Caspase cleavage releases a nuclear protein fragment that stimulates phospholipid scrambling at the plasma membrane

•cDNA library screening identified the constitutively active form of Xkr4•Revival using sgRNA XRCC4 as an Xkr4 activator•XRCC4 is cleaved by caspase to release its C-terminal fragment cytoplasm•Protein interaction showed that binds dimer Phospholipid scrambling in dying cells promotes phosphatidylserine exposure, a critical process for efferocytosis. We previously Xkr family protein phospholipid-scrambling protein, but activation mechanisms remain unknown. Here we show activated two steps: formation caspase-mediated cleavage and structural change caused activating factors. To identify factors, developed new system, “revival screening,” CRISPR library. Applying this nuclear single candidate activator. Upon apoptotic stimuli, XRCC4, contained DNA repair complex, caspases, with intrinsically disordered region released into cytoplasm. Protein interacts directly activate it. This study demonstrates releases direct regulation lipid dynamics on plasma membrane. Phospholipids membrane are distributed asymmetrically: (PS) located at inner layer membrane, whereas phosphatidylcholine (PC) outer (Leventis Grinstein, 2010Leventis P.A. Grinstein S. The distribution function cellular membranes.Annu. Rev. Biophys. 2010; 39: 407-427Crossref PubMed Scopus (593) Google Scholar; van Meer et al., 2008van G. Voelker D.R. Feigenson G.W. Membrane lipids: where they how behave.Nat. Mol. Cell Biol. 2008; 9: 112-124Crossref (4030) Scholar). However, asymmetry destroyed some physiological situations. For instance, PS externalized cell surface platelets functions scaffold coagulation factors be (Zwaal 2005Zwaal R.F. Comfurius P. Bevers E.M. Surface exposure pathological cells.Cell. Life Sci. 2005; 62: 971-988Crossref (608) A defect causes bleeding disorder Scott syndrome. In other situations, exposed “eat me” signal dead engulfed phagocytes; defects cause autoimmune diseases (Nagata, 2018Nagata Apoptosis Clearance Apoptotic Cells.Annu. 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Xk-related 8 CED-8 promote cells.Science. 341: 403-406Crossref (321) 2014Suzuki during apoptosis.J. 2014; 289: 30257-30267Abstract (84) major contributors scrambling. Among these, Xkr8, Xkr4, Xkr9 their cytoplasmic regions forms after upon stimuli expose 2016Suzuki complex exposure.Proc. Natl. Acad. USA. 113: 9509-9514Crossref (59) It remains whether or sufficient members. study, representative from family, not activity, suggesting required Xkr4. elucidate activation, performed three kinds unbiased screening: cDNA screening, mass spectrometry-based well newly system utilizing CRISPR-Cas9 library, call screening.” Based combination revealed involved caspases cytoplasm, which, turn, can regulate shows elicit concealed fragments death. C terminus apoptosis; however, alone enough living clarify role first investigated assaying incorporation extracellularly added fluorescent PC (NBD-PC). Although parental PLB cells, deficient scrambling, did incorporate treatment stimulus agent staurosporine (STS), those expressing full-length (FL) (?C) STS stimulation (Figures 1A S1A) activity-dependent manner (Figure S1B), events besides cleavage. Biochemical analysis blue native PAGE (BN-PAGE) Xkr4?C formed unstimulated S1C), necessary protein. understand sought obtain possibly mutations pathway. Two ubiquitous Scholar), were knocked out Ba/F3 (BDKO), Xkr4?C, tagged V5 N FLAG terminus, was electroporated stable transformants generated. BDKO stably (BDKO Xkr4?C) subjected uptake assay, high-lipid-scrambling (1%) collected flow cytometry expanded 1B S1D). When repeated 6 times, (PC6) obtained 1C Constitutive dependent because deleting against suppressed S1E). reveal constitutive libraries high weight (HMW; 2.5–6.0 kbps) low (LMW; 1.0–2.5 generated PC6 inserted directionally retroviral vector, HMW expressed cells. After third round sorting, populations (1LPC3) enriched 1D, left side), confirming positive cDNAs. 1LPC3 then genomic preparation, followed amplification integrated cDNAs PCR. Because PCR products appeared several bands S1F), prepared second 1E) sorting only once (2LPC1) right them analysis, nearly band vector analyzed sequencing. BLAST search all sequenced Xkr4?C. Unexpectedly, clones point mutation: I322S, L331F, Q332E 1F S1G), that, six-round shown Figure 1B, itself mutated acquire different idea supported independent expression LMW; same mutants. mutants without activities observed even any 2A, 2B, S2A). Insertion mutation Xkr4FL confer 2A 2B), indicating coupling deletion. BN-PAGE stayed monomer larger 2C), mutant and, possibly, S2B). also suggests dimer. hypothetical 1), context utilize general, enrichment target sgRNAs difficult such do proliferate. overcome difficulty, revival screening. With method, infected lentiviral revived generation next difficulty death, digested caspase-activated DNase CAD (Enari 1998Enari Sakahira Yokoyama Okawa Iwamatsu A. degrades apoptosis, inhibitor ICAD.Nature. 1998; 391: 43-50Crossref (2757) solve problem, knockout (KO) transformed transient CAD. resultant KO S3A–S3C) fused tagRFP (Xkr4?C-RFP), lentiviruses encoding (containing targets per gene) (Sanjana 2014Sanjana N.E. Shalem O. Zhang F. Improved vectors genome-wide screening.Nat. Methods. 11: 783-784Crossref (2236) stimulated 3 h, assay. Xkr4-expressing “RFP-positive” scrambling-defective “PC uptake-negative” cytometry, intact (because deficiency) used sgRNA-encoding amplified lentivirus production, 3A). repeating incorporation-defective (sgPC3 cells) 3B). another (sgPC4) subsequent next-generation sequencing (NGS) gene mapping. excluding had one targets, genes ranked based scores total reads 3C S3D; Table S1). Cytochrome c (CYCS) APAF1, both caspase-9 upstream caspase-3 S3E), found between 14th rankings, our functioning, further narrow down downstream caspase-3. genes, sgPC4 lower infectivity (10%) Xkr4?C-RFP process. Following this, sgPC5 again 10% infectivity, RFP-positive uptake-negative fixed paraformaldehyde (PFA), permeabilized methanol, stained anti-active antibody, caspase-3-positive From population (sgPC6), prepared, 3D). Genomic sgPC7 amplify regions, NGS, As result, gene, higher than CYCS APAF1 3E; S2), more 40-fold compared NGS data 3F). These results strongly indicate factor confirm caspase-3, introduced antibody. clearly affected (sgXRCC4) 4A, top panel), expected. assay sgXRCC4 lost bottom panel). Expression Q332E-expressing alter S4A), bypassed requirement activation. completely S4B S4C), Xkr4-mediated entirely absent 4C). been suggested possesses site amino acid 265 (Lee 2002Lee K.J. Dong X. Wang Takeda Y. Dynan W.S. 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Non-homologous end joining alternative pathways double-strand break repair.Nat. 2017; 18: 495-506Crossref (615) therefore questioned it investigate localization confocal microscopy. contrary expectation, containing (NLS) diffused cytoplasm N-terminal 4D). contrast, caspase-cleavage-deficient suggest location breaks. Therefore, examined effects WT repair. ? irradiation 10 Gy, cultured 12–24 h check viability. viability altered lack when checked 12 irradiation, reduced 24 5A). Exogenous restored viability, retains ability damaged DNA. 40 apoptosis 5B). Under condition, absence 5C) 5D). damage-induced apoptosis. generality XRCC4-mediated extrinsic pathway examined. express death receptor Fas, tried exogenously highly Fas-expressing generate. Even Fas expression, ligand, S5A). Lipid 5E). UV (2,000 J/m2) induce S5B). stimulation, 5F), fragment-mediated variety stimuli. recognized phagocytes, thioglycollate-elicited macrophages incubated irradiation-induced WT, 2DA, engulfment 5G S5C). various contributes determine series deletion constructed. part dimerization domain binding domains IV, important Grawunder 1997Grawunder U. Wilm Wu Kulesza Wilson T.E. Mann Activity mammalian cells.Nature. 1997; 388: 492-495Crossref (517) Junop 2000Junop M.S. Modesti Guarné Ghirlando R. Gellert Yang W. Crystal structure Xrcc4 implications joining.EMBO 19: 5962-5970Crossref (140) unnecessary sequential (XRCC4/116–336, XRCC4/156–336, XRCC4/204–336, XRCC4/248–336, XRCC4/256–336) still promoted 6A, 6B, S6A, S6B). Instead, complete up caspase-cleavage (XRCC4/266–336: XRCC4/C) failed fully S6C). Compared does have known partners (analyzed IUPred2A; Mészáros 2018Mészáros B. Erdos Dosztányi Z. IUPred2A: context-dependent prediction redox state binding.Nucleic Acids Res. 46: W329-W337Crossref (437) S6D). (XRCC4/1–285 XRCC4/1–305) (XRCC4/1–265: XRCC4/N) hardly S6C, S6E). mutants, XRCC4/256-285 (Mini), 20 acids (aa) XRCC4/C aa XRCC4/N, constructed, expressed, 6B S6F). results, speculated needs hydrophobic isoleucine fragment, conserved among species S6G) start codon methionine construct disrupts activity. I266G constructed expressed. expected, efficiently exposing S6H). (C20) XRCC4/C, C20 (C20/I266G), addition (C21) synthesized artificially 6C) electroporation. C20/I266G C21, 6D 6E), Xkr4FL-expressing peptides, activates amino-acid residues NLS, positively charged FL XRCC4. Four (R270A, K271A, R272A, R273A) except R275A enter localized 6F). (K271A, 6G S6I), entry On hand, R270A arginine residue peptide R270 K271 position Xkr4?C-expressing C20/R270A, C20/K271A, significance S6J). presence fragment. SPOT (Traenkle 2015Traenkle Emele Anton Poetz Haeussler R.S. Maier Kaiser P.D. Scholz A.M. Nueske Buchfellner al.Monitoring interactions endogenous beta-catenin intracellular nanobodies cells.Mol. Cell. Proteomics. 2015; 14: 707-723Abstract (49) tags (SPOT-Xkr4?C-FLAG), respectively, S7A). SPOT-Xkr4?C-FLAG efficient S7B). Then fractions solubilized detergent, immunoprecipitated anti-SPOT nanobody-conjugated magnetic agarose beads, trypsin, spectrometry. surprise, label-free quantification most apoptosis-dependent R270A-sensitive interactors 7A; S3). adjacent tryptic peptides spectrometry reside 7B). Targeted parallel reaction monitoring (PRM) increase WT- R270A-expressing 7C). detection XRCC4-RFP anti-RFP antibody immunoprecipitants lysates; could bind 7D). conjugated tetramethylrhodamine (C20-TMR) HCT116 Xkr4FL-GFP Xkr4?C-GFP. electroporation, C20-TMR retained Xkr4?C- 7E). association 7F). types infection retroviruses (?3–5 viral infections cell) ensure contains clones. many resulting PCR-mediated us