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Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems. NanoLuc substrates with improved solubility and bioavailability, hydrofurimazine and fluorofurimazine, strongly enhance bioluminescence signals in vivo and enable bright dual-color bioluminescent imaging with AkaLuc and AkaLumine.

G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease.

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Witho...

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and "off-targets" exhibiting a range of binding affinities, cellular abundances, and binding characteristics.

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT® SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354–359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two 15N-, 13C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline. Electronic supplementary material The online version of this article (doi:10.1007/s10969-010-9093-8) contains supplementary material, which is available to authorized users.

Cultured mammalian cells provide an environment ideal for producing functional recombinant mammalian proteins. However, low expression levels of recombinant proteins present a challenge for their detection and purification. This unit will focus on HaloTag, a protein fusion tag designed to bind selectively and covalently to a chloroalkane ligand that may be attached to a variety of functional groups, allowing both protein detection and immobilization. Detection of HaloTag-fusion protein is achieved through binding to a fluorescent chloroalkane ligand, enabling rapid optimization of expression levels. HaloTag-based purification uses covalent capture of the HaloTag fusion onto HaloLink resin coupled with proteolytic cleavage to release the protein of interest from the resin. Covalent binding provides efficient protein capture regardless of expression level and eliminates protein loss during washes of the resin and as a result, offers significant improvements in protein recovery and purity over traditional non-covalent approaches.

We have developed an assay for chaperone-mediated protein renaturation using thermally denatured Firefly luciferase. Dilution of denatured luciferase (> 99% loss of activity) into reticulocyte lysate typically results in recovery of 5-15% activity. Addition of an ATP-regenerating system increases yields to > 60%, while heat shock or the addition of denatured proteins inhibits the chaperoning activity. Reticulocyte lysate contains abundant quantities of the heat shock proteins, hsp90 and hsp70, and a 60-kDa protein homologous to the yeast stress protein, STI1. Immune isolated samples of these three proteins support recovery of up to 35% of luciferase activity in an ATP-dependent manner, suggesting that these or associated proteins are involved in the renaturation of luciferase. Furthermore, we observed a correlation between luciferase renaturation activity and the levels of hsp70 and hsp90 in reticulocyte lysate preparations. Purified hsp90 and hsp70, along with an ATP-regenerating system, are able to renature luciferase to greater than 20% of its original activity. This renaturation is most efficient when hsp90 and hsp70 are at about a 2:1 ratio and at concentrations similar to those found in reticulocyte lysate. This study provides evidence for an ATP-dependent chaperoning activity in reticulocyte lysate that involves a cooperative action of hsp70 and hsp90.

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)

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.

Protein arrays hold great promise for proteome-scale analysis of protein-protein interaction networks, but the technical challenges have hindered their adoption by proteomics researchers. The crucial issue of design and fabrication of protein arrays have been addressed in several studies, but the detection strategies used for identifying protein-protein interactions have received little attention. In this study, we evaluated six different detection strategies to identify four different protein-protein interaction pairs. We discuss each detection approach in terms of signal-to-background (S/B) ratio, ease of use, and adaptability to high-throughput format. Protein arrays for this study were made by expressing both the bait proteins (proteins captured at the surface) and prey proteins (probes) in cell-free rabbit reticulocyte lysate (RRL) systems. Bait proteins were expressed as HaloTag fusions that allow covalent capture on a HaloTag ligand-coated glass without any prior protein purification step. Prey proteins were expressed and modified with either tags (protein or peptides) or labels (fluorescent or radiometric) for detection. This simple method for creating protein arrays in combination with our analyses of several detection strategies should increase the usefulness of protein array technologies.

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

Wheat germ based eukaryotic cell-free systems have been shown to be applicable for both functional and structural analyses of proteins. However, the existing methods might require specialized instrumentation and/or a separate mRNA synthesis step. We have developed a DNA based, highly productive, coupled transcription/translation wheat germ cell-free system that incorporates the normally separate mRNA synthesis step and does not require specialized instrumentation. Using a small-volume batch reaction with fluorescence labeling, DNA templates predicted to encode proteins could be quickly screened for their ability to direct the expression of proteins of the appropriate size. Protein yield can be increased as much as 2 to 4-fold in this system using a dialysis reaction, reaching approximately 200-440 microg/ml in 10-20 h. Furthermore, enzyme activities can be assayed directly in the extract without further purification. Simple purification with affinity tags can be achieved in one-step and with minor modifications, efficient SeMet and [U-15N] labeling of95% can be accomplished in this system. Thus, this efficient cell-free expression system can facilitate both functional and structural proteomics.

Identifying the targets of a bioactive compound is often the rate limiting step toward understanding the molecular mechanism of drug action. Current approaches rely on linking the bioactive compound to a surface or an affinity handle, permitting selective capture of interacting proteins for identification by mass spectrometry. A major consideration with these methods is insuring that the chemical derivatization of the bioactive compound does not disrupt the binding interactions with the cellular targets. We have developed a method based on a novel chloroalkane capture tag that minimally affects compound potency and cell permeability. This allows verification of the pharmacological activity of the modified compound, thus increasing the confidence in the biological relevance of captured proteins. In addition, by allowing the chloroalkane-modified compound to bind the targets within living cells, the cellular architecture during the binding step is preserved and better represents the conditions that the unaltered compound would normally engage these targets. Following binding with the tagged compound in live cells, the cells are lysed and the chloroalkylated compound and its associated targets are rapidly captured onto immobilized HaloTag protein and then released by competitive elution. The identified targets are then validated for direct binding relationship with the bioactive compound by bioluminescence energy transfer. We tested this target capture/target validation work flow using the interaction of MAPK kinases with two allosteric kinase inhibitors (BIRB796 and a BIRB analog exhibiting 100-fold lower potency). RESULTS: Using the two BIRB-chloroalkane derivatives to selectively enrich for targets from HEPG2 cells, we identified and validated multiple relevant MAPK kinases as well as additional off-targets. Interestingly, all the discovered off-targets bind purines. Kinase inhibitors such as BIRB796 which acts by binding to the kinase ATP binding site can interact in a similar manner with other purine binding proteins. Using bioluminescence energy transfer we interrogated the affinity and residence time of the two BIRB compounds to multiple MAPK kinases inside living cells. Our results indicates that the BIRB796 analog exhibits 30-1000 fold reduced affinity to multiple MAPK kinases as well as significant shorter residence time compared to BIRB796. Taken together these results indicate that our workflow can reveal the direct binding relationships between bioactive compounds and their cellular targets and contribute to further understanding of these interactions. Citation Format: Rachel Friedman Ohana, Robin Hurst, Thomas Kirkland, Carolyn Woodroofe, Sergiy Levin, Paul Otto, Tetsuo Uyeda, Michael Ford, Richard Jones, Danette Daniels, Marjeta Urh, Keith Wood. Investigating the cellular interactions of BIRB796 analogues using a novel chloroalkane capture tag. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2015-2003

In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL-labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL-lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro-translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.

The conformationally constrained cyclic enkephalin analogs, [ 2- D - penicillamine , 5- L - cysteine ]- and [2- D - penicillamine , 5- D - cysteine]enkephalinamide were synthesized and their biological activities investigated. Both analogs effectively induced thermal analgesia as measured by the in vivo hot plate test. Both analogs were effective in inhibiting muscle contractions in the guinea pig ileum and mouse vas deferens assay systems and were shown to displace both [3H]naloxone and [3H] [ D - Ala 2 , D - Leu 5 ] enkephalin from rat brain receptor preparations. The analogs exhibited a significant preference for δ-receptors over μ-receptors, an unusual finding for enkephalinamide derivatives. In addition the 5- L - cysteine containing analog was more potent than the 5- D - cysteine analog in all the in vitro assays with the exception of the guinea pig ileum system. These uncommon results are attributed to the conformational constraints imposed by the cyclization via a disulfide and by the rigidizing effect of the penicillamine.

The rabbit reticulocyte heme-regulated eIF-2α kinase (HRI) utilizes adenosine-5′-0-(3-thiotriphosphate) (ATP-γ-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [ eIF-2(α-[ 35 S]P)·GDP ], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2·[3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(α-[ 35 S]P)·GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2·GDP alone, through the ability of eIF-2(α-P)·GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.

Inhibition of protein synthesis initiation in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation occurs due to the activation of a heme-regulated protein kinase (HRI), which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. How the activation of HRI in hemin-supplemented lysate occurs in response to oxidants and heat stress is not well understood. Recently, the 90-kDa heat shock protein (hsp 90) has been reported to co-purify with HRI activity. In this report, we have used monoclonal antibodies directed against hsp 90 to determine whether HRI and hsp 90 are functionally associated in the reticulocyte lysate in situ. The AC88 antibody recognizes only free hsp 90 and only bound significant amounts of hsp 90 upon prolonged incubation in the absence of heme or upon N-ethylmaleimide treatment of hemin-supplemented lysates. HRI activity is not absorbed by the AC88 antibody. The 8D3 monoclonal antibody, which binds to both free hsp 90 and hsp 90 complexed to steroid hormone receptors, absorbed the hsp 90 present in hemin-supplemented lysates and reduced the HRI activity by 70-95%. Progressively more HRI activity is not adsorbed by the 8D3 antibody the longer the reticulocyte lysate is incubated in the absence of hemin. The HRI that is adsorbed from heme-deficient lysates by the 8D3 antibody is also more active. The sedimentation rate of HRI was analyzed by glycerol gradient centrifugation. HRI present in hemin-supplemented lysate was found to have a sedimentation coefficient of approximately 7.5-8 S and was adsorbed from fractions by the 8D3 antibody in association with hsp 90. A second peak of HRI activity with a sedimentation coefficient of approximately 4.5-5 S was detected upon glycerol gradient centrifugation of heme-deficient lysates. Upon Western blot analysis, heme-deficient lysates were found to have less hsp 90 in the 7.5-8 S region of glycerol gradients than hemin-supplemented lysates. The data suggest that HRI is associated with hsp 90 in an inactive form in hemin-supplemented lysates and dissociates from hsp 90 upon activation. There also appears to be an intermediate of active HRI which is associated with hsp 90 or which can reversibly associate with hsp 90. Similarities between the stages of HRI activation and steroid hormone receptor activation and transformation are discussed.

The conformationally restricted, cyclic, disulfide-containing, enkephalin analogs [2-D-penicillamine, 5-L-penicillamine]enkephalin [(D-Pen2,L-Pen5]enkephalin) and [2-D-penicillamine, 5-D-penicillamine]enkephalin [(D-Pen2,D-Pen5]enkephalin) were synthesized by solid-phase methods. Selectivities of these analogs for a single class of opioid receptor were investigated by examining relative potencies in the mouse vas deferens assay, in which the functional receptor is the delta receptor, versus the guinea pig ileum assay, in which the mu receptor is the functional receptor, and by determining their relative abilities to displace the prototypical delta receptor ligand [D-Ala2, D-Leu5]enkephalin and the prototypical mu receptor ligand naloxone from rat brain membrane preparations. Based on these comparisons [D-Pen2,L-Pen5]- and [D-Pen2,D-Pen5]enkephalin exhibited delta receptor selectivities of 1,088 and 3,164, respectively, in the bioassays, and 371 and 175, respectively, in the binding assays. Compared with the previously reported delta receptor selective analogs, [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Thr2,Leu5,Thr6]enkephalin, the bis-Pen-containing analogs provide an order of magnitude increase in delta receptor selectivity.

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2.GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(alpha-32P). RF and eIF-2(alpha P) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(alpha-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F-, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(alpha P) in the absence of F-. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2.GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.