FicD sensitizes cellular response to glucose fluctuations in mouse embryonic fibroblasts

Significance The chaperone BiP plays a key quality control role in the endoplasmic reticulum, the cellular location for the production, folding, and transport of secreted proteins. The enzyme FicD regulates BiP’s activity through AMPylation and deAMPylation. Our study unveils the importance of FicD in regulating BiP and the unfolded protein response (UPR) during stress. We identify distinct BiP AMPylation signatures for different stressors, highlighting FicD’s nuanced control. Deletion of FicD causes widespread gene expression changes, disrupts UPR signaling, alters stress recovery, and perturbs protein secretion in cells. These observations underscore the pivotal contribution of FicD for preserving secretory protein homeostasis. Our findings deepen the understanding of FicD’s role in maintaining cellular resilience and open avenues for therapeutic strategies targeting UPR-associated diseases.


Cell Harvesting and Lysis
MEFs were grown in 6-well plates or 10cm dishes at 5% CO2 at 37 °C to 80% confluence.Treated MEFs were either harvested by trypsinization or transferred to ice and harvested by scraping and transferred to 1.5 mL reaction tubes on ice.Medium was aspirated after centrifugation at 1000xg for 5 min at 4 °C and cell pellet was washed with ice cold 1x PBS.MEFs were then frozen in liquid nitrogen and stored at 80 °C.To prepare cells for Western blotting, the cell pellet was resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.2% Triton, 1x Roche Complete EDTA free protease inhibitor cocktail, 1x Roche PhosSTOP, and approximately 0.5units/uL Novagen Benzonase nuclease) and chilled on ice for 5-10 minutes.Lysed MEFs were then resuspended in RIPA buffer (50mM Tris pH 7.4, 0.5% NP40, 0.5% DOC, 0.2% SDS, 1X Roche EDTA free protease inhibitor cocktail, 1X Roche PhosStop, 1mM DTT) before centrifugation at full speed for 10 min on a bench top Eppendorf 5424 R centrifuge at 4 °C.Clarified supernatants were transferred to fresh Eppendorf tube tubes.Lysate protein concentrations were determined by Pierce BCA Protein Assay (Thermo Scientific) and normalized in RIPA buffer before the addition of 0.5-1 volumes of 5X SDS sample buffer (250mM Tris pH 6.8, 10% SDS, 100mM DTT, 50% glycerol, 0.25% Bromophenol Blue).For mRNA isolation, frozen MEFs were lysed directly with QIAshredder columns (Qiagen).

Protein Synthesis Assay
MEFs were cultured and subjected to glucose starvation and refeeding as previously described.Thirty minutes prior to cell harvesting, the culture medium was replaced with a pre-warmed medium containing 1 μM Puromycin (Thermo Scientific), appropriate for the specific condition being tested (either regular or glucose-free medium).The cells were then incubated under 5% CO2 at 37°C for the final 30 minutes, followed by harvesting and lysis as previously described.Western blot analysis was conducted according to the protocol detailed in the subsequent section.

Analysis of RNA-seq
Fastq reads corresponding to each of four treatments (unstressed, glucose starved, 2-hour refed, and 4-hour refed) applied to FicD F/F and FicD -/-MEFs (all conditions in triplicate) were mapped to the mouse reference genome, and statistical analysis was performed using CLC Genomics Workbench software (version 9.5, CLC Bio, Aarhus, Denmark).Total counts and CPM for each mouse gene were generated for all conditions (8 conditions in triplicate, 24 samples).Principal Component Analysis (PCA) was calculated for all samples with ClustVis (60) using Log2 CPM counts with unit variance scaling and row centering.Principal components were calculated using Singular Value Decomposition (SVD) with imputation.Total counts mapped for each sample were used to calculate differential gene expression with four methods: EdgeR, DESeq2, limma, and NOISeq using the Integrative Differential Expression Analysis for Multiple EXperiments (IDEAMEX) server with chosen parameters (LogFC=0.75,FDR=0.05, and CPM=1) without batch effects (61).Differentially expressed genes (DEGs) were identified for various pairwise conditions (Fig. 2C).Differential expression results were integrated to observe the consistency of DEGs identified by each method (Fig. S3A and B).Ultimately, EdgeR (62) DEGs were chosen for subsequent GO term enrichment.
For GO term enrichment analysis, Ensemble IDs for upregulated EdgeR DEGs were submitted to the G:profiler server (63) limiting the statistical domain scope to annotated genes, using the G:SCS threshold to calculate adjusted P-values with a cutoff below 0.01, limiting pathway size to between 10 and 250 terms, and excluding electronic GO annotations.We removed redundant terms with identical lists of genes, keeping the top term ranked by the lowest P-value.We report the enriched GO biological process terms for upregulated DEGs from metabolic stress comparison of Fic FL/FL starved/FicD F/F unstressed MEFs and for enriched GO molecular function terms for upregulated DEGs from genotype comparison of FicD -/-with FicD F/F MEFs.PCA and heat maps were generated for mouse DEGs (identified an any of the RNA-seq comparisons from Fig3C, as well as DEGs from any of the conditions compared to the respective unstressed state) with GO terms related to unfolded protein response (UPR) as defined in UniProt (64).Genes were clustered in the heatmap by hierarchical clustering (Euclidean distance with Ward method) using clustvis with the same parameters as used for PCA (60).

Preparation of Secretomes
After overnight incubation in serum-containing media with or without glucose, the culture media of MEFs were replaced with serum-free media with or without glucose.Following an additional incubation period of 2-4-hours, the supernatants from the MEFs were collected into 1.5 mL tubes placed on ice.To remove cells and debris, the supernatants were centrifuged for 15 minutes at 3200 x g at 4 °C.Subsequently, the supernatants were filtered through a 0.22μm filter into fresh microcentrifuge tubes.To the filtered supernatants, a final concentration of 150 μg/ml sodium deoxycholate was added and incubated for 15 minutes on ice.Furthermore, a final concentration of 8% (v/v) trichloroacetic acid was added to each sample, followed by an overnight incubation at 4 °C.The precipitated proteins were resuspended and transferred to fresh microcentrifuge tubes that had been pre-rinsed with methanol to remove collagen contamination.These suspensions were then subjected to centrifugation for 1 hour at 27,000 x g at 4 °C.Afterward, the supernatants were discarded, and the pellets were washes twice with 1.5 ml of pre-cooled 100% acetone, with each wash involving centrifugation for 40 minutes at 27,000 x g at 4 °C.Following the second wash, the pellets were air-dried for 10 minutes and then resuspended in 1 ml of 10 mM Tris-HCl at pH 8.0.The resuspended samples were transferred to fresh microcentrifuge tubes in preparation for tryptic digestion.Tandem Mass Spectrometry Secreted protein samples were reduced with 10mM DTT for 1 hr at 56°C and alkylated with 50mM iodoacetamide for 45 min at room temperature in the dark.Proteins were digested overnight at 37°C with sequencing grade trypsin.Resulting peptides were then de-salted via solid phase extraction (SPE) prior to analysis.LC-MS/MS experiments were performed on a Thermo Scientific EASY-nLC 1200 liquid chromatography system coupled to a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer.To generate MS/MS spectra, MS1 spectra were first acquired in the Orbitrap mass analyzer (resolution 120,000).Peptide precursor ions were isolated and fragmented using highenergy collision-induced dissociation (HCD).The resulting MS/MS fragmentation spectra were acquired in the ion trap.Label-free quantitative searches were performed using Proteome Discoverer 2.2 software (Thermo Scientific).Samples were searched against all reviewed entries in the Mouse UniProt protein database.Searches included the following modifications: carbamidomethylation of cysteine residues (+57.021Da), oxidation of methionine (+15.995Da), and acetylation of peptide N-termini (+42.011Da).Precursor and product ion mass tolerances were set to 10 ppm and 0.6 Da, respectively.Peptide spectral matches were adjusted to a 1% false discovery rate (FDR) and proteins were filtered to a 5% FDR.All samples were run in biological triplicate.Data is reported in Supplemental Dataset S2.

Analysis of Secretomes
Proteins were mapped from the following treatment and genotype comparisons: starved FicD F/F / unstressed FicD F/F , starved FicD -/-/ unstressed FicD -/-, unstressed FicD -/-/ unstressed FicD F/F , and starved FicD -/-/ starved FicD F/F .Upregulated proteins were defined as having an abundance ratio fold change = >1.5 and p-value < 0.05, as well as proteins with missing values that were identified in at least 2 reps of the first condition (i.e.starved FicD F/F from starved FicD F/F / unstressed FicD F/F ) but 0 reps of the second (i.e.unstressed FicD F/F from starved FicD F/F / unstressed FicD F/F ).Proteins with low combined FDR confidence (<0.05) were excluded.The same cutoffs were used to identify downregulated proteins (abundance ratio = <1.5 and p-value < 0.05, as well as proteins with missing values that were identified in at least 2 reps the second condition but 0 reps of the first condition).Secreted proteins were identified from UniProt annotations for GO cellular component terms that include "extracellular" or for subcellular location terms that include "secreted".

Figure S2 .
Figure S2.Thapsigargin (TG), DTT, and Glucose Starvation induced ER stress of FicD F/F and FicD -/-MEFs.RT-qPCR showing relative expression levels of UPR marker genes ATF4, BiP, and FicD upon A) treatment of MEFs with Thapsigargin (1µM), or B) recovery of MEFs from 1h DTT (5mM) exposure, or C) glucose starvation (for 16h) and re-feeding of MEFs for indicated time points.Error bars represent the standard deviation of 3 biologically independent repeat of the experiment with 4 technical replicates.Genotypes are demonstrated by shades of blue for FicD F/F and shades of orange for FicD - /-MEFs.Two-way ANOVA with Tukey multiple comparison test is applied to determine the significance.P values: 0.1234 (ns), 0.0332 (*), 0.0021 (**), <0.0001 (****).Nonsignificant comparisons are not shown for clarity.
Fig. S3 Figure S4.A) PCA plot and B) heatmap (colored by Log2CPM Z-score) depict significant DEGs under any condition with Go term including "gluconeogenesis".Genes are clustered Euclidean distance and Ward linkage.C-E: Volcano plots of LogFC against -LogFDR (with maximim of 350 assigned to genes with FDR 0) for indicated comparison conditions (plot titles), up in FicD F/F (blue) and up in FicD -/-(orange).UPR genes are labeled.F) Venn diagram depicting DEG overlaps comparing FicD -/-to FicD F/F for the indicated conditions.G) Heatmap clustering Log2CPM (Z-score) of differentially expressed genes under any condition with GO BP term "response to hypoxia".

Figure S5 .
Figure S5.Heatmap (colored by Log2CPM Z-score) depicts significant DEGs under any condition with GO CC term including "Preribosome" and GO BP term including "Ribosomal large subunit biogenesis".Genes are clustered Euclidean distance and Ward linkage.

Figure S6 .
Figure S6.Nascent protein synthesis during glucose starvation and refeeding A) AMPylation and eIF2a phosphorylation of FicD F/F and FicD -/-MEFs during glucose starvation and refeeding.Bar graphs indicates the quantification of the rations of phospho-eIF2a/eIF2a and AMP-BiP/BiP.B) Cycloheximide control for puromycin treatment.Bar graph shows the quantification of western blot signals.C) Puromycin treatment assay during glucose starvation and refeeding.Two separate blots are shown.Bar graph indicates the quantification of western blot signals.Two-way ANOVA with Tukey multiple comparison test is applied to determine the significance.P values: 0.1234 (ns), 0.0332 (*), 0.0021 (**), <0.0001 (****).Non-significant comparisons are not shown for clarity.