Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.     

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s)

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.     

Topoisomerase IIB and an extracellular nuclease interact to digest sperm DNA in an apoptotic-like manner

We previously demonstrated that mammalian spermatozoa contain a nuclease activity that cleaves DNA into loop-sized fragments. We show here that this activity is mediated by a nuclear matrix-associated topoisomerase IIB (TOP2B) interacting with an extracellular Mn2+/Ca2+-dependent nuclease. Together, these enzymes cleave all of the DNA into fragments of 50 kb, and this cleavage can be reversed by EDTA. If dithiothreitol is included, the nuclease digests the DNA, and if the protamines are removed the DNA is completely digested. A similar, TOP2B-mediated, chromatin fragmentation, which is reversible, followed by digestion of the DNA by an intracellular nuclease occurs in somatic cells during apoptosis. The extracellular location of the sperm nuclease made it possible to reconstitute the fragmentation activity in isolated spermatozoa, thus allowing us to identify two novel aspects of the mechanism. First, the fragmentation of all of the DNA to 50 kb by TOP2B required the addition of the extracellular nuclease or factor. Second, the subsequent, complete digestion of the DNA by the nuclease could be inhibited by etoposide, suggesting that the nuclease digestion requires TOP2B religation of the cleaved DNA. These data are the first demonstration of an active TOP2B in spermatozoa, suggesting this inert chromatin may be more active than previously thought. They also show that the unique chromatin structure of spermatozoa may provide an important model to study the regulated degradation of chromatin by TOP2B and associated nucleases.     

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.     

Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.     

Compact structure of ribosomal chromatin in Xenopus laevis.

Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.     

Localization of phosphoproteins and of protein kinases in chromatin from butyrate treated HTC cells

We have recently shown that phosphoproteins are associated with the chromatin regions accessible to micrococcal nuclease. We have also shown that butyrate treatment modifies the accessibility of chromatin to the nuclease. In this work we have studied the effect of butyrate on the localization of phosphoproteins in chromatin. We observed a strong similarity between the effect of butyrate on the release of DNA fragments and on the release of phosphoproteins by the nuclease, which indicates that in butyrate treated as in control cells the released fragments include phosphoproteins. Butyrate treatment increases strongly chromatin protein kinase specific activity and modifies its localization in the released fragments; it is therefore likely that it modifies its localization in chromatin.     

Nucleosome organization during germ cell development in the sea cucumber Holothuria tubulosa.

Conformational changes that occur in chromatin from developing germ cells of the echinoderm Holothuria tubulosa have been probed with micrococcal nuclease. The results indicate that the extent of DNA degradation to acid-soluble nucleotides is highest in chromatin at the early stages of gonad growth, being drastically subdued in the mature sperm cell. Production of nucleosomal particles also varies with development, involving at least 70% of the chromatin at the final stage of maturation, whereas in immature germ cells it remains much lower. In contrast, electrophoretic analysis for DNA size has shown that the average nucleosome repeat length, about 227 base pairs, does not change throughout the maturation process. However, kinetics of the enzyme reaction have revealed that, although brief digestion of chromatin from both immature gonads and sperm yields comparable series of higher oligomers, extensive digest patterns differ widely. Sperm chromatin, highly protected, releases a 275 base pair intermediate fragment, wholly absent in immature gonads. The 145 base pair core released in both chromatins is not further digested in sperm. In comparison to sperm chromatin, that of immature germ cells is much more susceptible to fragmentation, yielding the usual set of smaller subnucleosomal fragments. These data suggest the induction of differential accessibilities of chromatin DNA with maturation, which is not accompanied by displacement of the histone complement. The histone variants present in this species may well be instrumental in the process.     

An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin

The single-stranded activity of S1-nuclease cleaves globin chromatin in red cell nuclei in specific regions. The cleavages are observed only in tissues in which the globin genes are active, and they "switch" to reflect the switching pattern of globin-gene expression in embryonic and adult red cells. The positions of the S1 cleavages in the beta- and alpha-globin chromatin correspond to the general region of known DNAase I-hypersensitive sites, but can be distinguished in detail. When DNA segments containing these regions are subcloned into pBR322 and the supercoiled molecules are treated with S1, similar sites are cleaved in the purified supercoiled (but not linear) recombinant plasmid DNA. However, the dominant S1 cutting sites are shifted in the plasmid vis-a-vis the chromatin. We believe that some aspect of DNA sequence is translated into an altered DNA structure in chromatin and that it is this altered structure that is recognized by s1 nuclease and possibly by certain chromosomal proteins. Several physical properties reflected in the S1 digestion of supercoiled plasmids suggest a mechanism for generating differences in daughter cells during development.     

Transition from Caspase-dependent to Caspase-independent Mechanisms at the Onset of Apoptotic Execution

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal “committed stage” cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the “execution phase” extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.     

Development of an apoptosis endonuclease assay.

A biochemical hallmark of cells undergoing programmed cell death, or apopotosis, is the endonucleolytic cleavage of genomic DNA at internucleosomal sites. To study further the nuclease involved in this process, an assay system was developed to measure internucleosomal DNA degradation. Micrococcal nuclease (MNase), a bacterial enzyme that cleaves chromatin at internucleosomal intervals, was used to validate the assay procedure. Thymocyte nuclear proteins obtained from glucocorticoid-treated chickens, a source of internucleosomal DNA-degrading activity, were incubated with chicken red blood cell nuclei, and genomic DNA was subsequently extracted and analyzed by agarose gel electrophoresis. Generation of internucleosomal DNA degradation products by the thymocyte protein extract required ATP and was both time and protein concentration dependent. This nuclease activity could be inhibited by EDTA, EGTA, alkylating agents, or heat denaturation. Addition of purified proteinases, RNases, or other types of nucleases to the assay failed to generate discrete internucleosomal lengths of DNA, thus confirming the nuclease specificity of this assay. On the basis of these data, we believe that this assay system will be instrumental in isolating and characterizing the nuclease(s) associated with apoptosis.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells

Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3-dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment.     

Topoisomerase activity in irradiated mammalian cells

The role of topoisomerase enzymes in the response of HeLa S3 cells to ionizing radiation was investigated. Exposure of cells to 100 Gy of X-radiation had no detectable effect either on the total cellular topoisomerase activity as measured by the relaxation of supercoiled plasmid DNA by cell sonicates or on the total cellular topoisomerase II activity as measured by plasmid DNA catenation. Total topoisomerase II activity remained constant for up to 90 min after cell irradiation. The effect of 2 drugs (caffeine and novobiocin) which inhibit topoisomerase II activity on the HeLa cell response to radiation was determined. Both drugs were found to inhibit topoisomerase II in vitro and to inhibit the recovery of nucleoid sedimentation in irradiated cells in vivo to the same extent. Topoisomerase II was inhibited by 50% by exposure to 10 mM caffeine and 0.79 mM novobiocin. At low concentrations neither drug affected the induction frequency, nor the rejoining rate, of DNA double-strand breaks. Caffeine (5 mM) inhibited the short-term recovery of cells from radiation while novobiocin (0.79 mM) had no detectable effect on the capacity of cells to recover from radiation exposure. The results indicate that topoisomerase II is not required for DNA double-strand break rejoining though it could be required for the recovery of DNA coiling in the irradiated cell. If topoisomerase II is involved at all in cell recovery from irradiation, this role does not apparently involve an ATP-dependent enzyme activity.