A Strain: fl/fl ∆hep Strain: fl/fl ∆hep AnalysisAAV: ofLuc the ironLuc chaperonePCBP1-WT PCBP1PCBP1-∆Fe interactome:PCBP1-∆RNA PCBP1 variant intersection of DNA repair and iron traffickingAAV: Luc Luc WT ∆Fe ∆RNA PCBP1 −40 Lorena Novoa-Aponte a*, Sarju J. Patel a, Olga Protchenko a, James Wohlschlegel b, Caroline C. Philpott a −40 PCBP1, IHC PCBP1, GAPDH - a Genetics and Metabolism Section,a NIDDK, NIH, Bethesda, MD, USA. b Department of Biological Chemistry, UCLA, Los Angeles, CA, USA C.C. Philpott, et al. *[email protected] 80 B P = 0.0262 60 40 1. Iron is essential but toxic 2. Increased DNA damage in cells lacking PCBP1 ns H&E ? + 20 C.C. Philpott, et al. Iron is used as an essential cofactor by several enzymes involved in DNA ⦿ Increased TUNEL in cells and tissue livers from mice lacking PCBP1. ? replication and repair. However, unchaperoned iron promotes redox ⦿ PCBP1 binds both, iron and single-stranded nucleicTG, nmol/mg protein 0 acids. stress that may affect DNA stability. Strain: Δhep Δhep Δhep ? ⦿ The iron binding activity of PCBP1 controls suppressionAAV: of DNA damage C WT ∆Fe ∆RNA PCBP1 variant ? Iron storage HEK293 cells Mouse Liver ? A 8 siNT+P1var 100 ? A 8 var siPCBP1 2 ? Mammals use the iron siNT+P1 ns var Fe-S cluster assembly 6 siPCBP1+P1siPCBP1 80 ADI1 TUNEL chaperone PCBP1 to var ? 6 siPCBP1+P1 metalate several iron- 60 = 0.0468 = 0.0354 = 0.0465 cell / mm population P P P population + dependent enzymes + 4 + 4 ? 40 Degradation of HIF1α Fig. 3. Iron chaperone-mediated handling of cytosolic labile iron pool. Divalent metal transporter 1 (DMT1) and Zip14, located on plasma membrane or endocytic vesicles, imports non-transferrin-bound or transferrin-bound iron, 20 respectively. Iron enters the cytosolic labile iron pool (LIP) as Fe(II) and is largely coordinated by reduced GSH and bound to iron chaperones, PCBPs. PCBP1 and 2 2 TUNEL TUNEL PCBP2 efficiently distribute iron for storage, non-heme iron cofactor assembly, or efflux through ferroportin (Fpn1). a, PCBP1 (and to a lesser extent, PCBP2) TUNEL % facilitates iron sequestration into ferritin,? which can be delivered to mitochondria by unknown mechanism for heme or FeeS cluster synthesis. b, PCBP1 form a TUNEL % 0 complex with cytosolic BolA2 via a bridging Fe-GS ligand. Iron provided by PCBP1-BolA2 complex can be combinedTUNEL with sulfur (S) compound, with the help of other Translation/ of polyproline motifs 0 cytosolic FeeS cluster assembly proteins and facilitateADI1 the [2Fee2S] cluster formation on BolA2-Glrx3 distribution system. c, PCBP1 delivers iron to mononuclear Strain: fl/fl Δhep Δhep Δhep Δhep P1var: WT - WT ∆RNA - ∆RNA ∆Fe - ∆Fe iron enzyme prolyl hydroxylase (PHD2), which regulates the degradation of hypoxia inducible factor 1-α (HIF1α). d, PCBP1 also metallates the dinuclear iron 0 AAV: Luc Luc WT ∆Fe ∆RNA enzyme deoxyhypusine hydroxylase (DOHH), which catalyzes the hydroxylation of hypusine (Hpu) on eukaryotic translation initiation factor 5A (elF5A) to promote P1var: WT - WT ∆RNA - ∆RNA ∆Fe - ∆Fe translation of polyproline motifs. DAPI Philpott, CC, et al. BBA - Molecular Cell Research (2020) Sarju, P. et al. PNAS (2021) PCBP1 variant state [31]. For this reason, PCBP1 cannot be delivering iron directly to PCBP1. Again, PCBP1 demonstrated iron-dependent binding to DOHH B siNT siPCBP1 Fig.the 3. Iron enzymatic chaperone-mediated active site handling but instead of cytosolic is likely labile releasing iron pool. iron to the in cells by coimmunoprecipitation. Because ferritin and the non-heme siNT + P1var siPCBP1 + P1var Divalentcarboxylate metal transporter residues that 1 (DMT1) line the and pore Zip14, of 3-fold located symmetry on plasma that membrane leads to or endocyticiron enzymes vesicles, targeted imports non-transferrin-bound by PCBP1 are structurally or transferrin-bound dissimilar, we iron, have B 150 siNT siPCBP1 respectively.the interior. Iron entersThis mechanism the cytosolic of labile associative iron pool transfer (LIP) as makes Fe(II) and the is role largely of a coordinatedproposed by reduced that the GSH interactions and bound are to iron largely chaperones, mediated PCBPs. through PCBP1 bridging and D 400 ns PCBP2bridging efficiently iron distribute ligand more iron likely.for storage, non-heme iron cofactor assembly, or effluxiron through ligands ferroportin that may (Fpn1). include a, PCBP1 GSH. (and Additional to a lesser electrostatic extent, PCBP2) interac- var var facilitates iron sequestration into ferritin, which can be delivered to mitochondria by unknown mechanism for heme or FeeS cluster synthesis. b, PCBP1 form a siNT + P1 PCBP1 can deliver iron to non-heme enzymes with mono- and di- tions, for example, cannot be ruled out, but may not be primarily re- 150 siPCBP1 + P1 complexnuclear with iron cytosolic centers BolA2 (Fig.via a 3 bridgingc, d) [32 Fe-GS]. This ligand. activity Iron provided was initially by PCBP1-BolA2 ob- sponsible complex can for be complex combined formation. with sulfur (S) compound, with the help of other cytosolic FeeS cluster assembly proteins and facilitate the [2Fee2S] cluster formation on BolA2-Glrx3 distribution system. c, PCBP1 delivers iron to mononuclear 300 served in the prolyl hydroxylases (PHDs) that regulate the degradation 100 iron enzyme prolyl hydroxylase (PHD2), which regulates the degradation of hypoxia inducible factor 1-α (HIF1α). d, PCBP1 also metallates the dinuclear iron of hypoxia inducible factor 1-α in cells [33]. Cells from which PCBP1 is e enzyme deoxyhypusine hydroxylase (DOHH), which catalyzes the hydroxylation of hypusine5. Interaction (Hpu) on eukaryotic of PCBP1 translation with cytosolic initiation factor Fe S 5A assembly (elF5A) to machinery promote translationdepleted of polyprolineexhibit loss motifs. of the iron cofactors from PHD2 and concomitant through BolA2 = 0.0159 = 0.0255 loss of prolyl hydroxylase activity, reduction of hydroxylated HIF1-α, 200 = 0.0079 P P P and accumulation of active HIF1-α. Similarly, PCBP1-depleted cells A proteomics analysis of the PCBP1 interactome indicated that 100 state [31]. For this reason, PCBP1 cannot be delivering iron directly to PCBP1. Again, PCBP1 demonstrated iron-dependent binding to DOHH 50 ALT, U/L ALT, exhibit loss of activity of the related asparagyl hydroxylase and PCBP1 PCBP1 was capable of binding many different protein components in % Cell Viability the enzymatic active site but instead is likely releasing iron to the in cells by coimmunoprecipitation. Because ferritin and the non-heme was found to directly interact with both of these mononuclear iron the cell, including ferritins, other PCBPs, and some non-heme iron en- 100 carboxylate residues that line the pore of 3-fold symmetry that leads to iron enzymes targeted by PCBP1 are structurally dissimilar, we have enzymes. A recent publication also indicates that acireductone dioxy- zymes. Inspection of the interactome for possible co-chaperone, rather the interior. This mechanism of associative transfer makes the role of a proposed that the interactions are largely mediated through bridging genase 1, a mono-nuclear iron enzyme involved in the methionine than client, proteins revealed BolA2 as a candidate (Fig. 3b) [26]. bridging iron ligand more likely. iron ligands that may include GSH. Additional electrostatic interac- salvage pathway, is also metallated by PCBP1 in cells [34]. BolA2 homologues have been characterized in budding and fission 0 0 PCBP1 can deliver iron to non-heme enzymes with mono- and di- tions, for example, cannot be ruled out, but may not be primarily re- 50 Deoxyhypusine hydroxylase (DOHH) is a di-nuclear iron enzyme yeast as components of a cytosolic/nuclear [2Fee2S] carrier complex EV P1-WT P1-∆RNA P1-∆Fe nuclear iron centers (Fig. 3c, d) [32]. This activity was initially ob- sponsible for complex formation. Strain: % Cell Viability that is structurally unrelated to PHDs or ferritins, but also depends on that includes the cytosolic monothiol glutaredoxin, Grx3 [36–40]. In fl/fl Δhep Δhep Δhep Δhep served in the prolyl hydroxylases (PHDs) that regulate the degradation PCBP1 for efficient metalation of its peroxo-bridged, di-nuclear iron yeast this complex is proposed to transfer [2Fee2S] clusters to iron- PCBP1-Flag Variants of hypoxia inducible factor 1-α in cells [33]. Cells from which PCBP1 is e AAV: Luc Luc WT ∆Fe ∆RNA center [35]. Cells lacking PCBP1 or PCBP2, but not wild type HEK cells, 5. Interactionregulatory transcription of PCBP1 with factors, cytosolic which Fe directlyS assembly affect machinery their activity depleted exhibit loss of the iron cofactors from PHD2 and concomitant lost DOHH activity when exposed to low levels of iron chelators. Iron through[41–44 BolA2]. In mammalian cells, BolA2-Glrx3 complexes also function as loss of prolyl hydroxylase activity, reduction of hydroxylated HIF1-α, cofactors were selectively lost from DOHH under these conditions and [2Fe-2S] chaperones; the only known recipient of these clusters in Fig. 5 | RNA or Iron binding activityPCBP1 of PCBP1 variant are associated with different phenotypes in PCBP1 and accumulation of active HIF1-α. Similarly, PCBP1-depleted cells 0 could be restored in vitro with iron or with cell lysates containing Amammalian proteomics cells analysis is Ciapin of 1, the an PCBP1early-acting interactome component indicated of the cytosolic that depleted cells. (A) Iron binding activity of PCBP1 is required to suppress DNA damage in PCBP1- exhibit loss of activity of the related asparagyl hydroxylase and PCBP1 PCBP1 was capable of binding many different protein components in EV P1-WT P1-∆RNA P1-∆Fe was found to directly interact with both of these mononuclear iron depleted cells. Tetracycline-inducible stable cell lines expressing PCBP1-Flag variants of wild type (WT), the cell, including ferritins, other PCBPs, and some non-heme iron en- enzymes. A recent publication also indicates that acireductone dioxy- zymes. Inspection of the interactome for possible co-chaperone, rather RNA-binding mutant (∆RNA) or iron-binding mutant (∆Fe)PCBP1-Flag were transfected Variants with nontargeting (NT) or genase 1, a mono-nuclear iron enzyme involved in the methionine than client, proteins revealed BolA2Fig.