Directed inhibition of nuclear import in cellular hypertrophy.

Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import.

Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia.

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

Stabilities of intrastrand pyrimidine motif DNA and RNA triple helices.

Nucleic acid triple helices have provoked interest since their discovery more than 40 years ago, but it remains unknown whether such structures occur naturally in cells. To pursue this question, it is important to determine the stabilities of representative triple helices at physiological temperature and pH. Previous investigations have concluded that while both DNA and RNA can participate in the pyrimidine triplex motif under mildly acidic conditions, these structures are often relatively unstable at neutral pH. We are now explorin g the stability of intrastrand DNA and RNA pyrimidine motif triplexes at physiological temperature and pH. Duplex and triplex formation were monitored by thermal denaturation analysis, circular dichroism spectroscopy and gel shift experi-ments. Short intrastrand triplexes were observed to form in the pyrimidine motif in both DNA and RNA. In the presence of physiological concentrations of Mg(2+)and at physiological pH, all detected triplexes were sufficiently stable to persist at physiological temperature. If sequences specifying such intrastrand triplexes are encoded in genomes, the potential exists for the formation of stable structures in RNA or DNA in vivo.

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca2+ homeostasis.

GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases.

We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

Influence of hairpins on template reannealing at trinucleotide repeat duplexes: a model for slipped DNA.

Hairpin stabilization of polymerase slippage has been proposed as part of the mechanism for large-scale expansion of CG-rich (CNG, where N = A, T, G, or C) trinucleotide repeats. However, hairpin formation does not entirely account for why long repeats but not short repeats or palindromes expand. Using ultraviolet spectroscopic methods, we examine the thermodynamic and kinetic properties of repeating trinucleotides to evaluate their behavior at a slippage site. We find that CNG trinucleotide repeats associated with expansion form stable hairpins whether they are short (with as few as 10 repeats) or long. However, long repeating stretches exist as single strands up to 2 orders of magnitude longer than sequences with either short repeats or random DNA. Thus, long hairpins have long lifetimes even in the presence of their complementary strands and inhibit duplex reannealing at a slippage site. The kinetic properties explain why expansion occurs with high frequency at long repeats but not at short repeats or palindromes.

DNA compression caused by an upstream point mutation.

We observed an apparent series of insertions and deletions beginning 5 bp downstream of an A-->G silent transition in exon 1 of the tumor necrosis factor receptor 1 gene. The apparent sequence anomaly was observed only in individuals carrying the transition. Formamide gel electrophoresis revealed that the apparent sequence anomaly was due to compression. The compression is plausibly explained by a hairpin in the reaction products in a region of trinucleotide CAG repeats. One should suspect the presence of DNA compression when a series of deletions and insertions follows a single base pair mutation that leads to a series of trinucleotide repeats.

Increased instability of intermediate alleles in families with sporadic Huntington disease compared to similar sized intermediate alleles in the general population.

We have directly compared intergenerational stability of intermediate alleles (IAs) derived from new mutation families (IANM) for Huntington disease (HD) with IAs in the general population (IAGP) which occur in approximately 1 in 50 persons. Analysis of meiotic events in blood and sperm reveals that IANM are significantly more unstable than IAGP despite similar size. However, for both IANM and IAGP CAG changes were small and risks for inheriting an expansion into the HD affected range were low. Sequence analysis reveals that the CAG tract is generally interrupted by a penultimate CAA in IAGP, IANM and alleles in the affected range. In one new mutation family, however, two A-->G mutations result in a pure CAG tract which is associated with very marked instability. These mutations alter the predicted DNA hairpin structure with a predicted increase in the likelihood of large expansion, supporting the model that hairpin loop formation plays an important role in trinucleotide instability.

Trinucleotide repeats that expand in human disease form hairpin structures in vitro.

We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly -50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMR1 alleles.

Hairpin formation within the human enkephalin enhancer region. 1. Kinetic analysis.

The 3'-5' cyclic AMP inducible enhancer region of the human enkephalin gene is located within an imperfect palindrome of 23 base pairs, located from -106 to -84 base pairs upstream of the transcriptional start site. Recent evidence has indicated that hairpin formation within this region may be involved in transcriptional regulation of the human proenkephalin gene. A 23-bp synthetic oligonucleotide of this region has been shown to undergo a reversible conformational change from a duplex to a cruciform structure of two hairpins [McMurray, C.T., Wilson, W.D., & Douglass, J.O. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 666]. Our current studies explore the kinetics and activation energies of the hairpin to duplex transitions of synthetic oligonucleotides under a variety of conditions. Ultraviolet spectroscopic data collected over a range of pH values, ionic strengths, and temperatures are used to determine the reaction rates and activation energies of the hairpin to duplex reaction. The rate of formation of a duplex from two hairpins is a slow second-order process, dependent on both pH and ionic strength. The return from the hairpin state to the duplex state occurs with a high activation energy of 22-41 kcal/mol strand, depending on the conditions. Pseudo-first-order reaction conditions indicate that one of the hairpins, the AC hairpin, is the rate-limiting reactant. These results suggest a model by which the formation of a cruciform might regulate transcription.