Fuzzy logic in mitochondrial locomotion.
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Fungal Proteins↗
Biomedical subjects
B H Robinson
Publications and source records attributed to B H Robinson.
Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry.
BACKGROUND: Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin. METHODS: Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy. RESULTS: Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy. CONCLUSIONS: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.
Adenosine Triphosphate↗
Expression and functional characterization of human protein X variants in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts.
To gain further insight into the nature and function of the domains of the human protein X (a pyruvate dehydrogenase complex component also known as the E3-binding protein), we expressed the wild-type as well as two artificially created variants, K37E and S422H, in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts. The former mutant does not carry the lipoic acid moiety, the latter mutant was designed to investigate the possibility that protein X could exhibit an intrinsic acetyltransferase activity and use either its own catalytic center or the catalytic center of E2. Similar experiments have been performed in the past using the Saccharomyces cerevisiae expression system. However, lack of sequence similarity between the mammalian and the yeast protein X homologues suggests they are not biochemically equivalent. Mutant cells transfected with the wild-type gene for protein X produced a PDH complex that exhibited about 50% overall activity of the control cells. None of the expressed protein X variants had an effect on the specific activity of the PDH complex, suggesting that the human protein X plays a purely structural role in the functioning of the pyruvate dehydrogenase complex.
Amino Acid Sequence↗
Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase.
In this report we show that ubiquinone cytochrome c reductase (complex III) from isolated rat heart mitochondria when inhibited with antimycin A, produces a large amount of superoxide as measured by the chemiluminescent probe coelenterazine. When mitochondria are inhibited with myxothiazol or stigmatellin, there is no detectable formation of superoxide. The antimycin A-sensitive free radical production can be dramatically reduced using either myxothiazol or stigmatellin. This suggests that the antimycin A-sensitive generation of superoxides originates primarily from the Q(o) semiubiquinone. When manganese superoxide dismutase depleted submitochondrial particles (SMP) were inhibited with myxothiazol or stigmatellin, a large superoxide signal was observed. These two inhibitors likely increase the concentration of the Q(i) semiquinone at the N center. The antimycin A-sensitive signal can, in the case of both the mitochondria and the SMP, be dissipated by the addition of copper zinc superoxide dismutase, suggesting that the measured coelenterazine signal was a result of superoxide production. Taken together, this data suggests that free radicals generated from the Q(i) species are more effectively eliminated by MnSOD in intact mitochondria.
Animals↗
Functional expression of four PDH-E(1)alpha recombinant histidine mutants in a human fibroblast cell line with zero endogenous PDH complex activity.
Conserved histidine residues have been implicated in the geometry and catalytic mechanism of the E(1)alpha proteins of the PDH complex. We constructed and expressed a series of PDH-E(1)alpha histidine mutants (H63, H84, H92, and H263) in a cell line with zero PDH complex activity due to a null E(1)alpha allele. Based on immunoblot and enzyme activity analyses, all introduced histidine mutations, with the exception of H92, affected the specific activity of the PDH complex. We showed that H63 and H263 are essential for the activity since mutations introduced at those sites produced a PDH complex with near-zero activity. Mutations introduced at H84 only partially reduced activity, implying that H84 may play a less critical role in the PDH complex. Mutations introduced at H92 caused the absence of immunoreactive material for both the E(1)alpha and E(1)beta subunits and may have impaired import or assembly of precursor peptides into the mature PDH complex.
Amino Acid Substitution↗
Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho(0) cells completely lacking mtdna.
The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho(0) cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F(1)F(0) were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F(0) part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F(1)F(0) has been purified for the first time from human cell lines. A partial complex was obtained from Rho(0) cells containing the F(1) subunits associated with several stalk, as well as F(0) subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein.
Adenosine Triphosphate↗
Sequential deletion of C-terminal amino acids of the E(1)alpha component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E(1)alpha(2)beta(2) tetramers: implications for patients with PDH deficiency.
Human pyruvate dehydrogenase (PDH) complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. We have characterized novel mutations that affect the C-terminal portion of the PDH-E(1)alpha-coding sequence. Although the molecular defects underlying these mutations are different, both effectively produce a stop codon prematurely three amino acids from the C-terminus. The clinical and biochemical consequences of these mutations are unusual in that the affected individuals are very long-term survivors with PDH complex deficiency despite having low (<20%) activity in skin fibroblasts. These findings prompted us to investigate the C-terminus of E(1)alpha in greater detail. We constructed and expressed a series of PDH-E(1)alpha deletion mutants in a cell line with zero PDH complex activity due to a null E(1)alpha allele. Sequential deletion of the C-terminus by one, two, three and four amino acids resulted in PDH complex activities of 100, 60, 36 and 14%, respectively, compared with wild-type E(1)alpha expressed in PDH complex-deficient cells. The immunodetectable protein was decreased by the same amount as the activity, suggesting that the stability and/or assembly of the E(1)alpha(2)beta(2)heterotetramer might depend on the intactness of the PDH-E(1)alpha C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E(1)alphaand concluded that they are biochemically equivalent.
Adult↗
Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency.
Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.
Amino Acid Sequence↗
Sequence-dependent dynamics in duplex DNA.
The submicrosecond bending dynamics of duplex DNA were measured at a single site, using a site-specific electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of the mean squared amplitude of bending relative to the end-to-end vector defined by the weakly bending rod model. The bending dynamics monitored at the single site varied when the length and position of a repeated AT sequence, distant from the spin probe, were changed. As the distance between the probe and the AT sequence was increased, the mean squared amplitude of bending seen by the probe due to that sequence decreased. A model for the sequence-dependent internal flexural motion of duplex DNA, which casts the mean squared bending amplitudes in terms of sequence-dependent bending parameters, has been developed. The best fit of the data to the model occurs when the (AT)(n) basepairs are assumed to be 20% more flexible than the average of the basepairs within the control sequence. These findings provide a quantitative basis for interpreting the kinetics of biological processes that depend on duplex DNA flexibility, such as protein recognition and chromatin packaging.
Animals↗
Mitochondria, oxygen free radicals, disease and ageing.
Superoxide is generated by the mitochondrial respiratory chain. The transformation of this superoxide into hydrogen peroxide and, under certain conditions, then into hydroxyl radicals is important in diseases where respiratory chain function is abnormal or where superoxide dismutase function is altered, as in amyotrophic lateral sclerosis. In addition, these reactive oxygen species can influence the ageing process through mechanisms involving mutagenesis of mtDNA or increased rates of shortening of telomeric DNA.
Aging↗
Human cytochrome oxidase deficiency.
The human cytochrome oxidase complex is a multisubunit assembly in the inner mitochondrial membrane responsible for the terminal event in electron transport in which molecular oxygen is reduced. Various phenotypic forms of cytochrome oxidase deficiency have been recognized, the major varieties involving degeneration of the brain stem and basal ganglia (Leigh syndrome) and lactic acidemia. Others include a fatal infantile form, a benign reversible form, and forms with cardiomyopathy. Early recognition of complementation groups within, for instance, the Leigh syndrome group has recently been followed up with a description of the gene defect for three of the nuclear-encoded forms of cytochrome c oxidase (COX) deficiency. The three genes indicted, SURF1 for Leigh syndrome, COX 10 for leukodystrophy and tubulopathy, and SCO2 for the cardiomyopathic form, all have a role in the assembly of the mature cytochrome oxidase complex. The description of these gene defects and the role these genes play are discussed in terms of what can be learned about COX assembly and about the etiology of the different phenotypic forms of the disease.
Alkyl and Aryl Transferases↗
Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.
The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.
Biological Transport↗
Pyruvate carboxylase deficiency: prenatal onset of ischemia-like brain lesions in two sibs with the acute neonatal form.
Pyruvate carboxylase (PC) is a key enzyme in the gluconeogenesis and anaplerotic metabolic pathways. PC deficiency is a rare autosomal recessive disorder with three clinical presentations: an infantile form, a severe neonatal form, and a benign form. We report brother and sister sibs with the severe form of PC deficiency. Both had macrocephaly and severe ischemia-like brain lesions at birth and died in the first week of life with intractable lactic acidemia. In the girl, increased head circumference and periventricular leukomalacia (PVL) were detected on fetal ultrasonography at 29.4 weeks of gestation. PC activity in cultured skin fibroblasts was <2% of control. This is the first reported case of ischemia-like brain lesions documented prenatally in PC deficiency. The lesions were detected at a time of maximal periventricular metabolic demand. We postulate that energy deprivation induced by PC deficiency impairs astrocytic buffering capacity against excitotoxic insult and compromises normal microvascular morphogenesis and autoregulation, both mechanisms leading to cystic degeneration of the periventricular white matter. Discovery of cystic PVL on cerebral ultrasound at birth in a newborn infant presenting with primary lactic acidemia is highly suggestive of PC deficiency. Moreover, PC deficiency should also be considered when ischemia-like brain lesions are documented by fetal ultrasonography.
Brain↗
Repopulation of rho0 cells with mitochondria from a patient with a mitochondrial DNA point mutation in tRNA(Gly) results in respiratory chain dysfunction.
Familial hypertrophic ventricular cardiomyopathy has been demonstrated to be associated with a number of mitochondrial DNA (mtDNA) mutations. A fibroblast cell line carrying a mutation in its mtDNA at position 9997 in the gene encoding tRNA glycine was obtained from a patient with hypertrophic cardiomyopathy. To demonstrate that the etiology of this disease was a result of the mtDNA mutation, cybrid clones were constructed by fusion of enucleated patient skin fibroblasts to rho0 osteosarcoma cells. Clones carrying high levels of mutant mtDNA showed predominantly cytochrome c oxidase and complex I deficiency, as well as an elevated lactate/pyruvate (L/P) ratio, a biochemical marker characteristic of respiratory chain deficiencies. Pulse-labeling experiments demonstrated a strong negative correlation between the levels of newly synthesized mtDNA-encoded polypeptides and glycine content. These data suggest that the T9997C mutation in mtDNA is causative of respiratory chain dysfunction when present at high levels of heteroplasmy.
Blotting, Southern↗
Linewidth analysis of spin labels in liquids. I. Theory and data analysis.
We present a method of simulating the EPR spectra of spin labels in liquids using direct convolution of hyperfine splitting with Lorentzian linewidths. The aim is to simulate the experimental lineshape by considering all spectrometer characteristics as well as inhomogeneous and homogeneous linewidth effects. A major advance in this method is the correction for the broadening produced by Zeeman modulation commonly used to obtain EPR signals; this allows experimenters much more freedom to optimize their experimental conditions for the best signal-to-noise ratio. Microwave power broadening (saturation) effects on the EPR lines are significant even at very low observer levels. Successful simulation requires that all contributions from unresolved hyperfine splittings be explicitly included. Inhomogeneous broadening is dealt with by including all spins that interact with the electron (as a set of superhyperfine interactions); there is no "effective Gaussian" to substitute for the correct superhyperfine interactions. The effects of spin exchange on the linewidth and lineshape can be observed and must be taken into account in order to extract the fundamental linewidths.
Computer Simulation↗
Linewidth analysis of spin labels in liquids. II. Experimental.
This work demonstrates that homogeneous linewidths can be extracted from continuous wave electron paramagnetic resonance spectra and that they quantitatively agree with the predictions of existing relaxation theory. We suggest that relaxation theory can be used to predict experimental lineshapes provided that the simulations properly include sources of broadening. We have found that the rotational correlation times for spin labels in different percentages of glycerol/water mixtures are best modeled by a power law treatment for the viscosity, similar to that for translational diffusion. The translational diffusion coefficients themselves also have a power law dependence on the viscosity for glycerol/water mixtures. The linewidths were linearly dependent upon both the oxygen and the spin label concentration. The hyperfine splittings of all nuclei were observed to decrease linearly with increasing spin label concentration, completely at odds with existing theory which predicts a quadratic dependence upon concentration. The linear dependence was independent of hyperfine splitting until the magnitude of the hyperfine splitting was less than the homogeneous linewidth.
Computer Simulation↗
Manifestations of slow site exchange processes in solution NMR: a continuous Gaussian exchange model.
The effects of site exchange due to slow conformational changes in rapidly rotating molecules in solution are examined in detail. Significant gaps in the currently available theory are filled. The effects of site exchange on the lineshape, decay of a simple spin-echo, decay of the even echoes in a Carr-Purcell-Meiboom-Gill (CPMG) pulse-sequence, and decay of the transverse magnetization in a resonant spin-locking field are investigated. Both trajectory and stochastic operator approaches are formulated and shown to be completely equivalent whenever the dynamics of population transfers among the inequivalent sites is governed by either a stationary or a nonstationary Markov process. A nonstationary Markov process may result from Brownian dynamics (a stationary Markov process) in a larger conformational space that contains the subspace of inequivalent sites. A continuous Gaussian exchange model is formulated in which a nucleus undergoes continuous one-dimensional motion in a harmonic potential well that is located in a linear chemical shift gradient. The effects of this Gaussian exchange model on the lineshape, simple spin-echo decay, and decay of the even echoes of a CPMG pulse train are treated rigorously via the trajectory approach. Compact analytical expressions are obtained for the relevant correlation functions in each case. The relevant decays are found to be exponential in the very short time and long time limits, which are not necessarily experimentally significant in any given case. In the fast exchange limit the relevant decays are exponential at all times, and explicit formulas are given for their decay rates. In the long time limit, all discrete multisite models with the same intrinsic Ro2 at every site are shown to be completely equivalent to a continuous Gaussian model with appropriate relaxation time and variance of the Larmor frequency. The effects of this Gaussian exchange model on the decay of the transverse magnetization in a resonant spin-locking field are treated heuristically by a trajectory approach. The intrinsic contribution (Ro1rho) of rapid rotations and dipole-dipole interactions to relax the transverse magnetizations of two nuclei of the same kind in the presence of a (nearly) resonant spin-locking field is also derived and found to be practically the same as the intrinsic contribution, Ro2, of those same rotations to the simple and CPMG spin-echo decay rates and linewidth. Literature data for the linewidth, decay rate of the CPMG even spin-echoes, and R(1rho) decay rate for the A9-H2 protons of adenines at the central TpA step in the sequence, 5'-GCAGGTTTAAACCTCG-3', are analyzed using the Gaussian exchange model to assess the time-scale and variance of the site exchange process as well as the intrinsic Ro2 rate. Although a single Gaussian exchange process with appropriate parameters can fit these three A9-H2 data rather well, this particular "solution" cannot be reconciled with NMR relaxation data on other protons in the same DNA molecule. Rather good agreement with all of the observations is obtained by using a model of two concurrent Gaussian exchange processes, whose relaxation times, tau = 7 and 460 micros, differ in time-scale by a factor of 65. The insensitivity of R1rho in the presence of a fast site exchange process to much slower concurrent site exchange processes is explicitly demonstrated. Protocols for detecting and characterizing a second slow site exchange process are suggested.
Adenine↗
Flexibility of duplex DNA on the submicrosecond timescale.
Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.
Base Sequence↗