ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson’s disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis.     

Synthesis,characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.     

THE MORPHOLOGY AND DEVELOPMENT OF SOME PROMINENTLY STALKED SOUTHERN AUSTRALIAN HALYMENIACEAE (CRYPTONEMIALES,RHODOPHYTA). II. THE SPONGE-ASSOCIATED GENERA THAMNOCLONIUM KUETZING AND CODIOPHYLLUM GRAY1

Three members of the red algal family Halymeniaceae (Thamnoclonium dichotomum (J. Ag.) J. Ag., Codiophyllum flabelliforme (Sond.) Schmitz, and C. decipiens (J. Ag.) Schmitz) are investigated. All are endemic to southern and southwestern Australia, possess basal stalks of substantial size and firmness, and are consistently associated with specific sponge taxa. In each case, the sponges are bonded by collagen-like fibrils to the host cuticle without modifying the algal tissue at the ultrastructural level. Secondary cortication and prominent growth rings occur in the stalks of all three species, and in each the pit plugs between cells become wider, more convoluted and less electron dense with increasing distance from the surface. Such pit plugs are apparently a unique attribute of the stalked Halymeniaceae. The three species share pit plug, sponge association and stalk morphological features but are not otherwise closely related, as they actually represent three distinct genera.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas

Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 °C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 °C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased, to varying extents, in a linear fashion relative to reduced pH with the rate of change greatest in leatherbacks>greenloggerhead turtles. All studies were conducted with reagents developed for human samples which would impact on the quantitative results with the turtle samples, but are not likely to alter the qualitative results. These comparative studies of the coagulation pathway in sea turtles and humans could enhance our knowledge of structure/function relationships and evolution of coagulation factors.     

Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.     

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.     

Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase

The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.