidase Y (CPY), in our reconstructed pgm1 strains. Invertase forms both a non-glycosylated homodimer (120 kDa) and a secreted homodimer that is heavily glycosylated (approximate range of 140–270 kDa) (Gascón et al., 1968; Zeng and Biemann, 1999). Mature CPY only exists in its glycosylated form, with a molecular weight of 61 kDa (Hasilik and Tanner, 1978). The ancestral pACT1-sec53-V238M strain shows underglycosylation of invertase and CPY. We find a reduced abundance of the higher-molecular-weight glycosylated form of invertase and mature CPY, accompanied by the appearance of underglycosylated forms of these proteins (Figure 3B). We find that evolved pgm1 mutations, pgm1Δ, and pgm1-S120A each restore glycosylation of these proteins to near-wild-type levels (Figure 3B, Figure 3—figure supplements 4 and 5). We do not observe rescue of protein glycosylation in pACT1-sec53-F126L strains carrying pgm1 mutations (Figure 3—figure supplement 6). Together, these suggest that pgm1-mediated rescue of pACT1-sec53-V238M fitness is due to restoration of protein glycosylation and the effect is specific for the pACT1-sec53-V238M background. Compensatory PGM1 mutations are dominant suppressors of sec53-V238M To further demonstrate that pgm1-mediated compensation is specific for sec53-V238M, we performed a genetic analysis by dissecting tetrads from strains that are heterozygous for both sec53 and pgm1. In the absence of a compensatory mutation, we expect 50% large colonies and all tetrads showing a 2:2 of due to the single locus if an evolved mutation is compensatory then we expect large colonies with three of and large to small These three are expected to a genetic (Figure For each genetic we tetrads and performed a test to determine whether the is more consistent with genetic than (Figure data 1). Figure 4 with 2 supplements see all Download asset Open asset pgm1 mutations are dominant suppressors of (A) Diagram of in the diploid strains could one of three based on allele (B) of in a sec53-V238M background and a sec53-F126L background are the probability and of based on the of growth to growth colonies or small (C) of in a sec53-V238M background and a sec53-F126L background of dominant of the pgm1 mutations in the sec53-V238M background and sec53-F126L background based on the Figure data 1 analysis of Download We find each of the evolved pgm1 mutations compensates for the allele of SEC53 (Figure supplement 1). For example, of a strain heterozygous for pACT1-sec53-V238M and show large colonies and nine with a of Figure These genetic the results from the fitness assays by showing that all five evolved pgm1 mutations, the pgm1Δ and pgm1-S120A mutations, as well as the evolved alg9 mutation all sec53-V238M (Figure supplement 1). We assayed each of the pgm1 mutations in a sec53-F126L background. In to the fitness we find several pgm1 alleles and that sec53-F126L (Figure supplement 1). It is that the of compensation is less than in the sec53-V238M background based on To test if pgm1 mutations are we a wild-type copy of PGM1 to each of the strains such that each haploid will either two wild-type of PGM1 or both wild-type PGM1 and mutant pgm1 We find that the five evolved pgm1 mutations and but not pgm1Δ, are dominant suppressors of sec53-V238M in the genetic (Figure Figure supplement 2). For the sec53-F126L background, we find dominant of Pgm1 activity deleterious effect of PMM2-CDG in yeast To determine how the evolved mutations Pgm1 enzymatic activity, we mutant and wild-type PGM1 alleles into expression purified enzyme, and assayed phosphoglucomutase activity using a standard enzymatic (Figure Figure supplement 1). We find that mutant Pgm1 have from near-wild-type to and (Figure The loss of activity of is a that an essential during (Stiers et al., and we that all the active forms show activity in the of (Figure data 1). not differ between with activity (Figure supplement 2). Figure with supplements see all Download asset Open asset loss of Pgm1 activity a fitness (A) structure of S. cerevisiae Pgm1 (Jumper et al., 2021). residues as (B) of Pgm1 as determined by a enzymatic ± 95% confidence (C) of wild-type and mutant Replicate measurements are plotted as circles. Pgm1 enzyme the fitness effect in the diploid pACT1-sec53-V238M background in Figure 3A). and 95% confidence fit as a = = = F = p = analysis of variance that we not measure activity and activity. Figure data 1 biochemical of mutant Download of the of active mutant shows a of between and relative to wild-type on the substrate of and are than wild-type Pgm1 = and = and p = and shows an increased compared to wild-type but this is not statistically significant = = p = these indicate that the five evolved pgm1 mutations have effects on enzymatic activity. The between pgm1 fitness and Pgm1 is best fit by a rather than a function (Figure fitness is by but not Pgm1 activity. Pgm1 does not have increased phosphomannomutase activity can levels of phosphomannomutase activity, and we that mutations in Pgm1 could from glucose to compensating for the loss of activity and 1969). We this in two we measured phosphomannomutase activity of wild-type Pgm1 and two mutant using a (Figure supplements 1 and 3A). We find that wild-type Pgm1 and show phosphomannomutase activity, for and of their phosphoglucomutase activity,
Source: eLife
Author: Wenying Shou
Institution: University College London
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4312968150