High-throughput identification of calcium-regulated proteins across diverse proteomes

Abstract

Calcium ions play important roles in nearly every biological process, yet whole-proteome analysis of calcium effectors has been hindered by a lack of high-throughput, unbiased, and quantitative methods to identify protein-calcium engagement. To address this, we adapted protein thermostability assays in budding yeast, human cells, and mouse mitochondria. Based on calcium-dependent thermostability, we identified 2,884 putative calcium-regulated proteins across human, mouse, and yeast proteomes. These data revealed calcium engagement of signaling hubs and cellular processes, including metabolic enzymes and the spliceosome. Cross-species comparison of calcium-protein engagement and mutagenesis experiments identified residue-specific cation engagement, even within well-known EF-hand domains. Additionally, we found that the dienoyl-coenzyme A (CoA) reductase DECR1 binds calcium at physiologically relevant concentrations with substrate-specific affinity, suggesting direct calcium regulation of mitochondrial fatty acid oxidation. These discovery-based proteomic analyses of calcium effectors establish a key resource to dissect cation engagement and its mechanistic effects across multiple species and diverse biological processes.

Keywords: CP: Cell biology; CP: Metabolism; TMTpro; calcium engagement; calcium ion engagement; calcium regulated proteins; cell signaling; magnesium engagement; oxidation of polyunsaturated fatty acids; sample multiplexed quantitation; thermal stability proteomics.

Figure 1:. PISA with isobaric sample multiplexing in three different proteomes identifies Ca²⁺-regulated proteins.

(A) Schematics of the experimental workflow to detect ion-induced changes in protein thermal stability. Lysates from yeast, human and isolated murine liver mitochondria were treated with chelator EGTA. After the addition of ions, a temperature gradient was applied to aliquoted samples, the samples were then mixed, and insoluble (heat denatured) material was precipitated. Soluble proteins were labeled with isobaric tags, and protein abundance was quantified. (B) Volcano plots show protein abundance in Ca²⁺ relative to EGTA, and their q-values. Proteins with a |log₂(FC)| ≥ 0.2, and q-value ≤ 0.05 (Welch’s t-test with multiple hypothesis correction) were considered to show significant thermal stability changes. Green dots indicate proteins with previous Ca²⁺ annotation. Dotted lines show log₂(FC) ± 0.2 and log₁₀(q-value) for a q-value of 0.05. (C) Venn diagrams show proteins with significant thermal stability changes in each category listed. Proteins that are in the shaded compartments represent CRPs. Proteins that do not show a significant thermal stability change between Mg²⁺ and Mg²⁺Ca²⁺ conditions were considered to be non-specific ion responders and excluded from the list of CRPs.

Figure 2:. Analysis of human and mouse datasets show significant enrichment of known Ca²⁺-regulated processes, proteins, and pathways.

(A,B) Gene set enrichment analysis (GSEA) of CRPs from mouse (A) and human (B) datasets show significant enrichment of molecular function terms related to Ca²⁺ binding and signaling (green text). (C) Ion-induced thermal stability changes of Annexin proteins, Ca²⁺-dependent phospholipid binding and Calcium ion binding proteins, from human and mouse datasets show a Ca²⁺-specific response. (D) GSEA of human dataset using KEGG annotations enriches Ca²⁺ signaling. Schematic representation of the KEGG Ca²⁺ signaling pathway. Proteins shown were quantified in the human lysates. Bolded proteins are identified as CRPs. (E) Ion-induced thermal stability changes of KEGG calcium signaling pathway proteins, relative to EGTA condition. Dotted lines show log2(FC) ± 0.2. Error bars: standard deviation of replicate measurements.

Figure 3:. Ion engagement comparison of yeast and human proteomes captured evolutionary differences in Ca²⁺ signaling and ion coordination.

(A) Ion-induced thermal stability changes of human and yeast OGDH and IDH homologs, relative to EGTA. Human TCA cycle dehydrogenases are CRPs, whereas yeast homologs do not engage Ca²⁺. Orthologs are indicated by the same color. Star denotes that the protein is a CRP in our dataset. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements. (B) Overlay of human OGDH (gray) and its yeast homolog Kgd1 (yellow) structures at the Ca²⁺ coordination site. Yeast Kgd1 lacks residues that coordinate Ca²⁺ in human OGDH. (C) Ion-induced thermal stability changes of human and yeast EPS15 homologs, relative to EGTA. Yeast homolog Ede1 changes its abundance with Ca²⁺ addition only, human homologs EPS15, ITSN1, ITSN2 show thermal stability changes with Ca²⁺ or Mg²⁺. REPS1 and EHD3 do not show ion engagement. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements. (D) Alignment of the second EF-hand domain of yeast and human EPS15 homologs. Yeast has a serine (S) where EPS15, ITSN1, ITSN2 have a conserved aspartic acid (D). REPS1 and EHD3 lack canonical EF-hand residues. (E) Overlay of EPS15^wt structure with a predicted EPS15^D177S structure. D177S mutation (stick representation, brown) is predicted to form a smaller coordination domain resulting in coordination of smaller Mg²⁺ ions. (F) Measurement of Ca²⁺ or Mg²⁺ binding to purified EPS15^wt and EPS15^D177 proteins based on microscale thermophoresis. Mammalian cells were transiently transfected with plasmids for expression and purification of EPS15-His proteins. Background refers to purification from cells that do not express tagged EPS15 proteins. ΔF_Norm refers to the change in normalized fluorescence induced by ligand binding (a zero value suggests no ligand binding). Significance based on Student’s t-test: ns – not significant, ** - p<0.005, **** - p<0.0001.

Figure 4:. DECR1 is a Ca²⁺ binding protein.

(A) Ion-induced thermal stability changes of mouse Decr1, relative to EGTA. Decr1 shows Ca²⁺-dependent stabilization. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements. (B) Analysis of Ca²⁺ or Mg²⁺ binding to purified DECR1 using MST shows that DECR1 is a Ca²⁺ -binding protein. ΔF_Norm refers to the change in normalized fluorescence induced by ligand binding (a zero value suggests no ligand binding). **** = p<0.0001. (C) Overlay of DECR1 structure with a Ca²⁺ liganded predicted model of DECR1 from AlphaFill. Placement of the Ca²⁺ ion (green) is based on a conserved binding pocket of a bacterial dehydrogenase that shows sequence homology to DECR1. The proximity of the Ca²⁺ ion to the NADP+ binding pocket and E310 of a neighboring DECR1 molecule within the tetramer suggests coordination.

Figure 5:. Enrichment of human CRPs identifies lipid-raft associated prohibitin/SPFH domain-containing proteins as calcium engagers.

(A) GSEA of mouse CRPs to identify enriched protein domains using SMART. (B) Ion-induced thermal stability changes of mouse prohibitin domain proteins, relative to EGTA. Stars indicate CRPs as called by our thermal stability analysis. Dotted lines show log₂(FC) ± 0.2. Error bars show standard deviation of replicate measurements. (C) Multiple sequence alignment of human prohibitin domain proteins using Constraint-based Multiple Alignment tool (COBALT). Red indicates high conservation; blue indicates lower conservation. The conserved prohibitin/SPFH domain is indicated under the alignment. (D) FLAG-tagged Podocin, Stoml1, Stoml3 or control GFP were transiently expressed in HEK293T cells. Their Ca²⁺-dependent thermal stability was assessed with PISA, followed by Western blotting. (E) Quantification of Western blot PISA results for Podocin, Stoml1, and Stoml3. Error bars show standard error of the mean of replicate measurements.

Figure 6:. Informatic analysis uncovers and stratifies calcium-specific engagement in murine mitochondria.

(A) Workflow and output from PCA to assess Ca²⁺ or Mg²⁺ binding. |log₂(FC)| was used based on the bidirectionality of thermal stability changes reflecting cation engagement. The resulting loadings were assessed for differentiation of the Ca²⁺ or Mg²⁺ conditions. PC2 separated proteins based on cation treatment and was used to index proteins in the subsequent analyses. (B) GSEA using PC2 loadings to index proteins showed strong enrichment for Ca²⁺ binding proteins at more negative loadings, consistent with separation of Ca²⁺ or Mg²⁺ binding specificity. (C) Cation-induced thermal stability changes of the top 4 proteins with the most negative and most positive PC2 loadings. Stability changes were consistent with specific engagement of either Ca²⁺ or Mg²⁺. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements. (D) GSEA across PC2-loading space reported enrichment of multiple calcium binding protein gene sets (green text) as well as several lipid or membrane associated gene sets (brown text). (E) Mapping of mouse CRPs to the ATP synthase structure (PDB: 8h9v) revealed calcium engagement with the F1 domains, axle, and stator. ATP synthase ligands are shown in magenta. (F) Cation-induced thermal stability changes for ATP synthase complex proteins annotated as CRPs. CRPs were observed in all major domains of the complex. Dotted lines show log₂(FC) ± 0.2. Error bars: standard deviation of replicate measurements.

Figure 7:. Exploration of calcium-specific engagement in murine mitochondria and oxidative phosphorylation complexes.

(A) From the GSEA in Figure 6D, proteins along the PC2 loadings were enriched for lipid binding and membrane associated functions. Based on these findings we investigated transmembrane proteins (“Tmem”) along PC2 and observed divergent engagement with cations for these proteins. (B) From (A), the top two ‘calcium specific’ proteins we observed were Tmem128 and Tmem165. The top ‘magnesium specific’ proteins we observed were Tmem41a and Tmem94. Dotted lines show log₂(FC) ± 0.2. Tmem94 (purple text) was recently identified as a magnesium transporter and Tmem165 (green text) was recently identified as a calcium importer. (C) Tmem128 protein interaction network established from the BioPlex interactome. Tmem128 interacts with at least four known endoplasmic reticulum (ER) proteins: RTN2, RTN4, REEP5, REEP6 (highlighted in grey).