Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

Effects of Doxorubicin on the Liquid-Liquid Phase Change Properties of Elastin-Like Polypeptides.

The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent ß-turn structures. Labeling with Dox further enhances ß-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets.

The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin.

M98Q amicyanin is isolated with zinc bound to its type 1 copper-binding site. The influence of the axial ligand of the type 1 copper site on metal specificity is strongest prior to the completion of protein folding and adoption of the final type 1 site geometry. The preference for zinc over copper correlated with the selectivity of apoamicyanin in vitro in the partially folded, rather than the completely folded state. These results suggest that metal incorporation in vivo occurs during protein folding in the periplasm and not to a preformed type 1 site.

Energetic basis of molecular recognition in a DNA aptamer.

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.

The ligand geometry of copper determines the stability of amicyanin.

Solution differential scanning calorimetry (DSC) of oxidized amicyanin, a Type I copper protein, at pH 7.5 reveals two thermal transitions. The major transition at 67.7 degrees C corresponds to the disruption of the Cys(92) thiolate to Cu(II) charge transfer as evidenced by a corresponding temperature-dependent loss of amicyanin visible absorbance. A minor transition at 75.5 degrees C describes the further irreversible protein unfolding. Reduced amicyanin exhibits a pH-dependent change of the copper ligand geometry. At pH 8.5 where the Type I tetrahedral geometry is maintained, DSC reveals two thermal transitions with T(m) values similar to that of oxidized amicyanin. At pH 6.2 where the Cu(I) coordination is trigonal planar, reduced amicyanin exhibits a single thermal transition with a lower T(m) of 64.0 degrees C. Apoamicyanin, from which copper has been removed, also exhibits a single thermal transition but with a much lower T(m) of 51.8 degrees C. Thus, the thermal stability of amicyanin is dictated both by the presence or absence of copper and its ligand geometry, but not its redox state. The physiological relevance of these data is discussed.

Probing the structure of DNA aptamers with a classic heterocycle.

DNA aptamers are synthetic, single-stranded DNA oligonucleotides selected by SELEX methods for their binding with specific ligands. Here we present ethidium binding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structure has been reported and each are found to be composed primarily of two domains consisting of a stem helical region and a loop domain that forms a binding pocket for the cognate ligand. Previous thermodynamic experiments demonstrated that the DNA aptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Here we extend those linkage binding studies by examining the binding of the heterocyclic intercalator ethidium to each of the three aptamers by fluorescence and absorption spectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with DeltaG degree's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for each aptamer and is quantitatively diminished in the presence of L-Arm as is the overall fluorescence intensity of ethidium. Together, these results demonstrate that a portion of the bound ethidium is excluded from the aptamer in the presence of a saturating amount of L-Arm. These results demonstrate the utility of ethidium and related compounds for the probing of non-conventional DNA structures and reveal an interesting fundamental thermodynamic linkage in DNA aptamers. Results are discussed in the context of the thermodynamic stability and structure of each of the aptamers examined.

Triplex selective 2-(2-naphthyl)quinoline compounds: origins of affinity and new design principles.

A novel competition dialysis assay was used to investigate the structural selectivity of a series of substituted 2-(2-naphthyl)quinoline compounds designed to target triplex DNA. The interaction of 14 compounds with 13 different nucleic acid sequences and structures was studied. A striking selectivity for the triplex structure poly dA:[poly dT](2) was found for the majority of compounds studied. Quantitative analysis of the competition dialysis binding data using newly developed metrics revealed that these compounds are among the most selective triplex-binding agents synthesized to date. A quantitative structure-affinity relationship (QSAR) was derived using triplex binding data for all 14 compounds used in these studies. The QSAR revealed that the primary favorable determinant of triplex binding free energy is the solvent accessible surface area. Triplex binding affinity is negatively correlated with compound electron affinity and the number of hydrogen bond donors. The QSAR provides guidelines for the design of improved triplex-binding agents.

Characterization of DNA structures by circular dichroism.

Circular dichroism (CD) of nucleic acids is commonly used to provide a signature for a given secondary structure. CD is particularly powerful for monitoring structural changes resulting from changes in environmental conditions such as temperature, ionic strength, and pH. A number of commercially available instruments are available for the measurement of CD spectra. Since the details of instrumental operation differ for each machine, this unit provides a descriptive guide, rather than a strict protocol. It describes considerations that the experimentalist will face when attempting to examine nucleic acid structures by CD spectropolarimetry. Specific details are also given for cell filling and cleaning, and for instrumental calibration. Finally, several examples of CD spectra are given to provide useful reference points for a variety of nucleic acid structures, ranging from single-stranded to triplex and quadruplex forms.

Mechanisms of nonspecific cytotoxic cell regulation of apoptosis: cytokine-like activity of Fas ligand.

The role of FasL/FasR pathways of immunoregulation of programmed cell death in teleost cytotoxic innate immunity has not been previously examined. In the present study, constitutive cytosolic soluble FasL (sFasL) was detected in anterior kidney (AK), peripheral blood (PBL) and liver NCC obtained from tilapia. Ligation of NCC by tumour cells caused the release of sFasL that was associated with lysis of HL-60 targets in 14 h killing assays. Evidence that sFasL mediated this activity was that anti-(human) FasL inhibited tilapia and catfish (cf.) NCC lysis of FasR+ HL-60 tumour cells. Inhibition was concentration dependent. Lysis of IM-9 targets (12% positive for FasR) by (cf.) anterior kidney and PBL NCC was only partially inhibited by anti-FasL mab. Activated NCC from both species were negative for the expression of membrane FasL and FasR. These data confirmed that NCC lyse sensitive tumour cells by multiple effector pathways. Pretreatment of (FasR+) HL-60 cells with anti-FasR mab completely inhibited cf. cytotoxicity at low (100:1) E:T ratios. Anti-FasR mab did not inhibit the lysis of IM-9 targets by cf. NCC. This study demonstrated that for catfish and tilapia, initial target cell conjugate formation was required; however, the terminal killing mechanism depended on at least two different pathways of cytotoxicity. One pathway depended on the release of preformed soluble FasL by activated NCC in the presence of FasR positive target cells. A second pathway has yet to be determined.