Activation Of Heme-regulated Inhibitor Research Articles

Abstract 4959: TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death

Abstract TRAIL-inducing compound TIC10/ONC201 binds and activates mitochondrial protease ClpP leading to integrated stress response (ISR) and ATF4 transcription factor activation. Our lab previously reported that ONC201 activates HRI (heme-regulated inhibitor) kinase but does not signal eIF2-alpha phosphorylation through PERK or GCN2. However, the connection between ClpP activation of HRI remains unknown. PG3, a prodigiosin analog that bypasses a defective p53 pathway potently induces ATF4 and pro-apoptotic PUMA. Our data indicate that PG3 activates ATF4 through ISR via HRI as knockdown of HRI but not PKR blocked upregulation of ATF4 and CHOP in several tumor cell lines. We have been exploring how ClpP activation leads to HRI and ISR activation, and the mechanism by which PG3 leads to HRI activation. We find that activation of HRI by PG3 or ONC201 is not through the OMA1/DELE1◊HRI activation pathway. Moreover, ROS and NO are not responsible for ONC201- or PG3-induced HRI activation. ALAS1 (5'-aminolevulinate synthase 1) catalyzes the first rate-limiting step in heme (Iron-protoporphyrin) biosynthesis. ONC201 treatment leads to potent downregulation and inhibition of ALAS1, indicating that ONC201 inhibits heme biosynthesis. It is well known that reduced heme results in activation of the HRI kinase. We show that an inhibitor of heme biosynthesis or knockdown of ALAS1 results in HRI activation, while knockdown of HRI blocks upregulation of ATF4 by ONC201. Knockdown of ClpP rescues ONC201-induced downregulation of ALAS1 which blocks ONC201-induced upregulation of CHOP. Our studies identify a novel link between ClpP activation induced by ONC201 treatment and ATF4 upregulation, via the ClpP/ALAS1◊HRI◊ATF4/CHOP pathway. However, PG3 does not inhibit ALAS1, reduce its expression or therefore signal through ClpP. PG3 treatment did not lead to degradation of ALAS1, indicating that PG3 does not activate ClpP as ALAS1 is a known ClpP direct substrate. We are further investigating the signaling pathway that leads to PG3-induced activation of HRI. Our results suggest that different small molecule inducers of the ISR such as ONC201 and PG3 can achieve an anti-tumor effect through different pathways involving kinase HRI ultimately leading to ATF4/CHOP activation and tumor cell death. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4959.

The heme-regulated inhibitor kinase requires dimerization for heme-sensing activity

The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells. Depletion of HRI increases HbF production, presenting a therapeutically exploitable target for the treatment of patients with sickle cell disease or thalassemia, which benefit from elevated HbF levels. HRI is known to be an oligomeric enzyme that is activated through autophosphorylation, although the exact nature of the HRI oligomer, its relation to autophosphorylation, and its mode of heme regulation remain unclear. Here, we employ biochemical and biophysical studies to demonstrate that HRI forms a dimeric species that is not dependent on autophosphorylation, the C-terminal coiled-coil domain in HRI is essential for dimer formation, and dimer formation facilitates efficient autophosphorylation and activation of HRI. We also employ kinetic studies to demonstrate that the primary avenue by which heme inhibits HRI is through the heme-binding site within the kinase domain, and that this inhibition is relatively independent of binding of ATP and eukaryotic initiation factor 2α substrates. Together, these studies highlight the mode of heme inhibition and the importance of dimerization in human HRI heme-sensing activity.

Heme-regulated inhibitor: an overlooked eIF2α kinase in cancer investigations.

Heme-regulated inhibitor (HRI) kinase is a serine-threonine kinase, controlling the initiation of protein synthesis via phosphorylating α subunit of eIF2 on serine 51 residue, mainly in response to heme deprivation in erythroid cells. However, recent studies showed that HRI is also activated by several diverse signals, causing dysregulations in intracellular homeostatic mechanisms in non-erythroid cells. For instance, it was reported that the decrease in protein synthesis upon the 26S proteasomal inhibition by MG132 or bortezomib is mediated by increased eIF2α phosphorylation in an HRI-dependent manner in mouse embryonic fibroblast cells. The increase in eIF2α phosphorylation level through the activation of HRI upon 26S proteasomal inhibition is believed to protect cells against the buildup of misfolded and ubiquitinated proteins, having the potential to trigger the apoptotic response. In contrast, prolonged and sustained HRI-mediated eIF2α phosphorylation can induce cell death, which may involve ATF4 and CHOP expression. Altogether, these studies suggest that HRI-mediated eIF2α phosphorylation may be cytoprotective or cytotoxic depending on the cells, type, and duration of pharmacological agents used. It is thus hypothesized that both HRI activators, inducing eIF2α phosphorylation or HRI inhibitors causing disturbances in eIF2α phosphorylation, may be effective as novel strategies in cancer treatment if the balance in eIF2α phosphorylation is shifted in favor of autophagic or apoptotic response in cancer cells. It is here aimed to review the role of HRI in various biological mechanisms as well as the therapeutic potentials of recently developed HRI activators and inhibitors, targeting eIF2α phosphorylation in cancer cells.

New activators of eIF2α Kinase Heme-Regulated Inhibitor (HRI) with improved biophysical properties

Heme-regulated inhibitor (HRI), a eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, is critically important for coupling protein synthesis to heme availability in reticulocytes and adaptation to various environmental stressors in all cells. HRI modifies the severity of several hemoglobin misfolding disorders including β-thalassemia. Small molecule activators of HRI are essential for studying normal- and patho-biology of this kinase as well as for the treatment of various human disorders for which activation of HRI or phosphorylation of eIF2α may be beneficial. We previously reported development of 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific HRI activators and demonstrated their potential as molecular probes for studying HRI biology and as lead compounds for treatment of various human disorders. To develop more druglike cHAUs for in vivo studies and drug development and to expand the chemical space, we undertook bioassay guided structure–activity relationship studies replacing cyclohexyl ring with various 4-6-membered rings and explored further substitutions on the N-phenyl ring. We tested all analogs in the surrogate eIF2α phosphorylation and cell proliferation assays, and a subset of analogs in secondary mechanistic assays that included endogenous eIF2α phosphorylation and expression of C/EBP homologous protein (CHOP), a downstream effector. Finally, we determined specificity of these compounds for HRI by testing their anti-proliferative activity in cells transfected with siRNA targeting HRI or mock. These compounds have significantly improved cLogPs with no loss of potencies, making them excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of β-thalassemia.

Anemia of β-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of β-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free α-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2α phosphorylation. The improvement of β-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of β-thalassemia.

Activation of HRI is mediated by Hsp90 during stress through modulation of the HRI-Hsp90 complex

Heme Regulated Inhibitor (HRI) is known to get activated in various stresses such as heme deficiency, heat shock, heavy metal toxicity etc. Heat shock protein 90 (Hsp90), a ubiquitous cytoplasmic protein interacts with HRI in order to regulate protein synthesis. However, it still remains to establish this interaction of HRI and Hsp90 at cellular levels and how this modulation of HRI activity is mediated by Hsp90 during stress. In the present report, using co-immunoprecipitation analysis we show that HRI interacts with Hsp90 and this association is independent of other co-chaperones in in vitro conditions. Further, analysis using truncated domains of HRI revealed that the K1 subdomain is essential for HRI - Hsp90 complex formation. Our in silico protein - protein interaction studies also indicated interaction of Hsp90 with K1 subdomain of HRI. Mammalian two hybrid assay validated this HRI - Hsp90 interaction at cellular levels. When the in vitro kinase assay was carried out with the co-immunoprecipitated complex of HRI - Hsp90, an increase in the kinase activity was observed resulting elevated levels of eIF2α phosphorylation upon heavy metal stress and heat shock. Thus, our results clearly indicate modulation of HRI kinase activity with simultaneous Hsp90 association under stress conditions.

Abstract 3014: ONC201 anti-cancer effects against solid tumors are mediated through eIF2α kinases HRI and PKR but are PERK-independent

Abstract ONC201 is a first-in-class small molecule inducer of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. We recently reported that ONC201-mediated induction of the TRAIL pathway is largely promoted by a preceding early activation of the integrated stress response pathway (ISR) (Kline et al., Sci. Sig., in press, 2015). ONC201 engages the ISR to exert its broad-spectrum anti-cancer effects through pro-apoptotic and anti-proliferative activities. ONC201 upregulates ATF4, the key indicator of ISR activation, in a manner that is dependent on two eIF2α kinases: heme-regulated inhibitor (HRI) and double-stranded RNA activated protein kinase (PKR). Of note, the activation of ATF4 and downstream cell death signaling by ONC201 is PERK-independent. We now further elucidate how the novel dual engagement of HRI and PKR by ONC201 contributes to the anti-cancer effects of ONC201. ONC201 treatment results in early phosphorylation of eIF2α in a manner that is dependent on HRI. On the other hand, we have shown that ONC201-induced downregulation of cyclin D1 is dependent, at least in part, on PKR, but not on eIF2 α phosphorylation per se. This occurs potentially via PKR-mediated ubiquitination and subsequent proteasome degradation of cyclin D1. Treating cells with ONC201 and the proteasome inhibitor MG132 prevents ONC201-induced decrease of cyclin D1. Despite distinctions in their regulatory protein domains, HRI and PKR have both been shown to respond to oxidative stress. Treating cells with ONC201 in the presence of the antioxidant N-acetylcysteine abrogated ATF4 induction. Our findings document a unique activity of ONC201 on protein translation, via activation of HRI and PKR, as well as effects on protein stability through the proteasome leading to anti-cancer effects. Citation Format: Christina Leah B. Kline, Wafik S. El-Deiry. ONC201 anti-cancer effects against solid tumors are mediated through eIF2α kinases HRI and PKR but are PERK-independent. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3014.

Role of HRI in apoptosis resistance

When exposed to environmental stresses, cells activate defence mechanisms to adapt stress and inhibit apoptotic pathways leading to their survival. Stressed cells also reduce their general metabolism in part by inhibiting mRNA translation, thereby saving energy needed to repair stress-induced damages. Under stress conditions, the inhibition of mRNA translation occurs mainly at its initiation step through the phosphorylation of the translation initiation factor eIF2α. One of the four kinases known to phosphorylate eIF2α is heme-regulated inhibitor (HRI). The activation of HRI occurs under conditions of heme deficiency, oxidative stress and treatment with anti-cancer drugs such as proteasome inhibitors. In this article, we discuss the role of HRI in promoting cell resistance to stress-mediated apoptosis.

Cytochrome P450 autophagy: Activation by acute hepatic heme depletion

We have previously reported that acute hepatic heme depletion activates the heme‐regulated inhibitor (HRI) eIF2α kinase resulting in the translational arrest of de novo synthesis of hepatic CYPs 2B. We now report that such hepatic heme depletion also results in the rapid autophagic‐lysosomal degradation (ALD) of existing hepatic P450s, as documented by its reversal with ALD‐inhibitors 3‐methyladenine (3MA) and NH4Cl. This is associated with the conversion of LC3‐I to its lipidated LC3‐II species as well as the accumulation and subsequent oligomerization of p62, hepatocellular hallmarks of ALD‐activation. These effects are enhanced by 3MA/NH4Cl, but more importantly reversed by hemin, thereby revealing their strategic placement in the P450 ALD cascade triggered by hepatic heme depletion. Activation of PERK and GCN2 eIF2α kinases results in the non‐canonical activation of nuclear factor kappa B (NF‐κB), a key transcription factor involved not only in inflammation, cancer, apoptosis and immunity, but also in ALD induction. Acute heme depletion we find also activates hepatic NF‐κB, and studies in HRI−/− mice are in progress to determine whether HRI activation is similarly responsible. Irrespective of HRI involvement, our findings rationalize the impaired P450‐dependent drug clearance clinically observed in the acute heme‐deficient states known as the hepatic porphyrias. Supported by NIH grants DK26506 and DK26743.

Oxidative Stress Signaling Pathway of Heme Regulated eIF2α Kinase in mitigating the Severity of β-Thalassemia

Maturation of erythroid precursors requires active synthesis of hemoglobin which consists of two pairs of α- and β-globin subunits with each monomer bound to a heme moiety. Heme Regulated Inhibitor (HRI) is the only eIF2αkinase responsible for the balanced synthesis of heme and globin at translational level in erythroid cells. Activation of HRI in heme deficiency leads to phosphorylation of the α-subunit of eukaryotic initiation factor (eIF2α) and inhibition of protein synthesis. HRI is also activated by denatured proteins and oxidative stress. In addition to general inhibition of protein synthesis, phosphorylation of eIF2α (eIF2αP) also leads to the induction of a stress signaling pathway. Activating transcription factor 4 (Atf4) mRNA is preferentially translated amidst global inhibition of protein synthesis. Atf4 activates transcription of stress response proteins, Chop (CCAAT/enhancer binding protein homologous protein-10) and the non-enzymatic cofactor of eIF2α phosphatase (PP1A) Gadd34. These stress response proteins help cells in mitigating the stress. While the role of HRI in translational regulation of non-nucleated reticulocytes is well established, the HRIdependent Atf4 stress signaling pathway of nucleated erythroid precursors is unknown.Sodium arsenite toxicity was used as a model system of oxidative stress to elucidate the HRI signaling pathway in Hri +/+ and −/− E14.5 mouse fetal liver erythroid precursors. In HRI deficiency, erythroid precursors were more sensitive to arsenite toxicity with decreased cell viability and increased apoptosis, by caspase 3 executed intrinsic apoptotic pathway. HRI was activated by autophosphorylation as early as 15 minutes following arsenite treatment. In addition to increased eIF2αP, there was induction of Atf4, Chop and Gadd34 in Hri+/+ fetal liver cells. Importantly, in Hri−/− cells neither the phosphorylation of eIF2α nor the expression of Atf4, Chop and Gadd34 was increased upon arsenite treatment. In addition, we also observed HRI dependent induction of Heme Oxygenase 1 (HO-1) that plays a pivotal role in adaptation to oxidative stress. These results demonstrate that HRI induces a signaling pathway for adaptive gene expression to protect the nucleated erythroid precursors from apoptosis upon oxidative stress.Iron overload, accumulation of unpaired α-globin and oxidative stress are well documented in β-thalassemia. Recently, HRI was discovered to be necessary for the survival of β-thalassemic mice. β-thalassemic mice lacking one copy of HRI (Hri+/− Hbb−/−) also manifest a more severe syndrome of the disease. We have investigated the activation of eIF2αP/Atf4 signaling pathway in Hri+/−Hbb−/− β-thalassemic erythroid cells using eIF2αP phosphatase (Gadd34) inhibitor salubrinal. Treatment of reticulocytes from Hri+/−Hbb−/− mice with salubrinal increased eIF2αP and resulted in inhibition of newly synthesized globin protein synthesis. The decreased globin protein synthesis also resulted in decreased aggregation of the unpaired α-globins. Furthermore, treatment of salubrinal in nucleated fetal liver erythroblasts also increased Chop expression and decreased apoptosis. Thus, activation of the eIF2αP/Atf4 pathway by small chemicals might be a novel pharmaceutical approach to decrease proteotoxicity and apoptosis for the treatment of β-thalassemia.

Nck‐1 selectively modulates eIF2αSer51 phosphorylation by a subset of eIF2α‐kinases

Phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51 is an early event associated with the down-regulation of protein synthesis at the level of translation and initiation of a transcriptional program. This constitutes a potent mechanism to overcome various stress conditions. In mammals, four eIF2alpha-kinases [PKR-like endoplasmic reticulum kinase (PERK), dsRNA-activated protein kinase (PKR), heme regulated inhibitor (HRI) and general control nonderepressible-2 (GCN2)], activated following specific stresses, have been shown to be involved in this process. In this article, we report that the ubiquitously expressed adaptor protein Nck, composed only of Src homology domains and classically implicated in cell signaling by activated plasma membrane receptor tyrosine kinases, modulates eIF2alpha-kinase-mediated eIF2alphaSer51 phosphorylation in a specific manner. Our results show that Nck not only prevents eIF2alpha phosphorylation upon PERK activation, as reported previously, but also reduces eIF2alpha phosphorylation in conditions leading to PKR and HRI activation. By contrast, the overexpression of Nck in mammalian cells fails to attenuate eIF2alphaSer51 phosphorylation in response to amino acid starvation, a stress well known to activate GCN2. This observation is further confirmed by showing that Nck fails to alter eIF2alphaSer51 phosphorylation in Saccharomyces cerevisiae, for which the sole eIF2alpha-kinase is Gcn2p. Our results suggest the existence of a novel mechanism that specifically modulates the phosphorylation of eIF2alpha on Ser51 under various stress conditions.

Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses.

Cytoplasmic stresses, including heat shock, osmotic stress, and oxidative stress, cause rapid inhibition of protein synthesis in cells through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) by eIF2alpha kinases. We have investigated the role of heme-regulated inhibitor (HRI), a heme-regulated eIF2alpha kinase, in stress responses of erythroid cells. We have demonstrated that HRI in reticulocytes and fetal liver nucleated erythroid progenitors is activated by oxidative stress induced by arsenite, heat shock, and osmotic stress but not by endoplasmic reticulum stress or nutrient starvation. While autophosphorylation is essential for the activation of HRI, the phosphorylation status of HRI activated by different stresses is different. The contributions of HRI in various stress responses were assessed with the aid of HRI-null reticulocytes and fetal liver erythroid cells. HRI is the only eIF2alpha kinase activated by arsenite in erythroid cells, since HRI-null cells do not induce eIF2alpha phosphorylation upon arsenite treatment. HRI is also the major eIF2alpha kinase responsible for the increased eIF2alpha phosphorylation upon heat shock in erythroid cells. Activation of HRI by these stresses is independent of heme and requires the presence of intact cells. Both hsp90 and hsc70 are necessary for all stress-induced HRI activation. However, reactive oxygen species are involved only in HRI activation by arsenite. Our results provide evidence for a novel function of HRI in stress responses other than heme deficiency.

Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.

Association of HSP90 with the heme-regulated eukaryotic initiation factor 2α kinase—A collaboration for regulating protein synthesis

Among the various heat shock proteins (HSPs), members of the HSP70 and HSP90 families have drawn particular attention due to their heat shock-unrelated functions. HSP90, an ubiquitous and abundant member of the HSP90 family has been shown to be associated with a large array of protein factors. These proteins reside in the nucleus as well as in the cytoplasm and are involved in various physiological processes, such as, regulation of chromatin structure, cell cycle, cytoskelelal architecture, protein trafficking and protein synthesis. In this article, we focus our interest on the role of HSP90 in protein synthesis. Recent data obtained from a few laboratories strongly suggest that HSP90 interacts with the heme-regulated eukaryotic initiation factor 2α (elF-2α) kinase, also called the heme-regulated inhibitor, and causes its activation which leads to inhibition of protein synthesis. On the basis of data reported from various laboratories, including our own, we propose a possible model on the mechanism of HSP90-mediated activation of heme-regulated inhibitor and regulation of protein synthesis.

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase.

The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp) and a 56-kDa protein in hemin-supplemented lysates [Matts, R.L., Xu, Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267m 18160-18167]. In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-2. This stimulation of eIF-2 alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed with denatured BSA, but not with not with native BSA. Hsp70 was co-adsorbed with HRI from lysate in the presence of native BSA, but not in the presence of denatured BSA.(ABSTRACT TRUNCATED AT 250 WORDS)