Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

Detection of rat Pneumocystis carinii proteinases and elastase and antipneumocystis activity of proteinase inhibitors in vitro

Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.     

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae)

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.     

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

In vitro binding of the purified hormone-binding subunit of the estrogen receptor to oligonucleotides containing natural or modified sequences of an estrogen-responsive element

Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.     

Purification and characterization of keratin hydrolase in psoriatic epidermis: application of keratin-agarose plate and keratin-polyacrylamide enzymography methods

Keratin-agarose plate and keratin-polyacrylamide enzymography methods were developed to demonstrate proteolytic digestion of epidermal keratin. By applying these methods, keratin hydrolase was purified from Tris-buffered saline extract of psoriatic scales by 50% ammonium sulfate precipitation, passage through a lysine-Sepharose column, DEAE-Sepharose, Sephacryl S-200, high-performance cation-exchange chromatography on Mono S, and aprotinin-Sepharose affinity chromatography. The final preparation demonstrated a single protein band at molecular weight 30,000 judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, in keratin-polyacrylamide slab gels, the purified enzyme preparation showed a translucent band at molecular weight 30,000, indicating keratin digestion. Keratin hydrolase digested reassembled epidermal keratin as well, whereas it had no effect on guinea pig hair keratin. The enzyme demonstrated a high level of hydrolytic activity on Ile-Pro-Arg-p-nitroanilide and other peptidyl arginine substrates, while it showed a low level of activity on Val-Leu-Lys-p-nitroanilide, and no activity on Arg-Pro-Tyr-p-nitroanilide, Glu-Pro-Val-p-nitroanilide, or Ala-Ala-Ala-p-nitroanilide. The keratin hydrolase was a serine proteinase, inactivated by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-lysyl-chloromethyl ketone, antipain, leupeptin, soybean trypsin inhibitor, aprotinin, and p-aminobenzamidine. The keratinolytic activity was not detected in normal epidermal extract.     

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3′, a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca²⁺-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3′ by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.     

Unintended molecular interactions in transgenic plants expressing clinically useful proteins: The case of bovine aprotinin traveling the potato leaf cell secretory pathway

We assessed the impact of subcellular targeting on the heterologous expression of a clinically useful protease inhibitor, bovine aprotinin, in leaves of potato, Solanum tuberosum. Transgenic potato lines targeting aprotinin to the cytosol, the ER or the apoplast were first generated, and then assessed for their ability to accumulate the recombinant protein. On‐chip detection and quantitation of aprotinin variants by SELDI TOF MS showed the inhibitor to be absent in the cytosol, but present under different forms in the ER and the apoplast. No visible phenotypic effects of aprotinin were observed for the transgenic lines, but aprotinin retention in the ER was associated with a significant decrease of leaf soluble protein content. A 2‐D gel assessment of control and transgenic lines revealed a possible link between this altered protein content and the down‐regulation of proteins implicated in protein synthesis and maturation. These observations, supported by complementary 2‐DE analyses with potato lines targeting aprotinin to the apoplast, suggest an aprotinin‐mediated feedback in planta negatively altering protein anabolism. From a practical viewpoint, these data illustrate the importance of taking into account not only the characteristics of recombinant proteins expressed in heterologous environments, but also their possible effects on protein accumulation in the host plant factory.     

Involvement of both heparanase and plasminogen activator in lymphoma cell-mediated degradation of heparan sulfate in the subendothelial extracellular matrix

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.     

The effect of trypsin inhibitors aprotinin (Trasylol) and TLCK administered locally by osmotic pumps on the gelatinolytic activity of acrosin and the transport of sperm cells in the female reproductive tract of rabbits

The trypsin inhibitors aprotinin (Trasylol) and TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone) were administered continuously into the lumen of the cervix uteri of sexually mature rabbits by means of surgically implanted osmotic minipumps. The doses were inseminated six days after implantation of the pumps, then sacrificed two to six hours after insemination and their reproductive tracts were prepared for gelatin substrate film test and scanning electron microscopy. At a pumping rate of 50 to 100 micrograms aprotinin/h neither gelatinolytic activity of acrosin nor sperm transport were visibly inhibited. TLCK, at a pumping rate of 10 micrograms/h, did not influence the proteolytic activity of acrosin; however it seemed, presumably for its toxicity, to destroy the fine structure of epithelial surfaces in the vagino-cervical region and to impair sperm transport. These results suggest, that acrosin, under these experimental conditions, is not inhibited by aprotinin and TLCK in vivo and may play no immediate role in sperm transport in the female reproductive tract.     

Preferential oxidation of zinc finger 2 in estrogen receptor DNA-binding domain prevents dimerization and, hence, DNA binding

For approximately one-third of estrogen receptor (ER)-positive breast cancer patients, extracted tumor ER is unable to bind to its cognate DNA estrogen response element (ERE), an effect that is partly reversible by the thiol-reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of structure and function by the action of oxidants such as diamide and hydrogen peroxide; however, prior DNA binding by ER or ER-DBD protects against this oxidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognition helix and a flexible helix-supported dimerization loop, respectively. Comparisons between synthetic peptide analogues of each zinc finger and recombinant ER-DBD in the presence of zinc by electrophoretic mobility shift assay, circular dichroism, and mass spectrometry confirm that cooperativity between these zinc fingers is required for both ER-DBD structure (alpha-helicity) and function (dimeric DNA binding). Rapid proteolytic digestion of monomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-binding finger, but not the finger supporting the flexible dimerization loop, which remains sensitive to internal disulfide formation. These findings indicate that the loss of ER DNA-binding function in extracts from some primary breast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation that prevents dimerization. Although ER-DBD contains several strategically located methionine residues, they are less susceptible to oxidation than the thiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.     

Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the unmutagenized ER. Several mutants displaying enhanced sensitivity to 2ME were isolated. We further characterized a mutant carrying the substitution L536P, which was located immediately N terminal to the AF-2-activating domain of the receptor. Amino acid 536 corresponds to a ligand contact residue in retinoic acid receptor gamma, suggesting that key contact points are conserved among receptors. Introduction of L536P into the original ER cDNA isolate HE0, which contains the substitution G400V, rendered the receptor more sensitive to a variety of agonists. When introduced into the wild-type ER HEG0, L536P also rendered the receptor more sensitive to agonists, and, in addition, induced high levels of constitutive activity that could be inhibited by antiestrogens. Estrogens containing a keto substitution in the steroid D ring, but not those containing a hydroxyl group, were full agonists of L536P-HEG0. Limited proteolytic analysis suggested that the L536P substitution, which is located immediately N terminal to the AF-2 domain, induces a conformational change in the ER that partially mimics binding by hormone. Both HEG0 and L536P-HEG0 formed complexes with hsp90 in vitro, indicating a lack of correlation between interaction with hsp90 in vitro and hormonal regulation of ER transactivation in vivo. This supports the idea that a factor(s) acting downstream of hsp90 is important for controlling activity of the hormone-free receptor.     

Relative binding affinity of norgestimate and other progestins for human sex hormone-binding globulin

The relative binding affinity of norgestimate for human sex hormone-binding globulin was compared with that of its metabolites and other progestins by measuring their abilities to displace [3H]testosterone from this carrier protein in vitro. Norgestimate and its 17-deacetylated and 3-keto metabolites did not significantly displace [3H]testosterone from sex hormone-binding globulin at concentrations up to 10,000 nM, whereas gestodene, levonorgestrel, and 3-keto desogestrel displaced [3H]testosterone from sex hormone-binding globulin with IC50 concentrations of 23.1, 53.4, and 91.0 nM, respectively. Since it is believed that a progestin may exert androgenic effects by displacing testosterone from sex hormone-binding globulin, thereby increasing circulating levels of free, active testosterone, these data are consistent with the results of preclinical and clinical studies demonstrating the selective progestational activity of norgestimate.     

A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding.

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

Analysis in vitro of uterine estrogen receptor conformation of young and old rats

The conformation of estrogen receptor (ER) and its in vitro transformation by RNase, Urea and ATP were analysed using the uteri of young (16 weeks) and old (92 weeks) rats. Following the digestion of ER with proteolytic enzymes like trypsin and chymotrypsin and the analysis of cleaved fragments by SDS-PAGE, similar pattern is observed in both ages. In vitro transformation of ER by RNase, Urea and ATP shows that the degree of transformation is lower in old than young. Furthermore, the transformed ER from old is less capable of binding to DNA than that from young. Thus our results show that the conformation of ER probably does not change with age, but the degree of transformation and the ability of transformed receptor to bind to DNA decrease with age.