Metabolic relevance of selenium in the insectCorcyra cephalonica

Requirement, uptake, and subcellular distribution of Na₂ ⁷⁵SeO₃ in the larvae of the insectC. cephalonica was investigated. That Se is well tolerated byC. cephalonica upto an added level of 2 ppm in the diet is suggested by the observed increase in body weight, total protein, and succinate dehydrogenase levels. Significant increases in the State 3 respiration ensued with Se supplementation up to 2 ppm in the mitochondrial oxidation of D-glycerol 1-phosphate, succinate and NADH, along with concomitant unaltered State 4 respiration, leading to enhanced RCR values. Maximal uptake of⁷⁵Se was registered in the larvae maintained on basal diet when subjected to short-term exposure to 0.5 ppm⁷⁵Se level. When exposure level was further increased up to 20 ppm, the observed decrease in the uptake of⁷⁵Se suggested that Se status of larvae itself controlled the tissue uptake. Subcellular distribution pattern revealed maximal incorporation of⁷⁵Se (cpm/g tissue) in the supernatant fraction, whereas, maximal specific⁷⁵Se activity (cpm/mg protein) was associated with the mitochondrial fraction. Autoradiography of the soluble fractions indicated the presence of single selenoprotein in the larval group with short term 2 ppm⁷⁵Se exposure. Inherent Se controls both the extent and the nature of distribution of mitochondrial⁷⁵Se incorporation. Uptake of⁴⁵Ca by the insect mitochondria was enhanced by dietary Se up to 2 ppm but was unaffected by addition ofin vitro ⁷⁵Se in the medium. A more fundamental role for Se in the mitochondrial energy metabolism emerges from these studies.     

The Positional Specificity of EXXK Motifs within an Amphipathic α-Helix Dictates Preferential Lysine Modification by Acrolein: Implications for the Design of High-Density Lipoprotein Mimetic Peptides

Despite the ability of acrolein to damage proteins, factors governing its reactivity with the ε-amino group of lysine are poorly understood. We used a small 26-mer α-helical peptide (ATI-5261) to evaluate the influence of acidic glutamate (E) residues on site-specific lysine modification by acrolein and if this targeting played a major role in inhibiting the cholesterol efflux activity of the peptide. Exposure of ATI-5261 to acrolein resulted in N-(3-formyl-3,4-dehydropiperidino) (FDP)-lysine adducts at positions 5 and 25 and led to a concentration-dependent reduction in cholesterol efflux activity (55 ± 7 and 83 ± 3% decrease with 5:1 and 20:1 acrolein:peptide molar ratios, respectively). Amino acid substitution (K → R) experiments and mass spectrometry revealed neither K5 nor K25 was preferentially modified by acrolein, despite the location of K5 within a putative EXXK motif. Moreover, both lysine residues remained equally reactive when the lipidated peptide was exposed to acrolein. In contrast, placement of EXXK in the center of ATI-5261 resulted in site-specific modification of lysine. The latter was dependent on glutamate, thus establishing that acidic residues facilitate lysine modification and form the molecular basis of the EXXK motif. Preferential targeting of lysine, however, failed to augment the inhibitory effect of the aldehyde. Overall, the inhibitory effects of acrolein on cholesterol efflux activity were largely dependent on the number of lysine residue modifications and cross-linking of α-helical strands that restricted dissociation of the peptide to active forms.     

Molecular Ageing of Alpha- and Beta-Synucleins: Protein Damage and Repair Mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[³H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated ³H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of ³H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.     

Pharmacological Inhibition of Caspase-2 Protects Axotomised Retinal Ganglion Cells from Apoptosis in Adult Rats

Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL⁺ RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3–4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.     

Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1(-/-)) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1(-/-) mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-(3)H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1(-/-) animals compared with Pcmt1(+/+) littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, beta-synuclein, and alpha-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1(-/-) brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.     

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.     

Targeted Intracellular Delivery of Resveratrol to Glioblastoma Cells Using Apolipoprotein E-Containing Reconstituted HDL as a Nanovehicle

The objective of this study is to transport and deliver resveratrol to intracellular sites using apolipoprotein E3 (apoE3). Reconstituted high-density lipoprotein (rHDL) bearing resveratrol (rHDL/res) was prepared using phospholipids and the low-density lipoprotein receptor (LDLr)-binding domain of apoE3. Biophysical characterization revealed that resveratrol was partitioned into the phospholipid bilayer of discoidal rHDL/res particles (~19 nm diameter). Co-immunoprecipitation studies indicated that the LDLr-binding ability of apoE3 was retained. Cellular uptake of resveratrol to intracellular sites was evaluated in glioblastoma A-172 cells by direct fluorescence using chemically synthesized NBD-labeled resveratrol (res/NBD) embedded in rHDL/res. Competition and inhibition studies indicate that the uptake is by receptor mediated endocytosis via the LDLr, with co-localization of apoE3 and res/NBD in late endosomes/lysosomes. We propose that rHDL provides an ideal hydrophobic milieu to sequester resveratrol and that rHDL containing apoE3 serves as an effective “nanovehicle” to transport and deliver resveratrol to targeted intracellular sites.     

HDL mimetic peptide ATI-5261 forms an oligomeric assembly in solution that dissociates to monomers upon dilution

ATI-5261 is a 26-mer peptide that stimulates cellular cholesterol efflux with high potency. This peptide displays high aqueous solubility, despite having amphipathic α-helix structure and a broad nonpolar surface. These features suggested to us that ATI-5261 may adopt a specific form in solution, having favorable structural characteristics and dynamics. To test this, we subjected ATI-5261 to a series of biophysical studies and correlated self-association with secondary structure and activity. Gel-filtration chromatography and native gel electrophoresis indicated ATI-5261 adopted a discrete self-associated form of low molecular weight at concentrations >1 mg/mL. Formation of a discrete molecular species was verified by small-angle X-ray scattering (SAXS), which further revealed the peptide formed a tetrameric assembly having an elongated shape and hollow central core. This assembly dissociated to individual peptide strands upon dilution to concentrations required for promoting high-affinity cholesterol efflux from cells. Moreover, the α-helical content of ATI-5261 was exceptionally high (74.1 ± 6.8%) regardless of physical form and concentration. Collectively, these results indicate ATI-5261 displays oligomeric behavior generally similar to native apolipoproteins and dissociates to monomers of high α-helical content upon dilution. Optimizing self-association behavior and secondary structure may prove useful for improving the translatability and efficacy of apolipoprotein mimetic peptides.     

Modification by acrolein, a component of tobacco smoke and age-related oxidative stress, mediates functional impairment of human apolipoprotein E

Oxidative damage to proteins such as apolipoprotein B-100 increases the atherogenicity of low-density lipoproteins (LDL). However, little is known about the potential oxidative damage to apolipoprotein E (apoE), an exchangeable antiatherogenic apolipoprotein. ApoE plays an integral role in lipoprotein metabolism by regulating the plasma cholesterol and triglyceride levels. Hepatic uptake of lipoproteins is facilitated by apoE's ability to bind with cell surface heparan sulfate proteoglycans and to lipoprotein receptors via basic residues in its 22 kDa N-terminal domain (NT). We investigated the effect of acrolein, an aldehydic product of endogenous lipid peroxidation and a tobacco smoke component, on the conformation and function of recombinant human apoE3-NT. Acrolein caused oxidative modification of apoE3-NT as detected by Western blot with acrolein-lysine-specific antibodies, and tertiary conformational alterations. Acrolein modification impairs the ability of apoE3-NT to interact with heparin and the LDL receptor. Furthermore, acrolein-modified apoE3-NT displayed a 5-fold decrease in its ability to interact with lipid surfaces. Our data indicate that acrolein disrupts the functional integrity of apoE3, which likely interferes with its role in regulating plasma cholesterol homeostasis. These observations have implications regarding the role of apoE in the pathogenesis of smoking- and oxidative stress-mediated cardiovascular and cerebrovascular diseases.     

Examination of lipid-bound conformation of apolipoprotein E4 by pyrene excimer fluorescence

Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wild-type apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another.