Hsp90 chaperones hemoglobin maturation in erythroidand nonerythroid cellsArnab Ghosha,1, Greer Gareea, Elizabeth A. Sweenya, Yukio Nakamurab, and Dennis J. Stuehra,1

aDepartment of Pathobiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195; and bCell Engineering Division, RIKEN BioResourceCenter, 305-0074 Ibaraki, Japan

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 27, 2017 (received for review October13, 2017)

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramersrequires that heme be incorporated into each globin. While hemo-globin alpha (Hb-α) relies on a specific erythroid chaperone (alphaHb-stabilizing protein, AHSP), the other chaperones that may helpmature the partner globins (Hb-γ or Hb-β) in erythroid cells, or mayenable nonerythroid cells to express mature Hb, are unknown. Weinvestigated a role for heat-shock protein 90 (hsp90) in Hb matu-ration in erythroid precursor cells that naturally express Hb-α witheither Hb-γ (K562 and HiDEP-1 cells) or Hb-β (HUDEP-2) and innonerythroid cell lines that either endogenously express Hb-αβ(RAW and A549) or that we transfected to express the globins.We found the following: (i) AHSP and hsp90 associate with distinctglobin partners in their immature heme-free states (AHSP withapo-Hbα, and hsp90 with apo-Hbβ or Hb-γ) and that hsp90 doesnot associate with mature Hb. (ii) Hsp90 stabilizes the apo-globinsand helps to drive their heme insertion reactions, as judged bypharmacologic hsp90 inhibition or by coexpression of an ATP-asedefective hsp90. (iii) In nonerythroid cells, heme insertion into allglobins became hsp90-dependent, which may explain how mixedHb tetramers can mature in cells that do not express AHSP. To-gether, our findings uncover a process in which hsp90 first binds toimmature, heme-free Hb-γ or Hb-β, drives their heme insertionprocess, and then dissociates to allow their heterotetramer forma-tion with Hb-α. Thus, in driving heme insertion, hsp90 works inconcert with AHSP to generate functional Hb tetramers duringerythropoiesis.

heme | erythropoiesis | hemoglobin | hemeprotein | nonerythroid

Hemoglobin (Hb) functions in oxygen delivery by virtue of itsheme prosthetic group (1–3). Its expression and maturation

is tightly coordinated during erythropoiesis and is subject tomultiple levels of regulation (4–6), with disregulation manifest-ing in some forms of β-thalassemia (7–9) and anemia (10–12). Inmammals, erythropoiesis involves a coordinated synthesis ofpartner globin chains, heme insertion, and globin interactionsteps that ultimately create functional fetal (α2γ2) Hb or adult(α2β2) Hb tetramers (13). Our understanding of Hb maturation isstill incomplete, and in particular, we do not know the details ofHb heme insertion or what partner proteins may assist the glo-bins during this process. Previous studies identified a role for cellchaperones, specifically hsp70, in stabilizing Hb-α and preventingits aggregation in cells during maturation (14, 15). In addition,Hb-α maturation in erythrocytes is aided by the erythroid-specific α-hemoglobin–stabilizing protein (AHSP), which bindsto nascent Hb-α to stabilize it against aggregation or degrada-tion and to down-regulate its superoxide production once it hasincorporated heme (16–18). However, what other chaperones,if any, might perform similar functions to assist maturation ofthe partner globin chains (Hb-γ and Hb-β), or might substi-tute for AHSP in nonerythroid cells that express Hb-α (19, 20),is unknown.To address these questions, we studied Hb maturation as it

occurs in the human erythroid leukemia cell line K562 (21, 22),in the human erythroid progenitor cells HiDEP-1 and HUDEP-2

during their differentiation, and in nonerythroid cell lines thateither naturally or transiently expressed Hb, keying on a possiblerole for the cell chaperone hsp90 (23, 24). Our findings revealthat hsp90 chaperones heme insertion into Hb-β and Hb-γ, andin some circumstances, into Hb-α, and thus plays a fundamentalrole in forming mature functional fetal (α2γ2) or adult (α2β2) Hbtetramers in both erythroid and nonerythroid cells.

ResultsHsp90 Associates with Hb in Erythroid-Like Cells and Is Required forHeme Insertion. We first examined if hsp90 associates with Hb inthe K562 human erythroid leukemia cell line (21, 22), whichconstitutively expresses fetal Hb (α2γ2) and can be induced toexpress greater levels when the cells are given hemin, whichpromotes their differentiation toward erythrocytes (21, 22). Cellswere given hemin over a 3-d period, and the cell supernatants weresubject to spectral analysis and immunoprecipitation (IP). Thespectral traces of clarified cell supernatants (equal protein amounts)in Fig. 1A show that Hb protein expression increased steadily overthe 3-d treatment and that this was associated with a twofold in-crease in hsp90 expression in the first 24 h (Inset). Hsp90 associatedwith fetal Hb in proportion to the level of Hb expression (Fig. 1B) inthe resting cells and during their heme-induced induction.We next determined if two hsp90 inhibitors that work by dif-

ferent mechanisms (25) (radicicol or novobiocin) would acutelyblock heme insertion into heme-free Hb that had accumulated inK562 cells that had been cultured with succinyl acetone (SA) tomake them heme-deficient (23, 24). The heme-deficient cellswere given hemin (5 μM) with or without either inhibitor for 3 h,and then the level of heme-replete Hb that formed was determined

Significance

Maturation of functional adult (α2β2) or fetal (α2γ2) hemoglobin(Hb) tetramers requires that a heme cofactor be incorporated intoeach globin. During erythropoiesis, Hb-α maturation is aided bythe alpha Hb-stabilizing protein (AHSP), but what enables thematuration and heme insertion of the other globins is unknown.We found that chaperone hsp90 stabilizes the immature, heme-free forms of Hb-β and Hb-γ and then drives their heme insertionreactions in an ATP-dependent process. This finding fills an im-portant gap in our understanding of hemoglobin maturationduring erythropoiesis and also helps to explain how functionalmature Hb can be expressed outside the circulation in non-erythroid cells and tissues that do not express AHSP.

Author contributions: A.G. and D.J.S. designed research; A.G., G.G., and E.A.S. performedresearch; Y.N. contributed new reagents/analytic tools; A.G. and D.J.S. analyzed data; andA.G. and D.J.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: stuehrd@ccf.org or ghosha3@ccf.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717993115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1717993115 PNAS | Published online January 22, 2018 | E1117–E1126

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by spectral analysis of the cell supernatants, which measured theheme content of Hb from the Soret absorbance peak at 414 nm(Fig. 1 C and D, equal amounts of total protein). The traces andassociated Western blot show that the heme-deficient cells con-tained Hb that was predominantly heme-free but could incorpo-rate the added heme over the 3-h period. Both hsp90 inhibitorsinhibited the heme insertion, indicating that active hsp90 was re-quired for the heme insertion into the apo-Hb.

Hsp90 Association Is Globin-Specific. Supernatants from K562 cellsthat were cultured without any added heme were subject to IPusing globin-specific antibodies, followed by Western analysis toobserve the globin hsp90 or AHSP associations (Fig. 2). Verylittle hsp90 was found associated with Hb-γ in the cells under thiscircumstance, but the level of hsp90 association increased if theK562 cells had been made heme-deficient before the experi-ment. This increased hsp90 association was then lost when the

Fig. 1. Hsp90 interacts with Hb and is needed for heme insertion during Hb maturation. (A and B) K562 cells were treated with 50 μM hemin over a 3-dperiod, and supernatants were generated every 24 h and used for absorption spectra and IPs. (A) Spectra show that Hb absorbance increases during the hemintreatment. (Inset) Western expression and cumulative densitometry data show that cell hsp90 expression increased after 24 h of hemin treatment. Valuesdepicted are mean ± SD of n = 3 experiments (*P < 0.05, by one-way ANOVA). (B) IP shows bound hsp90 and Hb-γ (input 20%) retained on the beads. Lane1 in the Upper two panels is a no-antibody control and indicates the input supernatant (Sup) from the 48-h time point minus the Hb(βγ) antibody (Ab). TheLower four panels show protein expression levels of hsp90 and Hb-γ (from 24 to 72 h of hemin treatment) from 8 and 15% SDS/PAGE, respectively, withβ-actin as the loading control. (C and D) K562 cells were heme-deprived by treating with SA for 3 d and then were incubated for 3 h with 5 μM of hemin in thepresence or absence of the hsp90 inhibitors radicicol or novobiocin. Cell supernatants (equal protein) were analyzed for absorption spectra and Hb-γ ex-pression. (C) UV-visible spectra of the supernatants and effect of hsp90 inhibition. (Inset) Hb protein expression levels. (D) The calculated Hb-heme content ofthe supernatants as depicted in A, using the Soret absorbance at 414 nm. Results are mean ± SD of n = 3 experiments (*P < 0.05, by one-way ANOVA).

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heme-deficient K562 cells were given heme for 3 h, which allowedthem to generate heme-replete Hb (Fig. 2 A and B). In compar-ison, hsp90 was not associated with Hb-α under any of the threeconditions (Fig. 2C) and was instead associated with AHSP, whichis known to bind exclusively to Hb-α (16–18) (Fig. 2 C and D). Asseen for the hsp90 association with Hb-γ, the AHSP associationwith Hb-α was strongest under the heme-deficient condition anddropped off in the heme-replenished cells. Protein expressionlevels of hsp90, AHSP, Hb-γ, and Hb-α were unchanged underthe different cell culture conditions (Fig. 2E). A specificity ofhsp90 toward Hb-γ was also seen during the hemin-induced dif-ferentiation in the K562 cells (Fig. 2 F and G). Thus, hsp90associated with Hb-γ only in its heme-free state and did notassociate with Hb-α under any condition.

Hsp90 Drives Globin Maturation During Erythropoiesis. We thenperformed experiments with the human erythroid progenitorcells HiDEP-1 and HUDEP-2, which can be induced by culturingin differentiation media with erythropoietin (EPO) and growthfactors to undergo full differentiation to erythroid cells thatproduce fetal (Hb-α/γ) or adult (Hb-α/β) hemoglobins, re-spectively (26). We studied the cells over a 16-d differentiationperiod (Fig. S1). Fig. 3 A–D and G–J shows spectroscopic andWestern results that document the time-dependent buildup ofheme-containing and total Hb proteins along with the expressionlevels of other proteins of interest in the HiDEP-1 and HUDEP-2cells. As expected, the levels of total and heme-replete globinsincreased during the differentiation period, with the heme-replete globin levels peaking between days 2–6 (Fig. 3 A, B, G,

and H and Figs. S2 and S3). The pulldown results in Fig. 3 E, F,K, and L show that the globins maintained their individualspecificities in associating with either AHSP or hsp90. Hsp90-associated Hb-γ or Hb-β and AHSP-associated Hb-α proteinswere observed over the entire course of the differentiation, al-though the chaperone association levels decreased with time inthe HUDEP-2 cells (Fig. S4). An association between hsp90 andHb-β could also be visualized in intact HUDEP-2 cells using theproximity ligation assay (PLA) protocol (Fig. 3 M–R).We next investigated if hsp90 inhibition would impact Hb-β heme

insertion and Hb maturation in HUDEP-2 cells. The cells weremade heme-deficient and then had hemin added with or withoutthe hsp90 inhibitors radicicol or AUY-922. Fig. 4 shows that theheme deficiency led to a buildup of apo-Hb–β and a diminishedlevel of mature Hb-α/β in the cells without altering the expressionlevels of either globin or related proteins of interest. Bothhsp90 inhibitors blocked heme insertion into apo-Hb–β and pre-vented the concomitant formation of mature Hb-α/β. IP experi-ments in Fig. 4D show that hsp90 dissociated from Hb-β after theheme reconstitution, but did not do so when radicicol was alsopresent to prevent heme insertion into Hb-β. AUY-922, unlikeradicicol, allowed dissociation of the hsp90–apo–Hbβ complex,despite preventing heme insertion (Fig. 4 A, C, and D). In all cases,the IP data (Fig. 4D) showed that the hsp90 association with Hb-βinversely correlated with an elevated Hb-αβ association, whichsignificantly fell in the presence of both hsp90 inhibitors. To-gether, our results establish that hsp90 chaperones Hb maturationin differentiating erythroid cells by exclusively associating with

Fig. 2. Hb-α and Hb-γ selectively interact with chaperone AHSP or hsp90. (A–E) K562 cells were cultured in normal media or were made heme-deficient bytreating with SA for 72 h before experiments. In some cases, the heme-deficient cells were then incubated for 3 h with 5 μM of hemin. (F and G) Hb expressionin K562 cells was induced by adding hemin (50 μM) for 72 h. Soluble cell supernatants were prepared every 24 h and analyzed by IP and Western blot todetermine Hb-α and Hb-γ interactions with chaperons (Hsp90 and AHSP). (A) IP of Hb-γ showing bound hsp90 and Hb-γ (input 20%) retained on the beads.(B) Densitometric quantification of bound hsp90. (C) IP of Hb-α comparing bound hsp90 and AHSP (input 20%). Input Sup-Ab is a no-antibody control andindicates the input supernatant (Sup) minus the Hb(α) antibody (Ab). (D) Densitometry of AHSP bound to Hb-α. (E) Expression levels derived from 8% (Uppertwo panels) and 15% (Lower four panels) SDS/PAGE, with β-actin as loading control. In B and D, values depicted are mean ± SD of three independent ex-periments (n = 3) (*P < 0.05, by one-way ANOVA). (F) IP of Hb-α showing bound AHSP and Hb-γ (input 20%) retained on the beads. (G) Expression levels in cellsupernatants as indicated from 8% (Upper two panels) and 15% (Lower four panels) SDS/PAGE.

Ghosh et al. PNAS | Published online January 22, 2018 | E1119

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Fig. 3. Distinct Hsp90 and AHSP globin interactions occur in erythroid progenitor cells during red blood cell differentiation. Erythroid progenitor cells HiDEP-1or HUDEP-2 were induced to differentiate for a 16-d period, cell aliquots were harvested before or during differentiation, and the soluble supernatants wereanalyzed. (A and G) Representative UV-visible spectra of cell supernatants (equal protein) created from cells before (day = 0) or during differentiation. (B andH) The calculated Hb heme content in HiDEP-1 and HUDEP-2 supernatants depicts a mean of n = 2 experiments. (C and I) Mean densitometry of Hb proteinexpression levels as depicted in D and J, from n = 2 experiments, during differentiation. (D and J) Hb, AHSP, and hsp90 expression levels depicted in 15 and 8%SDS/PAGE, respectively, during differentiation, with β-actin as loading control. (E, F, K, and L) IP showing AHSP bound to Hb-α or hsp90 bound to Hb-β and Hbαbound to Hbγ/Hbβ (input 20% in all cases). Western blots and IPs are representative, and the differentiation experiments were repeated two times withsimilar results. (M–R) Representative images from PLAs showing colocalization of hsp90 and Hb-β in undifferentiated (day = 0) HUDEP-2 cells. (M) Controlusing no primary antibody, (N) Hb-β antibody alone, (O) Hsp90 antibody alone, and (P–R) PLA images with both Hb-β and hsp90 antibodies with Insetsshowing close-ups. Red dots indicate the location and extent of interaction.

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either apo-Hb–γ or apo-Hb–β and by driving their heme insertionreactions in an ATP-dependent manner. The hsp90 then dissoci-ates after heme insertion takes place to allow heterotetramerformation between the heme-replete Hb-γ or Hb-β and Hb-α.

Hsp90 Drives Globin Maturation in Nonerythroid Cells.Hb-α and Hb-βare also expressed singly or in combination in diverse non-erythroid cell types, none of which naturally express AHSP (9,19, 20). To investigate hsp90 roles in this circumstance, we firstprobed the mouse macrophage (RAW 264.7) and human lungepithelium (A549) cell lines and confirmed that they expressheme-containing Hb-α and Hb-β (Fig. 5A and Fig. S5), eitherendogenously (A549) or after being immune-activated (RAW)(19, 27). AHSP protein was not expressed, and in this circum-stance the antibodies directed against either Hb-α or Hb-β pulleddown hsp90 in both cell types (Fig. 5B). The hsp90–globin in-teraction was confirmed by the proximity ligation assay (Fig. 5 C–F). These findings suggested that hsp90 may support maturationof both Hb-α and Hb-β in nonerythroid cells in the absence ofAHSP. To further investigate, we transfected HEK cells to ex-press Myc-tagged Hb-α or Hb-β, alone or in combination and withor without cotransfected AHSP. Individual transfection of Hb-α orHb-β led HEK cells to express similar levels of either globin (Fig. 6A and B). Cotransfection with AHSP boosted Hb-α expressionseveral fold, consistent with it stabilizing Hb-α (16–18), but re-pressed the expression of Hb-β (Fig. 6 A and B and Fig. S6). Whenthe HEK cells were cotransfected with Hb-α and Hb-β, their ex-pression levels increased beyond an additive effect (Fig. 6 A and B).Western analysis showed that a boost in Hb-α expression level wassimilar to when it was coexpressed with AHSP (Fig. 6A), suggestingthat there is a reduced reliance on AHSP when Hb-α and -β arecoexpressed in nonerythroid cells. Regarding hsp90, Fig. 6C showsthat hsp90 associated with Hb-β that was expressed either singly ortogether with Hb-α, whereas hsp90 did not associate with Hb-αwhen it was expressed alone or in combination with AHSP. This

suggests that the chaperone specificity toward individual globinsremains intact in nonerythroid cells.When the hsp90 inhibitors radicicol or AUY-922 were added

to the HEK cells during the 28-h cotransfection of Hb-α andAHSP, neither inhibitor altered the expression level of eitherprotein (Fig. 7A) or inhibited heme insertion into the Hb-α (Fig.7C). Likewise, adding either hsp90 inhibitor to heme-depletedHEK cells that were expressing apo-Hb–α and AHSP did notprevent the insertion of added hemin into the apo-Hb–α (Fig. 7B and D). In this circumstance, IP experiments showed thatAHSP was associated with Hb-α before and more weakly afterthe heme insertion took place, and no hsp90 was found associ-ated with Hb-α under any circumstance (Fig. 7E). This estab-lished that Hb-α heme insertion can remain hsp90-independentif AHSP is coexpressed along with it in nonerythroid cells.We next probed hsp90 involvement in heme-depleted HEK cells

that were cotransfected to express apo-Hb–α and apo-Hb–β in theabsence of AHSP. The heme-depleted cells expressed normal levelsof Hb-α and Hb-β proteins that, based on spectroscopic measures,were ≥60% heme-free, and a subsequent culture for 3 h with addedheme resulted in good heme incorporation into both apo-Hb–α andapo-Hb–β (Fig. 7 F and G). Heme incorporation restored the sta-bilization of a PAGE-resistant Hb dimer species that had been lostin the heme-depleted condition (Fig. 7F). Moreover, the spectralmeasures indicated that radicicol or AUY-922 inhibited the in-corporation of the added hemin into both apo-Hb–α and apo-Hb–βduring the 3-h reconstitution period (Fig. 7G). Thus, Hb-α hemeincorporation became hsp90-dependent in nonerythroid cells,where no AHSP is coexpressed. Pulldowns using an Hb-β–specificantibody showed that there was a strong hsp90 association with apo-Hb–β in the heme-depleted cells, which was lost after heme in-sertion took place, unless the hsp90 inhibitor radicicol was presentduring the heme reconstitution period (Fig. 7H, Upper, and Fig. 7I,Left). Also, the association of hsp90 with apo-Hb–β inversely cor-related with the stabilization of the PAGE-resistant Hb dimerspecies (Fig. 7H, Lower, and Fig. 7I, Right).

Fig. 4. Hsp90 inhibitors acutely block heme insertion and Hb maturation in HUDEP-2 cells. HUDEP-2 cells were made heme-deficient by a 5-d culture with SAand then were incubated for 3 h with 5 μM of hemin in the presence or absence of the hsp90 inhibitors radicicol (5 μM) or AUY-922 (300 nM), and the solublecell supernatants (equal protein) were analyzed. (A) UV-visible spectra of the supernatants. (B) Hb and hsp90 protein expression levels as analyzed in 15 and8% SDS/PAGE, respectively, with β-actin as loading control. (C) The Hb heme content of supernatants as calculated from spectra in A, mean ± SD of n =3 experiments (*P < 0.05, by one-way ANOVA). (D) IP of Hb-β in each supernatant compares levels of bound hsp90 or Hb-α (input 20% in all cases).

Ghosh et al. PNAS | Published online January 22, 2018 | E1121

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To investigate hsp90 involvement by an additional means, wecotransfected the HEK cells with Myc-tagged Hb-α and Hb-β,along with HA-tagged wild-type hsp90 or an ATPase-defectivehsp90 variant (D88N hsp90) that acts as a dominant negativeeffector of heme insertion into other hemeproteins (23, 24). Asshown in Fig. 8 A and B, the cotransfection with D88N hsp90, butnot with wild-type hsp90, severely diminished buildup of Hb-αand Hb-β proteins in the cells over the 42-h period. This suggeststhat active hsp90 helps to stabilize both globins in nonerythroidcells. Indeed, in HEK cells transfected to express Hb-β alone,overexpression of hsp90 aided Hb-β protein buildup (Fig. 8C)and also rescued its expression from the down-regulation causedby coexpression with AHSP, whereas coexpression of the D88Nhsp90 variant was ineffective at rescue (Fig. 8C). This shows thatthe ATPase activity of hsp90 plays a role in stabilizing Hb-β when

it is expressed in the absence of Hb-α or in the face of AHSPcoexpression.

DiscussionAlthough a role for AHSP in Hb-α maturation has been estab-lished (16–18), what chaperones might aid partner globin mat-uration (Hb-γ and Hb-β) has been unclear. Our study revealsthat hsp90 enables maturation of both Hb-γ and Hb-β by asso-ciating with and stabilizing their immature, heme-free forms andby driving their heme insertion reactions in an ATP-dependentmanner. This was demonstrated in the erythroid-like K562 cellline and in two human erythroid progenitor cell types (HiDEP-1and HUDEP-2) during their in vitro differentiation to matureerythroid cells, implying that hsp90’s role is relevant for globinmaturation during erythropoiesis. Thus, hsp90 appears to act as acounterpart to AHSP by chaperoning Hb-γ and Hb-β for theirheme insertion reactions, as is required to form functional fetal(α2γ2) and adult (α2β2) tetramers during erythropoiesis.Under all circumstances, the globin associations of hsp90 or

AHSP remained specific. Consider that heme insertion into Hb-βand Hb-γ was always hsp90-dependent whether AHSP wasexpressed or not and that Hb-β never associated with AHSPwhen they were coexpressed. However, we did find that Hb-βexpression was actually reduced when AHSP was coexpressedwith it in nonerythroid cells, and this effect could be preventedby hsp90 overexpression or by coexpression with Hb-α. Thus,AHSP antagonizes Hb-β expression when they are expressedalone together, which may explain why this expression pattern isnever observed in physiologic settings. In comparison, hsp90 wasnever found associated with Hb-α in cells that expressed Hb-αalone or together with AHSP, and Hb-α heme insertion underthese circumstances was immune to hsp90 inhibitors. Together,our findings support the view that Hb-α and Hb-β maturation areindependently chaperoned in erythroid cells (28) and confirmthat Hb-α maturation in erythroid cells is independent of hsp90.However, when Hb-α was coexpressed with Hb-β in nonerythroidcells either naturally or as a consequence of transfection, we sawthat its heme insertion then became hsp90-dependent, possiblyas a consequence of hsp90 enabling the Hb-β maturation. Thus,hsp90 is needed for heme insertion into at least two (Hb-β andHb-γ) and as many as three (Hb-α) globins, depending on thecircumstances under which they are expressed (i.e., with orwithout AHSP), and therefore hsp90 plays an unexpectedlybroad role in globin maturation. This concept may help explainhow Hb maturation can succeed in nonerythroid cells, which alllack AHSP expression. A model for Hb maturation that incor-porates our current findings is presented in Fig. 9.Hsp90-assisted heme insertion into Hb-β and Hb-γ appears to

follow the tenets established for maturation of other hemeproteins: Hsp90 associates with and stabilizes the immatureheme-free form of the protein, and after helping to drive hemeinsertion, dissociates and is replaced by a protein partner, whichgenerates a functional mature form (23, 24). This mechanismoperates in erythroid cells (HUDEP-2) that were made heme-deficient when expressing Hb-α and -β: The heme-free Hb-βassociated with hsp90 rather than Hb-α, and the Hb-α/β in-teraction significantly increased post heme insertion with aconcurrent hsp90 dissociation from the heme-replete complex.In another example (soluble guanylate cyclase β1 subunit,sGCβ1), evidence suggests that the exclusivity of its hsp90 versuspartner protein association arises in part from competition for acommon binding domain on sGCβ1 (29, 30). Similarly, there isoverlap between the interaction sites of AHSP and Hb-β withHb-α (13), and this likely explains why AHSP can bind only toheme-free or heme-replete Hb-α before it is has formed anoligomer with Hb-β (13). These events can be further driven byour finding that AHSP has an inhibitory effect on Hb-β expres-sion (Fig. 6 and Fig. S6), which can be protected and stabilized

Fig. 5. Hsp90-globin associations in nonerythroid cells that naturally ex-press Hb. RAW and A549 cell supernatants were analyzed for expression ofHb, hsp90, and ASHP, and the hsp90-globin associations were determined.The RAW cells were either resting or induced by a 16-h culture with IFN-γ +LPS before lysis. (A) Representative Western blots comparing protein ex-pression levels, derived from 15% (Upper four panels) or 8% (Lower twopanels) SDS/PAGE with β-actin as loading control. (B) IPs comparinghsp90 bound to Hbα or to Hb-β as indicated (input 20% in all cases). (C–F)Representative PLA images indicating colocalization of hsp90 and Hb-β inA549 cells. (C) PLA controls using no primary antibody, (D) Hb-β antibodyalone, (E) Hsp90 antibody alone, (F) PLA images with both Hb-β and hsp90antibodies with Insets showing close-ups. Red dots indicate the location andextent of the interaction.

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by hsp90, enabling Hb-α–ASHP and Hb-β–hsp90 to mature asparallel complexes before chaperon dissociation post heme in-sertion and subsequent Hb dimer and tetramer formation. Weare currently working to identify the interaction sites of hsp90and Hb-β to understand how hsp90 chaperones heme insertionduring its maturation.The discovery that hsp90 participates in Hb maturation

has several biomedical and therapeutic implications. Post-translational modifications that alter hsp90 function likephosphorylation (31, 32) and cysteine nitrosylation (32, 33)are known to undergo changes in inflammatory diseases orcancer (32, 34). Whether such modifications help hsp90 con-trol Hb maturation during erythropoiesis can now be in-vestigated. The hsp90 inhibitors that are being developed forcancer treatment might unintentionally block Hb maturationin the recipient. Indeed, anemia has been commonly reportedas a side effect during the clinical trials of hsp90 inhibitor drugcandidates (35, 36). Regarding Hb expression in nonerythroidcells, the Hb-α expressed in pulmonary endothelial cells wasrecently shown to regulate NO control of pulmonary bloodpressure and to present an effective target for therapeuticintervention (37). Hb-β expression in lung tissues was found tobe antimetastatic (38), while its expression in a variety of tu-mor cells increased their metastatic potential, possibly byenhancing tumor-cell survival during blood-born dissemina-tion (39, 40). In light of our current findings, hsp90 couldimpact Hb maturation under all of these circumstances andthus presents a locus for controlling the diverse biologicalroles of Hb.

Materials and MethodsReagents. All chemicals were purchased from Sigma, Fischer, and StemCellTechnologies. Hsp90 inhibitors (radicicol, novobiocin, and AUY-922), hemebiosynthesis inhibitor SA, protein synthesis inhibitor (cycloheximide), andheminwere all purchased from Sigma. Chemicals and growth factors requiredfor culture and differentiation of human erythroid progenitor cells werepurchased from StemCell Technologies and Sigma. Dexamethasone(DEX), doxicyclin (DOX), stem cell factor (SCF), and EPO were purchased fromStemCell Technologies while insulin, heparin, and holo-transferrin werepurchased from Sigma.Molecularmassmarkers were purchased fromBioRad.Human erythroid leukemia cells (K562), HEK293T, RAW 264.7, and A549 cellswere purchased from the American Type Culture Collection. The humanimmortalized erythroid progenitor cells HiDEP-1 and HUDEP-2 were obtainedfrom Y. Nakamura, RIKEN BioResource Center, Ibaraki, Japan. Dominant-

negative hsp90 mutant (D88N) and wild-type hsp90 constructs were giftsfrom Bill Sessa, Yale University, New Haven, CT.

Antibodies. Rabbit polyclonal hsp90 antibody was obtained from Cell Sig-naling Technology while mouse monoclonal hsp90 and rabbit polyclonalAHSP antibodies were obtained from Origene. Goat polyclonal Hb-α and Hb-βγ antibodies or mouse monoclonal Hb-α, Hb-β, and Hb-γ were purchasedfrom Santa Cruz Biotechnology. Rabbit polyclonal Hb-β and Hb-γ antibodieswere obtained from Sigma and Abcam, respectively. Mouse monoclonalMyc, HA, and β-actin antibodies were purchased from Sigma.

Cell Culture, Transient Transfection, and Growth/Differentiation of ErythroidProgenitor Cells. All cell lines were grown and harvested as previously de-scribed (23, 41). Cultures (50–60% confluent) of K562 cells expressing basallevels of Hb-γ were treated with SA for 72 h, pretreated with thehsp90 inhibitors radicicol (10 μM) or novobiocin (250 μM) for 30 min, alongwith cycloheximide (10 μg/mL), and then given hemin (5 μM) for an addi-tional 3 h before being harvested. In similar experiments, HEK cells pre-treated with SA (48 h) and transiently expressing (42 h) Myc-Hbα alone withAHSP or both Myc-Hbα and -β in the absence of AHSP were treated withhsp90 inhibitors, radicicol (5 μM), or the very potent water-soluble AUY-922(42) (300 nM) before hemin (5 μM for 3 h) addition. Control untreated cul-tures not receiving SA were included in all experimental setups. Other ex-periments included transfection of Myc-Hbα and AHSP in HEK cells in thepresence or absence of hsp90 inhibitors (radicicol, 5 μM, or AUY-922,300 nM), and cells were harvested by the 28th hour. In separate experi-ments, K562 cells were treated with hemin (50 μM) from 0 to 3 d to boostfetal Hb (γ) expression before being harvested. In other cases, HEK cells weretransfected in various combinations with Myc-Hbα, Myc-Hbβ, AHSP, D88N,and Hsp90 for 42 h before being harvested. For experiments involving en-dogenous Hb expression in nonerythroids, RAW and A549 cells were grownas previously described (23, 43). Macrophages (RAW) were induced by IFN-γ +LPS as previously mentioned (23), and all cells were harvested for biochemicalanalyses as indicated.

For the culture and differentiation of erythroid progenitor cells, immor-talized human erythroid progenitor cells HiDEP-1 and HUDEP-2 were culturedas described earlier (26) following protocols obtained from Y. Nakamura,RIKEN BioResource Center, Ibaraki, Japan. In brief, the HiDEP-1 and HUDEP-2cells were allowed to proliferate vigorously in Stem Span SFEM media(StemCell Technologies) containing EPO (3 IU/mL), SCF (50 ng/mL), DEX (10−6 M),and DOX (1 μg/mL). The cells were then split into the desired number ofT75 flasks and induced to differentiate by switching to differentiation media(IMDM media from Sigma, I:3390) containing 2% FBS + 3% AB human serum(Atlanta Biologicals), EPO (3 IU/mL), insulin (10 μg/mL), holo-transferrin(500 μg/mL), and heparin (3 U/mL). The cells were allowed to differentiateunder these conditions at intervals between 0 and 16 d and were harvestedfor various biochemical analyses. For heme-insertion studies involving

Fig. 6. AHSP and Hb-α have opposite impacts on Hb-β expression in nonerythroid cells. HEK cells transiently expressing Myc-tagged globins either individuallyor in combination with AHSP were analyzed for protein expression and protein associations. (A) Expression levels of Myc-tagged globins, AHSP, and loadingcontrol β-actin in HEK cells under conditions as indicated. (Right) Western blots probed with specific Hb antibodies. (B) Corresponding densitometry of proteinexpression levels. Values depicted are mean ± SD of three independent experiments (n = 3) (*P < 0.05, by one-way ANOVA). (C) IPs showing bound hsp90 andAHSP associated with Myc-Hb (input 10%) retained on the beads. (Lower three panels) The corresponding expression levels of individual globins and β-actin asindicated.

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Fig. 7. AHSP-supported heme insertion into Hb-α is immune to hsp90 inhibition, whereas Hb maturation in nonerythroid cells is hsp90-dependent. HEK cellswere transfected with Myc-Hb-α ± AHSP and ± radicicol (5 μM) or AUY-922 (300 nM) for 28 h, or HEK cells were heme-deprived by culturing with SA and thenwere transfected with Myc-Hb-α and AHSP or with Myc-Hb-α + Myc-Hb-β for 42 h, followed by hemin (5 μM) treatment for 3 h, in the presence or absence ofthe hsp90 inhibitors. Soluble cell supernatants (equal protein) were analyzed for protein expression, Hb heme content, and Hb protein associations. (A, B, andF) Protein expression levels under the indicated conditions. (C, D, and G) UV-visible spectra of the supernatants as indicated. Insets compare the calculated Hbheme contents. (E and H) IPs as indicated depicting AHSP or hsp90 bound to Hb-α and hsp90 bound to Hb-β, respectively (input 20% in all cases). (H) (Upper)Bound hsp90. (Lower) Myc-Hbβ retained on the beads. (I) Densitometric quantification of hsp90 and dimeric Myc-Hb protein bands as depicted in H. Valuesdepicted in all bar graphs are mean ± SD of three independent experiments (n = 3) (*P < 0.05, by one-way ANOVA; ns, not statistically significant). Molecularmarkers (kDa) indicate the position of protein standards on the gels, and the experimental conditions designated 1–5 are constant from F–I.

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HUDEP-2 cells expressing Hb-α and Hb-β, cells were grown in StemSpanmedia containing growth additives as described earlier and then heme-deprived by treating with SA (400 μM) for 5 d, pretreated with hsp90 in-hibitors radicicol (10 μM) or AUY 922 (300 nM) for 30 min, along withcycloheximide (10 μg/mL), and then given hemin (5 μM) for an additional 3 hbefore being harvested. In all cases wherever applicable, cell supernatantswere assayed for protein expression by Western blot, binding assays by IPs,heme insertion by heme staining, and soret absorption by UV-visible spec-troscopy, as indicated.

Western Blots, Heme Staining, and IPs. For Western blots, standard protocolswere followed as previously mentioned (23, 24, 29). Heme staining of cellsupernatants (50 μg) from differentiating HiDEP-1/HUDEP-2 or from RAW/A549 (150 μg) cells was done as previously described (41). For IPs, 500 μg ofthe total cell supernatant was precleared with 20 μL of protein G-Sepharosebeads (Amersham) for 1 h at 4 °C, beads were pelleted, and the supernatantswere incubated overnight at 4 °C with 3 μg of anti–Hb-βγ, Hb-α, or Mycantibodies. Protein G-Sepharose beads (20 μL) were then added and incubated

for 1 h at 4 °C. The beads were microcentrifuged (6,000 × g), washed threetimes with wash buffer (50 mM Hepes, pH 7.6, 100 mM NaCl, 1 mM EDTA, and0.5% Nonidet P-40) and then boiled with SDS buffer and centrifuged. Thesupernatants were then loaded on SDS/PAGE gels and Western-blotted withspecific antibodies. Band intensities on Western blots were quantified usingImage J quantification software (NIH).

UV-Visible Absorption Spectroscopy. UV-visible absorption spectra of cellsupernatants were recorded at room temperature between 350 and 700 nmon a Shimadzu spectrophotometer. Equal amounts of total protein super-natants were used for respective wavelength scans. The heme content for Hbwas determined from the Soret absorption peak at 414, using the extinctioncoefficient of 342.5 mM−1·cm−1 and a manipulation to account for the variableabsorbance contributions that were attributable to sample turbidity. Thisinvolved creating a baseline for each scan by drawing a line that con-nected the absorbance values at 380 and 470 nm. The additional Soretabsorbance at 414 nm above this baseline was then used to calculate theHb heme content.

Fig. 9. Model for chaperone involvement in Hb maturation. In erythroid cells, the immature, heme-free globins associate with their specific chaperones(AHSP or hsp90). Hsp90 helps drive heme insertion into its globin partner, and after the chaperones dissociate, the heme-replete globins interact to formfunctional hetero-tetramers. In nonerythroid cells, the heme-free globins associate in a complex with hsp90. In this case, heme insertion into the globins isentirely hsp90-dependent and allows them to form functional tetramers. See Discussion for details.

Fig. 8. Hsp90 ATPase activity is essential for Hb maturation in nonerythroid cells and can rescue Hb-β expression from inhibition by AHSP. HEK cells weretransfected with Myc-Hb-β alone or in combination with Myc-Hb-α and along with HA-tagged D88N or wild-type hsp90, and/or untagged AHSP, and analyzed forprotein expression. (A and C) Expression levels of proteins as indicated. (B) Corresponding densitometry of Myc-Hb or HA-tagged hsp90 protein expression levelsas shown in A. Western blots are representative, and values depicted are mean ± SD of three independent experiments (n = 3) (*P < 0.05, by one-way ANOVA).

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Proximity Ligation Assay. PLAs (Duolink; Sigma) for colocalization wereperformed according to the manufacturer’s protocol using primary anti-bodies (anti–Hb-β, 1:25; and anti–Hsp90, 1:200 for HUDEP-2 and 1:400 forA549), followed by a pair of oligonucleotide-labeled secondary antibodies.The assay detects positive signal only when the epitopes of the target pro-teins are in close proximity (<40 nm). The signal from each of the detectedpair of PLA probes was then imaged using fluorescence microscopy(excitation/emission for Duolink red: 594/624; excitation/emission for

Dapi: 360/460). One or both primary antibodies were omitted fornegative controls.

ACKNOWLEDGMENTS. This work was supported by National Institute ofHealth Grants GM 097041 (to D.J.S.) and HL081064 (to D.J.S. and A.G.); aResearch Centre for Excellence Grant from the Cleveland Clinic (to A.G.and D.J.S.); and an American Heart Association Postdoctoral Fellowship(to E.A.S.).

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