Skip to main content
PMC home page
Contact logoLink to Contact
. 2021 Nov 29;4:25152564211052392. doi: 10.1177/25152564211052392

Interactome Analysis of the ER Stress Sensor Perk Uncovers Key Components of ER-Mitochondria Contact Sites and Ca2+ Signalling

Maria Livia Sassano 1,2, Rita Derua 3,4, Etienne Waelkens 3,4, Patrizia Agostinis 1,2,✉, Alexander R van Vliet 1,*
PMCID: PMC10243573  PMID: 37366380

Abstract

We recently reported that the ER stress kinase PERK regulates ER-mitochondria appositions and ER– plasma membrane (ER-PM) contact sites, independent of its canonical role in the unfolded protein response. PERK regulation of ER-PM contacts was revealed by a proximity biotinylation (BioID) approach and involved a dynamic PERK-Filamin A interaction supporting the formation of ER-PM contacts by actin-cytoskeleton remodeling in response to depletion of ER-Ca2+ stores. In this report, we further interrogated the PERK BioID interactome by validating through co-IP experiments the interaction between PERK and two proteins involved in Ca2+ handling and ER-mitochondria contact sites. These included the vesicle associated membrane (VAMP)-associated proteins (VAPA/B) and the main ER Ca2+ pump sarcoplasmic/endoplasmic reticulum Ca ATPase 2 (SERCA2). These data identify new putative PERK interacting proteins with a crucial role in membrane contact sites and Ca2+ signaling further supporting the uncanonical role of PERK in Ca2+ signaling through membrane contact sites (MCSs).

Keywords: cell biology, endoplasmic reticulum, ER stress, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), mitochondrial associated membranes (MAM)

Introduction

The endoplasmic reticulum (ER) is a major site of protein folding in the cell, handling roughly one-third of all cellular proteins and all proteins destined for transport towards the plasma membrane (PM) or extracellular matrix (Brodsky & Wojcikiewicz, 2009). To cope with this demand, the ER has evolved to possess an intricate folding machinery and an ideal protein folding environment. However, when the ER can no longer match cellular protein folding demand, the resulting accumulation of unfolded proteins causes ER stress (Ron & Walter, 2007). The unfolded protein response (UPR) consists of the activation of a conserved signal transduction pathway, ultimately eliciting a transcriptional program that operates as a principal safeguard against the loss of ER homeostasis caused by ER stress. The UPR is launched by the activation of three ER membrane proteins. The ER stress kinase PKR-like endoplasmic reticulum kinase (PERK) is one of these three mediators and is activated upon ER stress (Ron & Walter, 2007). In homeostatic conditions, PERK is kept inactive through the binding of the ER chaperone BiP to its luminal domain, and is activated upon its release, resulting in the oligomerization of PERK, followed by its autophosphorylation. Activated PERK is then able to phosphorylate eukaryotic initiation factor 2 alpha (eIF2α), leading to a protein translation pause and giving the ER folding machinery time to deal with its protein burden (Ron & Walter, 2007).

In our previous studies (van Vliet et al., 2017; Verfaillie et al., 2012), we uncovered that independent of its UPR function, PERK moonlights at the ER-mitochondria contacts and aids apoptotic cell death by the transfer of ROS signals from the ER to the mitochondria. More recently, we showed that the activation of PERK can occur independently of its luminal domain and canonical ER stress, instead of being activated by a rise in cytosolic Ca2+.

To uncover the roles of PERK we carried out an unbiased proximity biotinylation (BioID) screen, using the promiscuous biotinylation enzyme BirA* tagged to PERK on its cytosolic side (Roux et al., 2012; van Vliet et al., 2017). Using this technique, we discovered the cytoskeletal protein Filamin A (FLNA) as a novel PERK interactor. The Ca2+ -mediated PERK-FLNA axis was found to be required to support the formation of ER-PM contacts and store-operated Ca2+ entry (SOCE) (van Vliet et al., 2017). These findings together hint at a broader cross-talk between ER stress proteins and the regulation of membrane contact sites (MCSs). However, many hits uncovered through our BioID experiment warrant further investigation in order to get a better picture of the potential additional roles of PERK. In this report, we reveal the full dataset of PERK interacting proteins identified through BioID analysis. We further confirmed by IP/Co-IP analysis, the physical interaction between PERK and sarcoplasmic/endoplasmic reticulum Ca ATPase 2 (SERCA2) and VAMP-associated protein A / B (VAPA/B) and show that for these interactions, PERK kinase activity is dispensable.

Results and Discussion

The PERK Proximity Interactome

We generated a C-terminally tagged PERK-BirA* construct that was well expressed in HEK293-T cells and showed the expected ER localization (van Vliet et al., 2017). Our BioID approach was designed and performed to closely match the original study reporting BioID (Roux et al., 2012). Similar to that study, our approach relied on the expression of PERK-BirA* in HEK293-T cells, using mock-transfected parental HEK293-T cells treated in the same way (50 µM biotin for 24 h), as a control. Only biotinylated protein hits identified by LC-MS/MS in the streptavidin pulldown from PERK-BirA* transfected cells and not from mock-transfected cells were taken into consideration as putative interaction partners. Table 1 shows a list of PERK proximity interactors showing no spectral counts in the pulldown from mock-transfected cells. Interestingly, while our previous research focused on one of the most prominent hits, Filamin A, which we further validated functionally, the BioID dataset showed several other potential interacting proteins (Table 1), ranging from more proteins involved in actin cytoskeleton maintenance (cofilin, profilin,...), proteins involved in ER trafficking (syntaxin 5, coatomer subunits), proteins involved in membrane contact sites (VAPA/B, E-Syt1, junctophilin) to proteins linked with metabolism (ATP-citrate synthase, acyl-protein thioesterase 2). Interestingly, PERK has been linked recently with metabolic regulatory networks (Balsa et al., 2019; Moncan et al., 2021; Sorge et al., 2020), although whether these potential interactors uncovered by our BioID are relevant remains to be tested. To gain more insight into the various groups of proteins in the dataset, we performed a gene ontology analysis using WEB-based GEne SeT AnaLysis Toolkit (http://www.webgestalt.org/, Figure 1). This analysis yielded a set of enriched biological processes in our dataset. As a validation of our approach, two of these groups are linked to PERK's main role in protein folding and ER stress response (Figure 1A and B).

Table 1.

List of identified proteins (Scaffold, FDR < 1%) resulting from the BioID interactome screen using PERK-BirA as bait. Protein hits were only detected using PERK-BirA as bait and not in control. Parental cells are shaded in yellow. Relative quantification of proteins is based on spectral counts (‘Total spectra’). Only proteins with atleast 2 exclusive unique peptides per protein are listed.

Molecular weight Quantitative value (total spectra) Exclusive unique peptide count Quantitative value (total spectra) Exclusive unique peptide count Protein identification probability Protein identification probability
Identified proteins (276) Accession number Alternate ID PERK-BirA PERK-BirA Control Control PERK-BirA Control
Eukaryotic translation initiation factor 2-alpha kinase 3 OS = Homo sapiens OX = 9606 GN = EIF2AK3 PE = 1 SV = 3 Q9NZJ5 EIF2AK3 125 kDa 415 70 0 0 100% 0
Filamin-A OS = Homo sapiens OX = 9606 GN = FLNA PE = 1 SV = 4 P21333 FLNA 281 kDa 94 66 0 0 100% 0
Coatomer subunit gamma-2 OS = Homo sapiens OX = 9606 GN = COPG2 PE = 1 SV = 1 Q9UBF2 COPG2 98 kDa 40 26 0 0 100% 0
Lamina-associated polypeptide 2, isoforms beta/gamma OS = Homo sapiens OX = 9606 GN = TMPO PE = 1 SV = 2 P42167 TMPO 51 kDa 28 16 0 0 100% 0
Keratin, type I cytoskeletal 16 OS = Homo sapiens OX = 9606 GN = KRT16 PE = 1 SV = 4 P08779 KRT16 51 kDa 26 4 0 0 100% 0
Kinectin OS = Homo sapiens OX = 9606 GN = KTN1 PE = 1 SV = 1 Q86UP2 KTN1 156 kDa 24 23 0 0 100% 0
RuvB-like 1 OS = Homo sapiens OX = 9606 GN = RUVBL1 PE = 1 SV = 1 Q9Y265 RUVBL1 50 kDa 20 13 0 0 100% 0
E3 SUMO-protein ligase RanBP2 OS = Homo sapiens OX = 9606 GN = RANBP2 PE = 1 SV = 2 P49792 RANBP2 358 kDa 20 19 0 0 100% 0
78 kDa glucose-regulated protein OS = Homo sapiens GN = HSPA5 PE = 1 SV = 2 P11021| GRP78_HUMAN HSPA5 72 kDa 16 13 0 0 100% 0
Double-strand break repair protein MRE11 OS = Homo sapiens OX = 9606 GN = MRE11 PE = 1 SV = 3 P49959 MRE11 81 kDa 15 14 0 0 100% 0
Zinc finger CCCH-type antiviral protein 1 OS = Homo sapiens OX = 9606 GN = ZC3HAV1 PE = 1 SV = 3 Q7Z2W4 ZC3HAV1 101 kDa 14 10 0 0 100% 0
Src substrate cortactin OS = Homo sapiens OX = 9606 GN = CTTN PE = 1 SV = 2 Q14247 CTTN 62 kDa 14 12 0 0 100% 0
Bifunctional glutamate/proline--tRNA ligase OS = Homo sapiens OX = 9606 GN = EPRS PE = 1 SV = 5 P07814 EPRS 171 kDa 12 11 0 0 100% 0
Lamin-B receptor OS = Homo sapiens OX = 9606 GN = LBR PE = 1 SV = 2 Q14739 LBR 71 kDa 11 7 0 0 100% 0
Transgelin-2 OS = Homo sapiens OX = 9606 GN = TAGLN2 PE = 1 SV = 3 P37802 TAGLN2 22 kDa 10 7 0 0 100% 0
eIF-2-alpha kinase activator GCN1 OS = Homo sapiens OX = 9606 GN = GCN1 PE = 1 SV = 6 Q92616 GCN1 293 kDa 10 10 0 0 100% 0
UBX domain-containing protein 4 OS = Homo sapiens OX = 9606 GN = UBXN4 PE = 1 SV = 2 Q92575 UBXN4 57 kDa 10 6 0 0 100% 0
Staphylococcal nuclease domain-containing protein 1 OS = Homo sapiens OX = 9606 GN = SND1 PE = 1 SV = 1 Q7KZF4 SND1 102 kDa 9 9 0 0 100% 0
Vesicle-associated membrane protein-associated protein A OS = Homo sapiens OX = 9606 GN = VAPA PE = 1 SV = 3 Q9P0L0 VAPA 28 kDa 8 4 0 0 100% 0
Histone H1.5 OS = Homo sapiens OX = 9606 GN = HIST1H1B PE = 1 SV = 3 P16401 HIST1H1B 23 kDa 7 3 0 0 100% 0
Sodium/potassium-transporting ATPase subunit alpha-1 OS = Homo sapiens OX = 9606 GN = ATP1A1 PE = 1 SV = 1 P05023 ATP1A1 113 kDa 7 7 0 0 100% 0
Eukaryotic translation initiation factor 5 OS = Homo sapiens OX = 9606 GN = EIF5 PE = 1 SV = 2 P55010 EIF5 49 kDa 7 6 0 0 100% 0
Cytoskeleton-associated protein 4 OS = Homo sapiens OX = 9606 GN = CKAP4 PE = 1 SV = 2 Q07065 CKAP4 66 kDa 7 7 0 0 100% 0
Protein ELYS OS = Homo sapiens OX = 9606 GN = AHCTF1 PE = 1 SV = 3 Q8WYP5 AHCTF1 253 kDa 7 7 0 0 100% 0
MKL/myocardin-like protein 2 OS = Homo sapiens OX = 9606 GN = MKL2 PE = 1 SV = 3 Q9ULH7 MKL2 118 kDa 7 7 0 0 100% 0
Neuroblast differentiation-associated protein AHNAK OS = Homo sapiens OX = 9606 GN = AHNAK PE = 1 SV = 2 Q09666 AHNAK 629 kDa 7 7 0 0 93% 0
Protein disulfide-isomerase A4 OS = Homo sapiens OX = 9606 GN = PDIA4 PE = 1 SV = 2 P13667 PDIA4 73 kDa 6 5 0 0 100% 0
Coronin-1B OS = Homo sapiens OX = 9606 GN = CORO1B PE = 1 SV = 1 Q9BR76 CORO1B 54 kDa 6 6 0 0 100% 0
Splicing factor, proline- and glutamine-rich OS = Homo sapiens OX = 9606 GN = SFPQ PE = 1 SV = 2 P23246 SFPQ 76 kDa 6 4 0 0 100% 0
Profilin-1 OS = Homo sapiens OX = 9606 GN = PFN1 PE = 1 SV = 2 P07737 PFN1 15 kDa 6 6 0 0 100% 0
Eukaryotic translation initiation factor 4 gamma 2 OS = Homo sapiens OX = 9606 GN = EIF4G2 PE = 1 SV = 1 P78344 EIF4G2 102 kDa 6 6 0 0 100% 0
Coiled-coil domain-containing protein 47 OS = Homo sapiens OX = 9606 GN = CCDC47 PE = 1 SV = 1 Q96A33 CCDC47 56 kDa 6 6 0 0 100% 0
Endoplasmin OS = Homo sapiens OX = 9606 GN = HSP90B1 PE = 1 SV = 1 P14625 HSP90B1 92 kDa 6 6 0 0 100% 0
Eukaryotic translation initiation factor 4B OS = Homo sapiens OX = 9606 GN = EIF4B PE = 1 SV = 2 P23588 EIF4B 69 kDa 6 6 0 0 100% 0
Multifunctional protein ADE2 OS = Homo sapiens OX = 9606 GN = PAICS PE = 1 SV = 3 P22234 PAICS 47 kDa 6 5 0 0 100% 0
Torsin-1A-interacting protein 1 OS = Homo sapiens OX = 9606 GN = TOR1AIP1 PE = 1 SV = 2 Q5JTV8 TOR1AIP1 66 kDa 6 6 0 0 100% 0
Threonylcarbamoyladenosine tRNA methylthiotransferase OS = Homo sapiens OX = 9606 GN = CDKAL1 PE = 1 SV = 1 Q5VV42 CDKAL1 65 kDa 6 5 0 0 100% 0
Receptor of activated protein C kinase 1 OS = Homo sapiens OX = 9606 GN = RACK1 PE = 1 SV = 3 P63244 RACK1 35 kDa 6 5 0 0 100% 0
Membrane-associated progesterone receptor component 2 OS = Homo sapiens OX = 9606 GN = PGRMC2 PE = 1 SV = 1 O15173 PGRMC2 24 kDa 5 3 0 0 100% 0
Chromodomain-helicase-DNA-binding protein 4 OS = Homo sapiens OX = 9606 GN = CHD4 PE = 1 SV = 2 Q14839 CHD4 218 kDa 5 5 0 0 100% 0
Afadin OS = Homo sapiens OX = 9606 GN = AFDN PE = 1 SV = 3 P55196 AFDN 207 kDa 5 5 0 0 100% 0
Adapter molecule crk OS = Homo sapiens OX = 9606 GN = CRK PE = 1 SV = 2 P46108 CRK 34 kDa 5 5 0 0 100% 0
Nucleophosmin OS = Homo sapiens OX = 9606 GN = NPM1 PE = 1 SV = 2 P06748 NPM1 33 kDa 5 5 0 0 100% 0
Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 OS = Homo sapiens OX = 9606 GN = ATP2A2 PE = 1 SV = 1 P16615 ATP2A2 115 kDa 5 5 0 0 100% 0
Peroxiredoxin-4 OS = Homo sapiens OX = 9606 GN = PRDX4 PE = 1 SV = 1 Q13162 PRDX4 31 kDa 5 2 0 0 100% 0
Eukaryotic translation initiation factor 4 gamma 1 OS = Homo sapiens OX = 9606 GN = EIF4G1 PE = 1 SV = 4 Q04637 EIF4G1 175 kDa 5 4 0 0 100% 0
Cytoskeleton-associated protein 5 OS = Homo sapiens OX = 9606 GN = CKAP5 PE = 1 SV = 3 Q14008 CKAP5 226 kDa 4 4 0 0 100% 0
PEST proteolytic signal-containing nuclear protein OS = Homo sapiens OX = 9606 GN = PCNP PE = 1 SV = 2 Q8WW12 PCNP 19 kDa 4 2 0 0 98% 0
Desmoplakin OS = Homo sapiens OX = 9606 GN = DSP PE = 1 SV = 3 P15924 DSP 332 kDa 4 4 0 0 100% 0
Ubiquitin-like modifier-activating enzyme 1 OS = Homo sapiens OX = 9606 GN = UBA1 PE = 1 SV = 3 P22314 UBA1 118 kDa 4 4 0 0 100% 0
Fatty aldehyde dehydrogenase OS = Homo sapiens OX = 9606 GN = ALDH3A2 PE = 1 SV = 1 P51648 ALDH3A2 55 kDa 4 4 0 0 100% 0
Creatine kinase B-type OS = Homo sapiens OX = 9606 GN = CKB PE = 1 SV = 1 P12277 CKB 43 kDa 4 4 0 0 100% 0
Syntaxin-5 OS = Homo sapiens OX = 9606 GN = STX5 PE = 1 SV = 2 Q13190 STX5 40 kDa 4 4 0 0 100% 0
Rab-like protein 3 OS = Homo sapiens OX = 9606 GN = RABL3 PE = 1 SV = 1 Q5HYI8 RABL3 26 kDa 4 3 0 0 100% 0
Regulator of chromosome condensation OS = Homo sapiens OX = 9606 GN = RCC1 PE = 1 SV = 1 P18754 RCC1 45 kDa 4 4 0 0 100% 0
Ran GTPase-activating protein 1 OS = Homo sapiens OX = 9606 GN = RANGAP1 PE = 1 SV = 1 P46060 RANGAP1 64 kDa 4 4 0 0 100% 0
Synaptobrevin homolog YKT6 OS = Homo sapiens OX = 9606 GN = YKT6 PE = 1 SV = 1 O15498 YKT6 22 kDa 4 3 0 0 100% 0
Cold shock domain-containing protein E1 OS = Homo sapiens OX = 9606 GN = CSDE1 PE = 1 SV = 2 O75534 CSDE1 89 kDa 4 4 0 0 100% 0
Vesicle-associated membrane protein-associated protein B/C OS = Homo sapiens OX = 9606 GN = VAPB PE = 1 SV = 3 O95292 VAPB 27 kDa 4 2 0 0 99% 0
Vigilin OS = Homo sapiens OX = 9606 GN = HDLBP PE = 1 SV = 2 Q00341 HDLBP 141 kDa 4 4 0 0 99% 0
Microtubule-associated protein 4 OS = Homo sapiens OX = 9606 GN = MAP4 PE = 1 SV = 3 P27816 MAP4 121 kDa 4 3 0 0 100% 0
TATA-binding protein-associated factor 2N OS = Homo sapiens OX = 9606 GN = TAF15 PE = 1 SV = 1 Q92804 TAF15 62 kDa 3 2 0 0 84% 0
Peptidyl-prolyl cis-trans isomerase A OS = Homo sapiens OX = 9606 GN = PPIA PE = 1 SV = 2 P62937 PPIA 18 kDa 3 3 0 0 100% 0
CAD protein OS = Homo sapiens OX = 9606 GN = CAD PE = 1 SV = 3 P27708 CAD 243 kDa 3 3 0 0 100% 0
Synapse-associated protein 1 OS = Homo sapiens OX = 9606 GN = SYAP1 PE = 1 SV = 1 Q96A49 SYAP1 40 kDa 3 3 0 0 100% 0
Leucine-rich repeat-containing protein 59 OS = Homo sapiens OX = 9606 GN = LRRC59 PE = 1 SV = 1 Q96AG4 LRRC59 35 kDa 3 3 0 0 100% 0
WW domain-containing oxidoreductase OS = Homo sapiens OX = 9606 GN = WWOX PE = 1 SV = 1 Q9NZC7 WWOX 47 kDa 3 3 0 0 99% 0
D-3-phosphoglycerate dehydrogenase OS = Homo sapiens OX = 9606 GN = PHGDH PE = 1 SV = 4 O43175 PHGDH 57 kDa 3 3 0 0 100% 0
40S ribosomal protein S3 OS = Homo sapiens OX = 9606 GN = RPS3 PE = 1 SV = 2 P23396 RPS3 27 kDa 3 3 0 0 100% 0
Voltage-dependent anion-selective channel protein 2 OS = Homo sapiens OX = 9606 GN = VDAC2 PE = 1 SV = 2 P45880 VDAC2 32 kDa 3 3 0 0 100% 0
Band 4.1-like protein 3 OS = Homo sapiens OX = 9606 GN = EPB41L3 PE = 1 SV = 2 Q9Y2J2 EPB41L3 121 kDa 3 3 0 0 100% 0
Junctophilin-1 OS = Homo sapiens OX = 9606 GN = JPH1 PE = 1 SV = 2 Q9HDC5 JPH1 72 kDa 3 2 0 0 100% 0
Emerin OS = Homo sapiens OX = 9606 GN = EMD PE = 1 SV = 1 P50402 EMD 29 kDa 3 2 0 0 100% 0
Thioredoxin-dependent peroxide reductase, mitochondrial OS = Homo sapiens OX = 9606 GN = PRDX3 PE = 1 SV = 3 P30048 PRDX3 28 kDa 3 3 0 0 100% 0
Eukaryotic translation initiation factor 5B OS = Homo sapiens OX = 9606 GN = EIF5B PE = 1 SV = 4 O60841 EIF5B 139 kDa 3 3 0 0 99% 0
Clathrin heavy chain 1 OS = Homo sapiens OX = 9606 GN = CLTC PE = 1 SV = 5 Q00610 CLTC 192 kDa 3 3 0 0 99% 0
Stromal interaction molecule 1 OS = Homo sapiens OX = 9606 GN = STIM1 PE = 1 SV = 3 Q13586 STIM1 77 kDa 3 2 0 0 98% 0
Splicing factor 3B subunit 3 OS = Homo sapiens OX = 9606 GN = SF3B3 PE = 1 SV = 4 Q15393 SF3B3 136 kDa 3 3 0 0 96% 0
Nuclear migration protein nudC OS = Homo sapiens OX = 9606 GN = NUDC PE = 1 SV = 1 Q9Y266 NUDC 38 kDa 3 3 0 0 93% 0
Proliferation marker protein Ki-67 OS = Homo sapiens OX = 9606 GN = MKI67 PE = 1 SV = 2 P46013 MKI67 359 kDa 3 2 0 0 84% 0
Chloride channel CLIC-like protein 1 OS = Homo sapiens OX = 9606 GN = CLCC1 PE = 1 SV = 1 Q96S66 CLCC1 62 kDa 3 3 0 0 99% 0
Far upstream element-binding protein 2 OS = Homo sapiens OX = 9606 GN = KHSRP PE = 1 SV = 4 Q92945 KHSRP 73 kDa 3 3 0 0 96% 0
Clathrin interactor 1 OS = Homo sapiens OX = 9606 GN = CLINT1 PE = 1 SV = 1 Q14677 CLINT1 68 kDa 3 3 0 0 91% 0
Eukaryotic translation initiation factor 2A OS = Homo sapiens OX = 9606 GN = EIF2A PE = 1 SV = 3 Q9BY44 EIF2A 65 kDa 3 3 0 0 61% 0
Junction plakoglobin OS = Homo sapiens OX = 9606 GN = JUP PE = 1 SV = 3 P14923 JUP 82 kDa 3 3 0 0 40% 0
Pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 OS = Homo sapiens OX = 9606 GN = DHX15 PE = 1 SV = 2 O43143 DHX15 91 kDa 2 2 0 0 100% 0
T-complex protein 1 subunit zeta OS = Homo sapiens OX = 9606 GN = CCT6A PE = 1 SV = 3 P40227 CCT6A 58 kDa 2 2 0 0 100% 0
Cytoplasmic dynein 1 heavy chain 1 OS = Homo sapiens OX = 9606 GN = DYNC1H1 PE = 1 SV = 5 Q14204 DYNC1H1 532 kDa 2 2 0 0 100% 0
60S ribosomal protein L15 OS = Homo sapiens OX = 9606 GN = RPL15 PE = 1 SV = 2 P61313 RPL15 24 kDa 2 2 0 0 100% 0
Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3B OS = Homo sapiens OX = 9606 GN = STT3B PE = 1 SV = 1 Q8TCJ2 STT3B 94 kDa 2 2 0 0 100% 0
Protein SGT1 homolog OS = Homo sapiens OX = 9606 GN = SUGT1 PE = 1 SV = 3 Q9Y2Z0 SUGT1 41 kDa 2 2 0 0 100% 0
Protein 4.1 OS = Homo sapiens OX = 9606 GN = EPB41 PE = 1 SV = 4 P11171 EPB41 97 kDa 2 2 0 0 100% 0
Heterogeneous nuclear ribonucleoprotein D0 OS = Homo sapiens OX = 9606 GN = HNRNPD PE = 1 SV = 1 Q14103 HNRNPD 38 kDa 2 2 0 0 100% 0
Chloride intracellular channel protein 1 OS = Homo sapiens OX = 9606 GN = CLIC1 PE = 1 SV = 4 O00299 CLIC1 27 kDa 2 2 0 0 100% 0
Signal recognition particle subunit SRP68 OS = Homo sapiens OX = 9606 GN = SRP68 PE =1 SV= 2 Q9UHB9 SRP68 71 kDa 2 2 0 0 100% 0
Signal recognition particle 54 kDa protein OS = Homo sapiens OX= 9606 GN= SRP54 PE = 1 SV = 1 P61011 SRP54 56 kDa 2 2 0 0 100% 0
Polyadenylate-binding protein 1 OS = Homo sapiens OX = 9606 GN = PABPC1 PE = 1 SV = 2 P11940 PABPC1 71 kDa 2 2 0 0 100% 0
T-complex protein 1 subunit epsilon OS = Homo sapiens OX = 9606 GN = CCT5 PE = 1 SV = 1 P48643 CCT5 60 kDa 2 2 0 0 100% 0
C-1-tetrahydrofolate synthase, cytoplasmic OS = Homo sapiens OX = 9606 GN = MTHFD1 PE = 1 SV = 3 P11586 MTHFD1 102 kDa 2 2 0 0 100% 0
Voltage-dependent anion-selective channel protein 3 OS = Homo sapiens OX = 9606 GN = VDAC3 PE = 1 SV = 1 Q9Y277 VDAC3 31 kDa 2 2 0 0 100% 0
RNA-binding protein 26 OS = Homo sapiens OX = 9606 GN = RBM26 PE = 1 SV = 3 Q5T8P6 RBM26 114 kDa 2 2 0 0 99% 0
Ran-binding protein 3 OS = Homo sapiens OX = 9606 GN = RANBP3 PE = 1 SV = 1 Q9H6Z4 RANBP3 60 kDa 2 2 0 0 99% 0
60S ribosomal protein L10 OS = Homo sapiens OX = 9606 GN = RPL10 PE = 1 SV = 4 P27635 RPL10 25 kDa 2 2 0 0 99% 0
Protein RCC2 OS = Homo sapiens OX = 9606 GN = RCC2 PE = 1 SV = 2 Q9P258 RCC2 56 kDa 2 2 0 0 98% 0
Extended synaptotagmin-1 OS = Homo sapiens OX = 9606 GN = ESYT1 PE = 1 SV = 1 Q9BSJ8 ESYT1 123 kDa 2 2 0 0 97% 0
PC4 and SFRS1-interacting protein OS = Homo sapiens OX = 9606 GN = PSIP1 PE = 1 SV = 1 O75475 PSIP1 60 kDa 2 2 0 0 94% 0
SAFB-like transcription modulator OS = Homo sapiens OX = 9606 GN = SLTM PE = 1 SV = 2 Q9NWH9 SLTM 117 kDa 2 2 0 0 92% 0
Probable rRNA-processing protein EBP2 OS = Homo sapiens OX = 9606 GN = EBNA1BP2 PE = 1 SV = 2 Q99848 EBNA1BP2 35 kDa 2 2 0 0 80% 0
Cysteine and histidine-rich domain-containing protein 1 OS = Homo sapiens OX = 9606 GN = CHORDC1 PE = 1 SV = 2 Q9UHD1 CHORDC1 37 kDa 2 2 0 0 79% 0
ATP-dependent RNA helicase DDX39A OS = Homo sapiens OX = 9606 GN = DDX39A PE = 1 SV = 2 O00148 (+1) DDX39A 49 kDa 2 2 0 0 100% 0
Heterogeneous nuclear ribonucleoprotein H3 OS = Homo sapiens OX = 9606 GN = HNRNPH3 PE = 1 SV = 2 P31942 HNRNPH3 37 kDa 2 2 0 0 100% 0
Peroxisomal multifunctional enzyme type 2 OS = Homo sapiens OX = 9606 GN = HSD17B4 PE = 1 SV = 3 P51659 HSD17B4 80 kDa 2 2 0 0 98% 0
Eukaryotic translation initiation factor 3 subunit D OS = Homo sapiens OX = 9606 GN = EIF3D PE = 1 SV = 1 O15371 EIF3D 64 kDa 2 2 0 0 96% 0
Tyrosine-protein phosphatase non-receptor type 1 OS = Homo sapiens OX = 9606 GN = PTPN1 PE = 1 SV = 1 P18031 PTPN1 50 kDa 2 2 0 0 92% 0
Jupiter microtubule associated homolog 2 OS = Homo sapiens OX = 9606 GN = JPT2 PE = 1 SV = 1 Q9H910 JPT2 20 kDa 2 2 0 0 90% 0
U5 small nuclear ribonucleoprotein 200 kDa helicase OS = Homo sapiens OX = 9606 GN = SNRNP200 PE = 1 SV = 2 O75643 SNRNP200 245 kDa 2 2 0 0 89% 0
PDZ and LIM domain protein 5 OS = Homo sapiens OX = 9606 GN = PDLIM5 PE = 1 SV = 5 Q96HC4 PDLIM5 64 kDa 2 2 0 0 86% 0
Coatomer subunit alpha OS = Homo sapiens OX = 9606 GN = COPA PE = 1 SV = 2 P53621 COPA 138 kDa 2 2 0 0 86% 0
Activator of 90 kDa heat shock protein ATPase homolog 1 OS = Homo sapiens OX = 9606 GN = AHSA1 PE = 1 SV = 1 O95433 AHSA1 38 kDa 2 2 0 0 76% 0
Asparagine synthetase [glutamine-hydrolyzing] OS = Homo sapiens OX = 9606 GN = ASNS PE = 1 SV = 4 P08243 ASNS 64 kDa 2 2 0 0 69% 0
Transitional endoplasmic reticulum ATPase OS = Homo sapiens OX = 9606 GN = VCP PE = 1 SV = 4 P55072 VCP 89 kDa 2 2 0 0 62% 0
RNA cytidine acetyltransferase OS = Homo sapiens OX = 9606 GN = NAT10 PE = 1 SV = 2 Q9H0A0 NAT10 116 kDa 2 2 0 0 54% 0
Protein arginine N-methyltransferase 5 OS = Homo sapiens OX = 9606 GN = PRMT5 PE = 1 SV = 4 O14744 PRMT5 73 kDa 2 2 0 0 38% 0
60S ribosomal protein L38 OS = Homo sapiens OX = 9606 GN = RPL38 PE = 1 SV = 2 P63173 RPL38 8 kDa 2 2 0 0 37% 0
Angiomotin OS = Homo sapiens OX = 9606 GN = AMOT PE = 1 SV = 1 Q4VCS5 AMOT 118 kDa 2 2 0 0 29% 0
Segment polarity protein dishevelled homolog DVL-2 OS = Homo sapiens OX = 9606 GN = DVL2 PE = 1 SV = 1 O14641 DVL2 79 kDa 2 2 0 0 21% 0
DDRGK domain-containing protein 1 OS = Homo sapiens OX = 9606 GN = DDRGK1 PE = 1 SV = 2 Q96HY6 DDRGK1 36 kDa 2 2 0 0 41% 0
Calponin-3 OS = Homo sapiens OX = 9606 GN = CNN3 PE = 1 SV = 1 Q15417 CNN3 36 kDa 2 2 0 0 19% 0
Synaptosomal-associated protein 29 OS = Homo sapiens OX = 9606 GN = SNAP29 PE = 1 SV = 1 O95721 SNAP29 29 kDa 2 2 0 0 16% 0
T-complex protein 1 subunit theta OS = Homo sapiens OX = 9606 GN = CCT8 PE = 1 SV = 4 P50990 CCT8 60 kDa 58 32 1 1 100% 55%
Fatty acid synthase OS = Homo sapiens OX = 9606 GN = FASN PE = 1 SV = 3 P49327 FASN 273 kDa 32 27 1 1 100% 82%
Protein LYRIC OS = Homo sapiens OX = 9606 GN = MTDH PE = 1 SV = 2 Q86UE4 MTDH 64 kDa 24 15 1 1 100% 11%
Plasminogen activator inhibitor 1 RNA-binding protein OS = Homo sapiens OX = 9606 GN = SERBP1 PE = 1 SV = 2 Q8NC51 SERBP1 45 kDa 12 8 1 1 100% 77%
Ubiquitin-40S ribosomal protein S27a OS = Homo sapiens OX = 9606 GN = RPS27A PE = 1 SV = 2 P62979 RPS27A 18 kDa 16 7 1 1 100% 15%
Heterogeneous nuclear ribonucleoprotein L OS = Homo sapiens OX = 9606 GN = HNRNPL PE = 1 SV = 2 P14866 HNRNPL 64 kDa 9 9 1 1 100% 94%
Poly(rC)-binding protein 1 OS = Homo sapiens OX = 9606 GN = PCBP1 PE = 1 SV = 2 Q15365 PCBP1 37 kDa 7 5 1 1 100% 98%
Eukaryotic initiation factor 4A-I OS = Homo sapiens OX = 9606 GN = EIF4A1 PE = 1 SV = 1 P60842 EIF4A1 46 kDa 6 6 1 1 100% 81%
Heterogeneous nuclear ribonucleoprotein A3 OS = Homo sapiens OX = 9606 GN = HNRNPA3 PE = 1 SV = 2 P51991 HNRNPA3 40 kDa 5 5 1 1 100% 98%
GTP-binding nuclear protein Ran OS = Homo sapiens OX = 9606 GN = RAN PE = 1 SV = 3 P62826 RAN 24 kDa 5 5 1 1 100% 93%
ADP/ATP translocase 2 OS = Homo sapiens OX = 9606 GN = SLC25A5 PE = 1 SV = 7 P05141 SLC25A5 33 kDa 4 4 1 1 100% 92%
Splicing regulatory glutamine/lysine-rich protein 1 OS = Homo sapiens OX = 9606 GN = SREK1 PE = 1 SV = 1 Q8WXA9 SREK1 59 kDa 4 4 1 1 100% 47%
60S ribosomal protein L12 OS = Homo sapiens OX = 9606 GN = RPL12 PE = 1 SV = 1 P30050 RPL12 18 kDa 3 2 1 1 100% 76%
Fructose-bisphosphate aldolase A OS = Homo sapiens OX = 9606 GN = ALDOA PE = 1 SV = 2 P04075 ALDOA 39 kDa 4 4 1 1 100% 20%
60S ribosomal protein L3 OS = Homo sapiens OX = 9606 GN = RPL3 PE = 1 SV = 2 P39023 RPL3 46 kDa 5 4 1 1 100% 8%
Heterogeneous nuclear ribonucleoprotein H OS = Homo sapiens OX = 9606 GN = HNRNPH1 PE = 1 SV = 4 P31943 HNRNPH1 49 kDa 3 3 1 1 100% 98%
Desmoglein-1 OS = Homo sapiens OX = 9606 GN = DSG1 PE = 1 SV = 2 Q02413 DSG1 114 kDa 2 2 1 1 100% 97%
40S ribosomal protein S2 OS = Homo sapiens OX = 9606 GN = RPS2 PE = 1 SV = 2 P15880 RPS2 31 kDa 2 2 1 1 100% 96%
Dermcidin OS = Homo sapiens OX = 9606 GN = DCD PE = 1 SV = 2 P81605 DCD 11 kDa 2 2 1 1 100% 94%
40S ribosomal protein S16 OS = Homo sapiens OX = 9606 GN = RPS16 PE = 1 SV = 2 P62249 RPS16 16 kDa 2 2 1 1 100% 92%
Heterogeneous nuclear ribonucleoprotein M OS = Homo sapiens OX = 9606 GN = HNRNPM PE = 1 SV = 3 P52272 HNRNPM 78 kDa 2 2 1 1 100% 92%
Serine/arginine-rich splicing factor 7 OS = Homo sapiens OX = 9606 GN = SRSF7 PE = 1 SV = 1 Q16629 SRSF7 27 kDa 3 3 1 1 99% 77%
40S ribosomal protein S15a OS = Homo sapiens OX = 9606 GN = RPS15A PE = 1 SV = 2 P62244 RPS15A 15 kDa 3 3 1 1 100% 74%
T-complex protein 1 subunit alpha OS = Homo sapiens OX = 9606 GN = TCP1 PE = 1 SV = 1 P17987 TCP1 60 kDa 3 3 1 1 100% 56%
Pre-mRNA-processing factor 40 homolog A OS = Homo sapiens OX = 9606 GN = PRPF40A PE = 1 SV = 2 O75400 PRPF40A 109 kDa 3 3 1 1 99% 50%
Glyceraldehyde-3-phosphate dehydrogenase OS = Homo sapiens OX = 9606 GN = GAPDH PE = 1 SV = 3 P04406 GAPDH 36 kDa 2 2 1 1 100% 31%
RNA-binding protein 39 OS = Homo sapiens OX = 9606 GN = RBM39 PE = 1 SV = 2 Q14498 RBM39 59 kDa 3 3 1 1 99% 28%
Probable ATP-dependent RNA helicase DDX46 OS = Homo sapiens OX = 9606 GN = DDX46 PE = 1 SV = 2 Q7L014 DDX46 117 kDa 3 3 1 1 100% 8%
Filaggrin-2 OS = Homo sapiens OX = 9606 GN = FLG2 PE = 1 SV = 1 Q5D862 FLG2 248 kDa 3 3 1 1 85% 23%
Cofilin-1 OS = Homo sapiens OX = 9606 GN = CFL1 PE = 1 SV = 3 P23528 CFL1 19 kDa 3 3 1 1 100% 7%
Heterogeneous nuclear ribonucleoprotein A/B OS = Homo sapiens OX = 9606 GN = HNRNPAB PE = 1 SV = 2 Q99729 HNRNPAB 36 kDa 2 2 1 1 98% 98%
Pre-mRNA-processing-splicing factor 8 OS = Homo sapiens OX = 9606 GN = PRPF8 PE = 1 SV = 2 Q6P2Q9 PRPF8 274 kDa 4 4 1 1 85% 31%
Elongation factor Tu, mitochondrial OS = Homo sapiens OX = 9606 GN = TUFM PE = 1 SV = 2 P49411 TUFM 50 kDa 9 8 2 2 100% 95%
RNA-binding protein FUS OS = Homo sapiens OX = 9606 GN = FUS PE = 1 SV = 1 P35637 FUS 53 kDa 11 6 2 2 100% 30%
Peroxiredoxin-2 OS = Homo sapiens OX = 9606 GN = PRDX2 PE = 1 SV = 5 P32119 PRDX2 22 kDa 6 3 2 1 100% 73%
ADP-ribosylation factor-like protein 6-interacting protein 4 OS = Homo sapiens OX = 9606 GN = ARL6IP4 PE = 1 SV= 2 Q66PJ3 ARL6IP4 45 kDa 6 4 2 1 100% 98%
Nucleolar RNA helicase 2 OS = Homo sapiens OX = 9606 GN = DDX21 PE= 1 SV = 5 Q9NR30 DDX21 87 kDa 7 7 2 2 100% 96%
Fibronectin OS = Homo sapiens OX = 9606 GN = FN1 PE = 1 SV = 4 P02751 FN1 263 kDa 4 3 2 2 100% 100%
Serine/arginine-rich splicing factor 1 OS = Homo sapiens OX = 9606 GN = SRSF1 PE = 1 SV = 2 Q07955 SRSF1 28 kDa 5 4 2 2 100% 66%
Serine/threonine-protein kinase PRP4 homolog OS = Homo sapiens OX = 9606 GN = PRPF4B PE = 1 SV = 3 Q13523 PRPF4B 117 kDa 4 4 2 2 100% 100%
Nucleolar protein 58 OS = Homo sapiens OX = 9606 GN = NOP58 PE = 1 SV = 1 Q9Y2X3 NOP58 60 kDa 4 4 2 2 100% 100%
Alpha-enolase OS = Homo sapiens OX = 9606 GN = ENO1 PE = 1 SV = 2 P06733 ENO1 47 kDa 4 3 2 2 100% 98%
Histone H2A type 1 OS = Homo sapiens OX = 9606 GN = HIST1H2AG PE = 1 SV = 2 P0C0S8 (+6) HIST1H2AG 14 kDa 2 2 2 2 100% 100%
Serine/arginine-rich splicing factor 2 OS = Homo sapiens OX = 9606 GN = SRSF2 PE = 1 SV = 4 Q01130 SRSF2 25 kDa 2 2 2 2 100% 100%
60S ribosomal protein L36a OS = Homo sapiens OX = 9606 GN = RPL36A PE = 1 SV = 2 P83881 (+1) RPL36A 12 kDa 2 2 2 2 99% 93%
Poly(rC)-binding protein 2 OS = Homo sapiens OX = 9606 GN = PCBP2 PE = 1 SV = 1 Q15366 PCBP2 39 kDa 4 1 2 2 40% 41%
Proliferation-associated protein 2G4 OS = Homo sapiens OX = 9606 GN = PA2G4 PE = 1 SV = 3 Q9UQ80 PA2G4 44 kDa 4 4 2 2 100% 71%
40S ribosomal protein S26 OS = Homo sapiens OX = 9606 GN = RPS26 PE = 1 SV = 3 P62854 RPS26 13 kDa 2 2 2 1 100% 67%
Keratinocyte proline-rich protein OS = Homo sapiens OX = 9606 GN = KPRP PE = 1 SV = 1 Q5T749 KPRP 64 kDa 2 2 2 2 73% 85%
ATP-dependent RNA helicase DDX3X OS = Homo sapiens OX = 9606 GN = DDX3X PE = 1 SV = 3 O00571 DDX3X 73 kDa 2 2 2 2 100% 27%
ATP-citrate synthase OS = Homo sapiens OX = 9606 GN = ACLY PE = 1 SV = 3 P53396 ACLY 121 kDa 5 5 2 2 100% 16%
60S ribosomal protein L4 OS = Homo sapiens OX = 9606 GN = RPL4 PE = 1 SV = 5 P36578 RPL4 48 kDa 3 3 2 2 100% 15%
60S ribosomal protein L7a OS = Homo sapiens OX = 9606 GN = RPL7A PE = 1 SV = 2 P62424 RPL7A 30 kDa 2 2 2 1 92% 10%
60S ribosomal protein L8 OS = Homo sapiens OX = 9606 GN = RPL8 PE = 1 SV = 2 P62917 RPL8 28 kDa 1 1 2 2 83% 98%
Uncharacterized protein NKAPD1 OS = Homo sapiens OX = 9606 GN = NKAPD1 PE = 1 SV = 2 Q6ZUT1 NKAPD1 34 kDa 2 2 2 2 60% 66%
Glutathione S-transferase P OS = Homo sapiens OX = 9606 GN = GSTP1 PE = 1 SV = 2 P09211 GSTP1 23 kDa 1 1 2 2 71% 46%
Interleukin enhancer-binding factor 3 OS = Homo sapiens OX = 9606 GN = ILF3 PE = 1 SV = 3 Q12906 ILF3 95 kDa 0 0 2 2 0 100%
Non-histone chromosomal protein HMG-14 OS = Homo sapiens OX = 9606 GN = HMGN1 PE = 1 SV = 3 P05114 HMGN1 11 kDa 1 1 2 2 9% 31%
ATP-dependent RNA helicase A OS = Homo sapiens OX = 9606 GN = DHX9 PE = 1 SV = 4 Q08211 DHX9 141 kDa 20 15 3 2 100% 98%
Peroxiredoxin-1 OS = Homo sapiens OX = 9606 GN = PRDX1 PE = 1 SV = 1 Q06830 PRDX1 22 kDa 13 11 3 3 100% 100%
Retinitis pigmentosa 9 protein OS = Homo sapiens OX = 9606 GN = RP9 PE = 1 SV = 2 Q8TA86 RP9 26 kDa 11 8 3 3 100% 100%
Non-POU domain-containing octamer-binding protein OS = Homo sapiens OX = 9606 GN = NONO PE = 1 SV = 4 Q15233 NONO 54 kDa 12 9 3 2 100% 100%
L-lactate dehydrogenase B chain OS = Homo sapiens OX = 9606 GN = LDHB PE = 1 SV = 2 P07195 LDHB 37 kDa 7 5 3 2 100% 100%
Vimentin OS = Homo sapiens OX = 9606 GN = VIM PE = 1 SV = 4 P08670 VIM 54 kDa 7 6 3 2 100% 100%
40S ribosomal protein S3a OS = Homo sapiens OX = 9606 GN = RPS3A PE = 1 SV = 2 P61247 RPS3A 30 kDa 3 3 3 3 100% 100%
THO complex subunit 4 OS = Homo sapiens OX = 9606 GN = ALYREF PE = 1 SV = 3 Q86V81 ALYREF 27 kDa 2 2 3 2 100% 100%
Histone H1x OS = Homo sapiens OX = 9606 GN = H1FX PE = 1 SV = 1 Q92522 H1FX 22 kDa 3 2 3 2 100% 100%
40S ribosomal protein S14 OS = Homo sapiens OX = 9606 GN = RPS14 PE = 1 SV = 3 P62263 RPS14 16 kDa 2 2 3 2 100% 97%
60S ribosomal protein L13 OS = Homo sapiens OX = 9606 GN = RPL13 PE = 1 SV = 4 P26373 RPL13 24 kDa 2 2 3 2 100% 100%
Serum albumin OS = Homo sapiens OX = 9606 GN = ALB PE = 1 SV = 2 P02768 ALB 69 kDa 2 1 3 3 96% 100%
Elongation factor 1-gamma OS = Homo sapiens OX = 9606 GN = EEF1G PE = 1 SV = 3 P26641 EEF1G 50 kDa 1 1 3 3 97% 100%
60S ribosomal protein L10a OS = Homo sapiens OX = 9606 GN = RPL10A PE = 1 SV = 2 P62906 RPL10A 25 kDa 1 1 3 2 41% 98%
ATP synthase subunit alpha, mitochondrial OS = Homo sapiens OX = 9606 GN = ATP5F1A PE = 1 SV = 1 P25705 ATP5F1A 60 kDa 1 1 3 3 8% 100%
Heat shock protein HSP 90-alpha OS = Homo sapiens OX = 9606 GN = HSP90AA1 PE = 1 SV = 5 P07900 HSP90AA1 85 kDa 12 9 4 3 100% 100%
Pyruvate kinase PKM OS = Homo sapiens OX = 9606 GN = PKM PE = 1 SV = 4 P14618 PKM 58 kDa 15 15 4 4 100% 100%
Heat shock protein HSP 90-beta OS = Homo sapiens OX = 9606 GN = HSP90AB1 PE = 1 SV = 4 P08238 HSP90AB1 83 kDa 10 4 4 1 100% 8%
Splicing factor, arginine/serine-rich 19 OS = Homo sapiens OX = 9606 GN = SCAF1 PE = 1 SV = 3 Q9H7N4 SCAF1 139 kDa 8 7 4 3 100% 100%
Heterogeneous nuclear ribonucleoprotein K OS = Homo sapiens OX = 9606 GN = HNRNPK PE = 1 SV = 1 P61978 HNRNPK 51 kDa 5 5 4 4 100% 100%
Trypsin-1 OS = Homo sapiens OX = 9606 GN = PRSS1 PE = 1 SV = 1 P07477 PRSS1 27 kDa 4 2 4 2 100% 100%
40S ribosomal protein S30 OS = Homo sapiens OX = 9606 GN = FAU PE = 1 SV = 1 P62861 FAU 7 kDa 3 1 4 2 97% 100%
DNA topoisomerase 1 OS = Homo sapiens OX = 9606 GN = TOP1 PE = 1 SV = 2 P11387 TOP1 91 kDa 6 5 4 4 100% 97%
60S ribosomal protein L7 OS = Homo sapiens OX = 9606 GN = RPL7 PE = 1 SV = 1 P18124 RPL7 29 kDa 4 3 4 3 100% 100%
40S ribosomal protein S8 OS = Homo sapiens OX = 9606 GN = RPS8 PE = 1 SV = 2 P62241 RPS8 24 kDa 3 3 4 2 100% 100%
60S ribosomal protein L5 OS = Homo sapiens OX = 9606 GN = RPL5 PE = 1 SV = 3 P46777 RPL5 34 kDa 1 1 4 2 95% 80%
Ribosomal RNA processing protein 1 homolog B OS = Homo sapiens OX = 9606 GN = RRP1B PE = 1 SV = 3 Q14684 RRP1B 84 kDa 0 0 4 4 0 100%
Elongation factor 2 OS = Homo sapiens OX = 9606 GN = EEF2 PE = 1 SV = 4 P13639 EEF2 95 kDa 31 19 5 5 100% 100%
Tubulin beta chain OS = Homo sapiens OX = 9606 GN = TUBB PE = 1 SV = 2 P07437 TUBB 50 kDa 16 11 5 4 100% 100%
Tubulin alpha-1B chain OS = Homo sapiens OX = 9606 GN = TUBA1B PE = 1 SV = 1 P68363 (+2) TUBA1B 50 kDa 13 8 5 4 100% 100%
L-lactate dehydrogenase A chain OS = Homo sapiens OX = 9606 GN = LDHA PE = 1 SV = 2 P00338 LDHA 37 kDa 7 6 5 5 100% 100%
E3 ubiquitin-protein ligase RBBP6 OS = Homo sapiens OX = 9606 GN = RBBP6 PE = 1 SV = 1 Q7Z6E9 RBBP6 202 kDa 11 10 5 5 100% 100%
Protein LLP homolog OS = Homo sapiens OX = 9606 GN = LLPH PE = 1 SV = 1 Q9BRT6 LLPH 15 kDa 7 4 5 3 100% 100%
Probable ATP-dependent RNA helicase DDX17 OS = Homo sapiens OX = 9606 GN = DDX17 PE = 1 SV = 2 Q92841 DDX17 80 kDa 4 3 5 4 100% 100%
Protein SREK1IP1 OS = Homo sapiens OX = 9606 GN = SREK1IP1 PE = 1 SV = 1 Q8N9Q2 SREK1IP1 18 kDa 4 3 5 3 100% 100%
RNA-binding motif protein, X chromosome OS = Homo sapiens OX = 9606 GN = RBMX PE = 1 SV = 3 P38159 RBMX 42 kDa 8 6 5 4 100% 97%
Putative RNA-binding protein Luc7-like 2 OS = Homo sapiens OX = 9606 GN = LUC7L2 PE = 1 SV = 2 Q9Y383 LUC7L2 47 kDa 2 2 5 4 99% 100%
60S ribosomal protein L37 OS = Homo sapiens OX = 9606 GN = RPL37 PE = 1 SV = 2 P61927 RPL37 11 kDa 0 0 5 2 0 100%
Heat shock 70 kDa protein 1A OS = Homo sapiens OX = 9606 GN = HSPA1A PE = 1 SV = 1 P0DMV8 (+1) HSPA1A 70 kDa 20 16 6 6 100% 100%
Heat shock cognate 71 kDa protein OS = Homo sapiens OX = 9606 GN = HSPA8 PE = 1 SV = 1 P11142 HSPA8 71 kDa 18 8 6 4 100% 100%
Heterogeneous nuclear ribonucleoproteins A2/B1 OS = Homo sapiens OX = 9606 GN = HNRNPA2B1 PE = 1 SV = 2 P22626 HNRNPA2B1 37 kDa 16 11 6 4 100% 100%
Histone H4 OS = Homo sapiens OX = 9606 GN = HIST1H4A PE = 1 SV = 2 P62805 HIST1H4A 11 kDa 9 7 6 5 100% 100%
AP-3 complex subunit delta-1 OS = Homo sapiens OX = 9606 GN = AP3D1 PE = 1 SV = 1 O14617 AP3D1 130 kDa 9 7 6 4 100% 100%
Nucleolin OS = Homo sapiens OX = 9606 GN = NCL PE = 1 SV = 3 P19338 NCL 77 kDa 8 8 6 6 100% 100%
A-kinase anchor protein 17A OS = Homo sapiens OX = 9606 GN = AKAP17A PE = 1 SV = 2 Q02040 AKAP17A 81 kDa 11 8 6 6 100% 100%
60S ribosomal protein L6 OS = Homo sapiens OX = 9606 GN = RPL6 PE = 1 SV = 3 Q02878 RPL6 33 kDa 2 2 6 4 95% 100%
Heterogeneous nuclear ribonucleoprotein A1 OS = Homo sapiens OX = 9606 GN = HNRNPA1 PE = 1 SV = 5 P09651 HNRNPA1 39 kDa 16 11 7 4 100% 100%
La-related protein 7 OS = Homo sapiens OX = 9606 GN = LARP7 PE = 1 SV = 1 Q4G0J3 LARP7 67 kDa 6 6 7 6 100% 100%
G patch domain-containing protein 4 OS = Homo sapiens OX = 9606 GN = GPATCH4 PE = 1 SV = 2 Q5T3I0 GPATCH4 50 kDa 5 5 7 6 100% 100%
NF-kappa-B-activating protein OS = Homo sapiens OX = 9606 GN = NKAP PE = 1 SV = 1 Q8N5F7 NKAP 47 kDa 8 6 8 5 100% 100%
60S ribosomal protein L23a OS = Homo sapiens OX = 9606 GN = RPL23A PE = 1 SV = 1 P62750 RPL23A 18 kDa 8 6 8 4 100% 100%
Histone H2B type 1-K OS = Homo sapiens OX = 9606 GN = HIST1H2BK PE = 1 SV = 3 O60814 (+8) HIST1H2BK 14 kDa 7 5 8 4 100% 100%
Histone H3.1 OS = Homo sapiens OX = 9606 GN = HIST1H3A PE = 1 SV = 2 P68431 (+3) HIST1H3A 15 kDa 6 4 8 4 100% 100%
Transcription initiation factor TFIID subunit 3 OS = Homo sapiens OX = 9606 GN = TAF3 PE = 1 SV = 1 Q5VWG9 TAF3 104 kDa 6 6 8 7 100% 100%
Keratin, type I cytoskeletal 17 OS = Homo sapiens OX = 9606 GN = KRT17 PE = 1 SV = 2 Q04695 KRT17 48 kDa 12 3 9 2 100% 76%
Heterogeneous nuclear ribonucleoproteins C1/C2 OS = Homo sapiens OX = 9606 GN = HNRNPC PE = 1 SV = 4 P07910 HNRNPC 34 kDa 9 6 9 4 100% 100%
Hornerin OS = Homo sapiens OX = 9606 GN = HRNR PE = 1 SV = 2 Q86YZ3 HRNR 282 kDa 12 9 10 7 100% 100%
Heterogeneous nuclear ribonucleoprotein U OS = Homo sapiens OX = 9606 GN = HNRNPU PE = 1 SV = 6 Q00839 HNRNPU 91 kDa 17 12 11 6 100% 100%
Serine/arginine-rich splicing factor 11 OS = Homo sapiens OX = 9606 GN = SRSF11 PE = 1 SV = 1 Q05519 SRSF11 54 kDa 11 8 11 6 100% 100%
Serine/arginine repetitive matrix protein 2 OS = Homo sapiens OX = 9606 GN = SRRM2 PE = 1 SV = 2 Q9UQ35 SRRM2 300 kDa 15 7 11 6 100% 100%
Poly [ADP-ribose] polymerase 1 OS = Homo sapiens OX = 9606 GN = PARP1 PE = 1 SV = 4 P09874 PARP1 113 kDa 29 22 12 12 100% 100%
Elongation factor 1-alpha 1 OS = Homo sapiens OX = 9606 GN = EEF1A1 PE = 1 SV = 1 P68104 (+1) EEF1A1 50 kDa 21 14 13 9 100% 100%
Nucleolar protein 56 OS = Homo sapiens OX = 9606 GN = NOP56 PE = 1 SV = 4 O00567 NOP56 66 kDa 16 11 13 10 100% 100%
U2 snRNP-associated SURP motif-containing protein OS = Homo sapiens OX = 9606 GN = U2SURP PE = 1 SV = 2 O15042 U2SURP 118 kDa 25 19 14 10 100% 100%
Guanine nucleotide-binding protein-like 3 OS = Homo sapiens OX = 9606 GN = GNL3 PE = 1 SV = 2 Q9BVP2 GNL3 62 kDa 9 7 16 9 100% 100%
Actin, cytoplasmic 1 OS = Homo sapiens OX = 9606 GN = ACTB PE = 1 SV = 1 P60709 (+1) ACTB 42 kDa 22 12 17 9 100% 100%
Multiple myeloma tumor-associated protein 2 OS = Homo sapiens OX = 9606 GN = MMTAG2 PE = 1 SV = 1 Q9BU76 MMTAG2 29 kDa 21 13 17 8 100% 100%
Keratin, type II cytoskeletal 5 OS = Homo sapiens OX = 9606 GN = KRT5 PE = 1 SV = 3 P13647 KRT5 62 kDa 27 15 20 11 100% 100%
Histone H1.0 OS = Homo sapiens OX = 9606 GN = H1F0 PE = 1 SV = 3 P07305 H1F0 21 kDa 16 9 20 9 100% 100%
Nucleolar protein of 40 kDa OS = Homo sapiens OX = 9606 GN = ZCCHC17 PE = 1 SV = 1 Q9NP64 ZCCHC17 28 kDa 19 9 20 10 100% 100%
60S ribosomal protein L29 OS = Homo sapiens OX = 9606 GN = RPL29 PE = 1 SV = 2 P47914 RPL29 18 kDa 10 2 21 5 100% 100%
Keratin, type I cytoskeletal 14 OS = Homo sapiens OX = 9606 GN = KRT14 PE = 1 SV = 4 P02533 KRT14 52 kDa 32 16 22 7 100% 100%
Protein SON OS = Homo sapiens OX = 9606 GN = SON PE = 1 SV = 4 P18583 SON 264 kDa 15 15 23 17 100% 100%
Keratin, type II cytoskeletal 6A OS = Homo sapiens OX = 9606 GN = KRT6A PE = 1 SV = 3 P02538 KRT6A 60 kDa 30 8 25 7 100% 100%
Transcription termination factor 1 OS = Homo sapiens OX = 9606 GN = TTF1 PE = 1 SV = 3 Q15361 TTF1 103 kDa 31 23 25 17 100% 100%
Treacle protein OS = Homo sapiens OX = 9606 GN = TCOF1 PE = 1 SV = 3 Q13428 TCOF1 152 kDa 26 17 27 17 100% 100%
Methylcrotonoyl-CoA carboxylase subunit alpha, mitochondrial OS = Homo sapiens OX = 9606 GN = MCCC1 PE = 1 SV = 3 Q96RQ3 MCCC1 80 kDa 50 25 40 19 100% 100%
Lysine-rich nucleolar protein 1 OS = Homo sapiens OX = 9606 GN = KNOP1 PE = 1 SV = 1 Q1ED39 KNOP1 52 kDa 61 33 42 23 100% 100%
Arginine and glutamate-rich protein 1 OS = Homo sapiens OX = 9606 GN = ARGLU1 PE = 1 SV = 1 Q9NWB6 ARGLU1 33 kDa 29 17 46 20 100% 100%
Cell growth-regulating nucleolar protein OS = Homo sapiens OX = 9606 GN = LYAR PE = 1 SV = 2 Q9NX58 LYAR 44 kDa 50 24 50 23 100% 100%
Keratin, type I cytoskeletal 9 OS = Homo sapiens OX = 9606 GN = KRT9 PE = 1 SV = 3 P35527 KRT9 62 kDa 98 34 54 19 100% 100%
Keratin, type I cytoskeletal 10 OS = Homo sapiens OX = 9606 GN = KRT10 PE = 1 SV = 6 P13645 KRT10 59 kDa 60 27 54 24 100% 100%
Keratin, type II cytoskeletal 2 epidermal OS = Homo sapiens OX = 9606 GN = KRT2 PE = 1 SV = 2 P35908 KRT2 65 kDa 64 29 60 24 100% 100%
Histone H1.2 OS = Homo sapiens OX = 9606 GN = HIST1H1C PE = 1 SV = 2 P16403 HIST1H1C 21 kDa 72 2 69 2 100% 100%
Acetyl-CoA carboxylase 1 OS = Homo sapiens OX = 9606 GN = ACACA PE = 1 SV = 2 Q13085 ACACA 266 kDa 41 33 69 45 100% 100%
Histone H1.4 OS = Homo sapiens OX = 9606 GN = HIST1H1E PE = 1 SV = 2 P10412 HIST1H1E 22 kDa 74 13 75 18 100% 100%
Keratin, type II cytoskeletal 1 OS = Homo sapiens OX = 9606 GN = KRT1 PE = 1 SV = 6 P04264 KRT1 66 kDa 135 45 94 31 100% 100%
Propionyl-CoA carboxylase alpha chain, mitochondrial OS = Homo sapiens OX = 9606 GN = PCCA PE = 1 SV = 4 P05165 PCCA 80 kDa 117 49 114 45 100% 100%
Pyruvate carboxylase, mitochondrial OS = Homo sapiens OX = 9606 GN = PC PE = 1 SV = 2 P11498 PC 130 kDa 122 54 121 49 100% 100%
Open in a new tab

Figure 1.

Figure 1.

Open in a new tab

(A) Gene ontology analysis of the protein hits discovered using PERK-BirA* obtained using WEB-based GEne SeT AnaLysis toolkit (http://www.webgestalt.org/). The minimum number of IDs in each category was required to be 5, the maximum was 2000, the false-discovery rate (FDR) correction used was Benjamini and Hochberg (BH). The categories were first ranked based on FDR and after that the topmost significant categories were selected. 10 categories of biological processes were identified, many related to the function of PERK. The enrichment ratio indicates the ratio between the fraction of proteins belonging to each biological process in our dataset over the fraction of these proteins expected If our dataset was completely random. (B) Two specific categories are highlighted, response to endoplasmic reticulum stress, and protein folding, indicating an enrichment in the dataset of proteins and processes linked to PERK's role in the unfolded protein response and ER stress.

Considering our previous and ongoing studies, we decided to focus on validating the interactions of PERK with proteins with a known tethering role and/or with a relevant function in membrane contact sites and ER Ca2+ homeostasis. Two hits that were interesting in this regard were SERCA1/2 and VAPA/B, found at positions 47 and 62, respectively, on our list (Table 1).

PERK Interaction With VAPB

The isoforms VAPA and B are members of a small VAP protein family and have broadly similar structures and functions (Murphy & Levine, 2016). They are tail-anchored ER membrane proteins that are central to the formation of MCSs. VAPs act as tethers for a growing group of proteins, including protein tyrosine phosphatase interacting protein 51 (PTPIP51), StAR Related Lipid Transfer Domain Containing 3 (STARD3), oxysterol-binding protein (OSBP), Nir2, and Sorting nexin 2 (SNX2), among others (Alpy et al., 2013; Amarilio et al., 2005; De Vos et al., 2012; Dong et al., 2016; Wyles et al., 2002). VAPB is an important mediator of both ER-mitochondria contact sites and ER-plasma membrane contact sites, where it can interact with various proteins responsible for tethering and lipid trafficking at these contact sites, including PTPIP51 on mitochondria and Nir2 at the ER-PM contacts. Given the role of PERK in modulating both ER-mitochondria and indirectly ER-PM contacts, we then explored the possibility that VAPA/B is a bona fide interacting partner of PERK.

In our dataset, we picked up unique peptides for both VAPA and VAPB, indicating that both proteins might interact with PERK. Because VAPB has traditionally been the more studied of the two at MCS, and because both proteins are highly similar, for simplicity, here we focused on VAPB. We tested the possible interaction between VAPB and PERK by co-immunoprecipitation (co-IP) experiments and successfully confirmed that immunoprecipitation of wild type PERK-myc (PERKWT) expressed in HEK293-T cells, pulled down VAPB (Figures 2 and S2). In previous studies, we showed that the function of PERK required at the ER-mitochondria and ER-PM contacts was independent of its kinase function (van Vliet et al., 2017; Verfaillie et al., 2012). Here, to investigate whether PERK's kinase activity was dispensable for PERK-VAPB interaction, we performed the IP in cells expressing either a PERKWT or a kinase dead mutant of PERK (PERKKD). Interestingly, the expression of the PERKKD mutant yielded no difference in VAPB binding (Figures 2 and S2). Together these observations suggest that PERK interacts with VAPB constitutively, and in the absence of a signal, evoking its UPR activation. It is possible that VAPB could recruit PERK to specific MCS upon certain stresses, or vice versa; further research would be needed to validate this possibility.

Figure 2.

Figure 2.

Open in a new tab

Identification of VAPB and SERCA2 as putative PERK interactors. HEK293-T cells were transfected with myc-tagged expression vectors for PERK WT (PERKWT) and PERK Kinase dead (PERKKD). After 48 h of transfection, PERKWT and PERKKD were immunoprecipitated (PERK IP) and interactors were detected using antibodies against VAPB and SERCA2 by immunoblot. Input was 10% of the total protein amount used for the IP (50 µg of protein was loaded as an input versus 500 µg of total protein used for the IP). Unspecific anti-mouse antibody was used as a negative control (Mouse IP). Data show are representative of N = 3 (VAPB), N = 2 (SERCA2) biologically independent experiments.

Proteins that interact with VAPs usually do so through a specific domain, termed a FFAT motif (two phenylalanines (FF) in an acidic tract) that binds the major sperm protein domain (MSP) on VAP (Murphy & Levine, 2016). However, using a previously published algorithm (Murphy & Levine, 2016) we did not find any robust FFAT motif in the PERK primary sequence. This suggests that the PERK-VAPB interaction may involve either another FFAT containing protein or may be mediated through another binding in cis, as they are both ER membrane proteins. This would leave the MSP of VAPB free to bind FFAT motifs in other proteins. A recent publication has explored the human “VAPome” in a systematic way using BioID and found that PERK was a hit for at least VAPA (Cabukusta et al., 2020). This same study also reports the putative interaction between PERK and MOSDP1 and 3. These proteins are VAP-like but bind to a slight variation of the FFAT motif, the FFNT motif (two phenylalanines (FF) in a neutral tract). Their analysis indicates that PERK contains an FFNT motif at location 283 of the protein, which is located in the lumen of the ER. This study, along with ours, does give support for a functional role between PERK and VAPA/B and other MCS proteins.

PERK Interaction With SERCA2

SERCA1/2 are ATPases located in the ER membrane that pump Ca2+ ions from the cytosol into the ER lumen, counteracting the Ca2+ leak from various sources (translocon, inositol 1,4,5-trisphosphate receptor) and maintaining a high level of Ca2+ in the ER lumen with a steady-state Ca2+ concentration of approximately 1 mM (de la Fuente et al., 2013).

In contrast to SERCA2, no unique peptides for SERCA1 could be identified in the BioID data set, so the presence of the latter isoform in the PERK interactome could not be proven. Neither SERCA1 nor SERCA2 have been detected at the ER-mitochondria contact sites, but a truncated version of SERCA1, S1 T (truncated after amino acid 395) has been shown to play a role at ER-mitochondria contact sites (Chami et al., 2008). As with VAPB, we confirmed the interaction of SERCA2 with PERK through co-IP, which was again detected independently of its kinase activity (Figures 2 and S2). Our previous results indicated that PERK is strongly activated by increases in cytosolic Ca2+ levels, and we speculated that this is linked to the depletion of ER luminal Ca2+ leading to ER stress, as shown also in a recent study (Preissler et al., 2020). Furthermore, we reported that PERK is involved in SOCE, by regulating the ER-PM contacts through its binding to FLNA (van Vliet et al., 2017). SOCE is induced by ER Ca2+ depletion, which leads to oligomerization of STIM1 and its translocation to the PM where it interacts with the PM localized ORAI1 protein. The interaction of STIM1 with ORAI1 then allows Ca2+ to enter the cytosol from the extracellular medium through the opening of the ORAI1 channel (Zhang et al., 2005). PERK's interaction with SERCA2 is an intriguing link since, although ORAI1 allows Ca2+ to enter the cytosol, the refilling of ER-Ca2+ store requires the activity of the Ca2+ pump SERCA. A previous study has indicated a close relationship between STIM1/ORAI1 driven SOCE and SERCA (Courjaret & Machaca, 2014). We can then speculate that PERK may be located close to the site of STIM1/ORAI1 contact, playing a part in the mechanism that ensures Ca2+ entering the cytosol is rapidly internalized into the ER lumen through SERCA2. Further study is needed to investigate a possible role of PERK in regulating SERCA2 activity, and therefore, influencing this mechanism.

Conclusions and Limitations of the Study

In this report, we show a BioID dataset obtained by tagging the promiscuous biotin ligase BirA* to PERK, in order to map its close interactors. We highlight and validate two of these, VAPB and SERCA2, which lead us to further speculate about the role of PERK in ER MCS formation and Ca2+ signaling.

Our study was performed when BioID was just emerging as a tool, and at the time of our planning, only the original study had been published (Roux et al., 2012). We, therefore, closely modeled our experimental setup on this study. It is important to note when interpreting this dataset that later studies using BioID (and derivatives of BioID like TurboID, where the BirA* ligase has been mutated to biotinylate at a much higher rate), have implemented more stringent controls to detect false positives (inherent with using BioID as an approach to screen protein-protein interactions). For example, the initial study reporting BioID used only parental cells (mock-transfected) as a control, while some recent studies used BirA* (or TuboID) not tagged to their protein of interest to control for non-specific biotinylation events (Szczesniak et al., 2021; Vermehren-Schmaedick et al., 2021; Zhang et al., 2019). Our reasoning for using only parental cells as control was that by using free BirA* as a control, there was a risk that this enzyme might biotinylate bona fide hits randomly, leading to false negatives. Since the initial study reporting BioID, the original authors have published numerous extensive updates on how to set up a BioID study, incorporating more appropriate or bespoke controls and comparing the different new techniques (May et al., 2020; May & Roux, 2019; Roux et al., 2018; Sears et al., 2019). In summary, we have identified 129 potential interacting partners of PERK, of which we have tested and confirmed two, SERCA2 and VAPB. We hope that this dataset can yield new insights concerning PERK and cellular signaling.

Materials and Methods

Cell Lines and Transfection:

HEK293-T cells have been maintained in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose and 0.11 g/l sodium pyruvate and supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) (all added, AA medium). Cells were transiently transfected with different PERK constructs encoding PERKK618A (Addgene plasmid 21815) or PERKFL (Addgene plasmid 21814), both myc tagged, using Trans-IT X2 transfection reagent (Mirus Bio LLC, Science Dr.Madison, WI USA).

BioID, Biotinylation Assay, and Mass Spectrometry

BioID was performed as described previously (Roux et al., 2012), with minor modifications. Overexpression of PERK-BioID was achieved by transiently transfecting Hek293-T cells using X-tremegene 9 (Roche, Germany). A 6 μl X-tremegene9 was mixed with a 2 μg PERK-BioID plasmid and dispersed on cultured cells. A 30 h post-transfection medium containing 50 μM biotin was added to the cells for 24 h. After incubation with biotin, cells were lysed in 1 mL lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM DTT, and 1x complete protease inhibitor [Roche]) and sonicated. Triton X-100 was added to a 2% final concentration. After further sonication, an equal volume of 4 °C 50 mM Tris (pH 7.4) was added before additional sonication (subsequent steps at 4 °C) and centrifugation at 16,000 g. Supernatants were incubated with 600 μl Dynabeads (50% slurry) (MyOne Streptavidin C1; Invitrogen) overnight. Beads were collected and washed twice for 8 min at 25 °C (all subsequent steps at 25 °C) in 1 mL wash buffer 1 (2% SDS in dH2O). This was repeated once with wash buffer 2 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES, pH 7.5), once with wash buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1), and twice with wash buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). For western blot analysis, bound proteins were removed from the magnetic beads with 50 μl of Laemmli SDS-sample buffer saturated with biotin at 98 °C. For mass spectrometry analysis, beads were washed repeatedly in MQ water containing 50 mM ammonium bicarbonate (AmBic) before being incubated for 30 min at 37 °C in 50 mM AmBic containing 5 mM DTT. Beads were further washed in 50 mM AmBic and incubated with 25 mM iodoacetamide in the dark for 30 min at 37 °C. After reduction/alkylation, beads were washed in 50 mM AmBic and incubated with 1:20 (w/w) modified trypsin (Pierce) ON at 37 °C in 50 mM AmBic containing 5% acetonitrile. After removal of beads by magnetic separation, formic acid was added to the peptide solution (to 2%) before desalting by C18 Micro Spin Columns (Harvard Apparatus). The resulting peptide mixture was analyzed by nano LC-MS on a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific). Peptides were identified by MASCOT 2.2 (Matrix Science) using the SwissProt database (taxonomy Homo sapiens, 20231 entries) adopting the following MASCOT search parameters: trypsin/P, two missed cleavages allowed, variable modification oxidation (M), fixed modification carbamidomethylation (C). The mascot was searched with a fragment ion mass tolerance of 0,02 Da and a parent ion mass tolerance of 10 ppm.

Scaffold 4 (Proteome Software Inc.) was used to validate MS/MS-based peptide and protein identifications. Peptide and protein identifications were accepted to achieve an FDR less than 10%. Standard protein grouping was adopted. The presence of at least 2 exclusive unique peptides per protein was required.

Immunoprecipitation

After 48 h of transfection with selected plasmids cells, they were collected through scraping and lysed in lysis buffer (1% CHAPS, 100 mM KCl, 150 mM NaCl, 1x protease inhibitor (Pierce Protease Inhibitor Tablets, Thermo Fisher Scientific Inc.)) for 30 min at 4 °C. Cells were centrifuged at 13.000 g for 15 min to remove debris and unbroken cells. From the supernatant, 500 μg of proteins were combined with primary antibodies overnight (ON) at 4 °C, against PERK and using a non-specific Mouse IgG as a control. Protein-antibody complexes were captured by the addition of Protein AG magnetic beads (Pierce) for 1.5 h at room temperature (RT). Protein AG magnetic beads with captured protein-antibody complexes were washed three times with lysis buffer. Proteins were eluted with sample buffer (62.5 μM Tris-HCl, 10% glycerol, 2% SDS, 1x protease inhibitor, 1x phosphatase inhibitor (Pierce phosphatase Inhibitor Tablets, Thermo Fisher Scientific Inc.) in MQ water) and loaded on a gel for western blot analysis.

Western Blotting

Samples were separated by SDS-PAGE on the Criterion system (Bio-Rad Laboratories, Hercules, CA, USA) on a 4%–12% Bis-TRIS gel and electrophoretically transferred to Protran 2 μm-pored nitrocellulose paper (PerkinElmer, Wellesley, MA, USA). The blots were blocked for 1 h at RT in TBS-T buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk and then incubated with selected antibody solutions. Samples were processed and enhanced chemiluminescence using Pierce ECL Western Blotting Substrate was used for western blot detection and membranes were scanned using the Bio-Rad Chemidoc Imager (Bio-Rad Laboratories N.V.3, Winninglaan, Temse, Belgium).

Antibodies

Antibodies used were mouse monoclonal anti-c-Myc (Sigma, Cat# M4439), Control normal mouse IgG (sc-2025), anti-ATP2A2/SERCA2 (Cell signaling 388S), anti-VAPB (Invitrogen, PA5-53023), and an HRP-based detection using as a secondary antibody, the Veriblot antibody (#ab131366, Abcam).

Footnotes

Author Contributions: Experiment design, analysis, and manuscript writing: MLS, PA, ARVV. Performing and analyzing western blots: MLS. Mass spectrometry analysis: RD, EW. Project and funding: PA.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the EOS consortium (grant number 30837538, FAF-F/2018/1252, G049817N, G070115N, G076617N).

References

  1. Alpy F., Rousseau A., Schwab Y., Legueux F., Stoll I., Wendling C., Spiegelhalter C., Kessler P., Mathelin C., Rio M. C., Levine T. P., Tomasetto C. (2013). STARD3 Or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER. Journal of Cell Science, 126(Pt 23), 5500–5512. 10.1242/jcs.139295 [DOI] [PubMed] [Google Scholar]
  2. Amarilio R., Ramachandran S., Sabanay H., Lev S. (2005). Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction. Journal of Biological Chemistry, 280(7), 5934–5944. 10.1074/jbc.M409566200 [DOI] [PubMed] [Google Scholar]
  3. Balsa E., Soustek M. S., Thomas A., Cogliati S., Garcia-Poyatos C., Martin-Garcia E., Jedrychowski M., Gygi S. P., Enriquez J. A., Puigserver P. (2019). ER and nutrient stress promote assembly of respiratory chain supercomplexes through the PERK-eIF2alpha axis. Molecular Cell, 74(5), 877–90e6. 10.1016/j.molcel.2019.03.031 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brodsky J. L., Wojcikiewicz R. J. (2009). Substrate-specific mediators of ER associated degradation (ERAD). Current Opinion in Cell Biology, 21(4), 516–521. 10.1016/j.ceb.2009.04.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cabukusta B., Berlin I., van Elsland D. M., Forkink I., Spits M., de Jong A. W. M., Akkermans Jjll, Wijdeven R. H. M., Janssen G. M. C, van Veelen P. A., Neefjes J. (2020). Human VAPome analysis reveals MOSPD1 and MOSPD3 as membrane contact site proteins interacting with FFAT-related FFNT motifs. Cell Reports, 33(10), 108475. 10.1016/j.celrep.2020.108475 [DOI] [PubMed] [Google Scholar]
  6. Chami M., Oules B., Szabadkai G., Tacine R., Rizzuto R., Paterlini-Brechot P. (2008). Role of SERCA1 truncated isoform in the proapoptotic calcium transfer from ER to mitochondria during ER stress. Molecular Cell, 32(5), 641–651. 10.1016/j.molcel.2008.11.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Courjaret R., Machaca K. (2014). Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release. Nature Communications, 5, 3916. 10.1038/ncomms4916 [DOI] [PubMed] [Google Scholar]
  8. de la Fuente S., Fonteriz R. I., Montero M., Alvarez J. (2013). Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin. Cell Calcium, 54(1), 37–45. 10.1016/j.ceca.2013.04.001 [DOI] [PubMed] [Google Scholar]
  9. De Vos K. J., Morotz G. M., Stoica R., Tudor E. L., Lau K. F., Ackerley S., Warley A., Shaw C. E., Miller C. C. (2012). VAPB Interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis. Human Molecular Genetics, 21(6), 1299–1311. 10.1093/hmg/ddr559 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dong R., Saheki Y., Swarup S., Lucast L., Harper J. W., De Camilli P. (2016). Endosome-ER contacts control actin nucleation and retromer function through VAP-dependent regulation of PI4P. Cell, 166(2), 408–423. 10.1016/j.cell.2016.06.037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. May D. G., Roux K. J. (2019). BioID: A method to generate a history of protein associations. Methods in Molecular Biology, 2008, 83–95. 10.1007/978-1-4939-9537-0_7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. May D. G., Scott K. L., Campos A. R., Roux K. J. (2020). Comparative application of BioID and TurboID for protein-proximity biotinylation. Cells, 9(5). 10.3390/cells9051070 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moncan M., Mnich K., Blomme A., Almanza A., Samali A., Gorman A. M. (2021). Regulation of lipid metabolism by the unfolded protein response. Journal of Cellular and Molecular Medicine, 25(3), 1359–1370. 10.1111/jcmm.16255 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Murphy S. E., Levine T. P. (2016). VAP, a Versatile access point for the endoplasmic Reticulum: Review and analysis of FFAT-like motifs in the VAPome. Biochimica et Biophysica Acta, 1861(8 Pt B), 952–961. 10.1016/j.bbalip.2016.02.009 [DOI] [PubMed] [Google Scholar]
  15. Preissler S., Rato C., Yan Y., Perera L. A., Czako A., Ron D. (2020). Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes. Elife, 9. 10.7554/eLife.62601 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ron D., Walter P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nature Reviews Molecular Cell Biology, 8(7), 519–529. 10.1038/nrm2199 [DOI] [PubMed] [Google Scholar]
  17. Roux K. J., Kim D. I., Burke B., May D. G. (2018). BioID: A screen for protein-protein interactions. Current Protocols in Protein Science, 91, 19231–192315. 10.1002/cpps.51 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Roux K. J., Kim D. I., Raida M., Burke B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. Journal of Cell Biology, 196(6), 801–810. 10.1083/jcb.201112098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sears R. M., May D. G., Roux K. J. (2019). BioID as a tool for protein-proximity labeling in living cells. Methods in Molecular Biology, 2012, 299–313. 10.1007/978-1-4939-9546-2_15 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sorge S., Theelke J., Yildirim K., Hertenstein H., McMullen E., Muller S., Altburger C., Schirmeier S., Lohmann I. (2020). ATF4-induced Warburg metabolism drives over-proliferation in drosophila. Cell Reports, 31(7), 107659. 10.1016/j.celrep.2020.107659 [DOI] [PubMed] [Google Scholar]
  21. Szczesniak L. M., Bonzerato C. G., Wojcikiewicz R. J. H. (2021). Identification of the Bok interactome using proximity labeling. Frontiers in Cell and Developmental Biology, 9, 689951. 10.3389/fcell.2021.689951 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. van Vliet A. R., Giordano F., Gerlo S., Segura I., Van Eygen S., Molenberghs G., Rocha S., Houcine A., Derua R., Verfaillie T., Vangindertael J., De Keersmaecker H., Waelkens E., Tavernier J., Hofkens J., Annaert W., Carmeliet P., Samali A., Mizuno H., Agostinis P. (2017). The ER stress sensor PERK coordinates ER-plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling. Molecular Cell, 65(5), 885–99e6. 10.1016/j.molcel.2017.01.020 [DOI] [PubMed] [Google Scholar]
  23. Verfaillie T., Rubio N., Garg A. D., Bultynck G., Rizzuto R., Decuypere J. P., Piette J., Linehan C., Gupta S., Samali A., Agostinis P. (2012). PERK Is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress. Cell Death & Differentiation, 19(11), 1880–1891. 10.1038/cdd.2012.74 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vermehren-Schmaedick A., Huang J. Y., Levinson M., Pomaville M. B., Reed S., Bellus G. A., Gilbert F., Keren B., Heron D., Haye D., Janello C., Makowski C., Danhauser K., Fedorov L. M., Haack T. B., Wright K. M., Cohen M. S. (2021). Characterization of PARP6 function in knockout mice and patients with developmental delay. Cells, 10(6). 10.3390/cells10061289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wyles J. P., McMaster C. R., Ridgway N. D. (2002). Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum. Journal of Biological Chemistry, 277(33), 29908–29918. 10.1074/jbc.M201191200 [DOI] [PubMed] [Google Scholar]
  26. Zhang S. L., Yu Y., Roos J., Kozak J. A., Deerinck T. J., Ellisman M. H., Stauderman K. A., Cahalan M. D. (2005). STIM1 Is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature, 437(7060), 902–905. 10.1038/nature04147 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zhang Y., Song G., Lal N. K., Nagalakshmi U., Li Y., Zheng W., Huang P. J., Branon T. C., Ting A. Y., Walley J. W., Dinesh-Kumar S. P. (2019). TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity. Nature Communications, 10(1), 3252. 10.1038/s41467-019-11202-z [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Contact are provided here courtesy of SAGE Publications

RESOURCES