N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

The Parkinson disease protein α-synuclein is N-terminally acetylated, but most in vitro studies have been performed using unacetylated α-synuclein. Binding to lipid membranes is considered key to the still poorly understood function of α-synuclein. We report the effects of N-terminal acetylation on α-synuclein binding to lipid vesicles of different composition and curvature and to micelles composed of the detergents β-octyl-glucoside (BOG) and SDS. In the presence of SDS, N-terminal acetylation results in a slightly increased helicity for the N-terminal ∼10 residues of the protein, likely due to the stabilization of N-terminal fraying through the formation of a helix cap motif. In the presence of BOG, a detergent used in previous isolations of helical oligomeric forms of α-synuclein, the N-terminally acetylated protein adopts a novel conformation in which the N-terminal ∼30 residues bind the detergent micelle in a partly helical conformation, whereas the remainder of the protein remains unbound and disordered. Binding of α-synuclein to lipid vesicles with high negative charge content is essentially unaffected by N-terminal acetylation irrespective of curvature, but binding to vesicles of lower negative charge content is increased, with stronger binding observed for vesicles with higher curvature. Thus, the naturally occurring N-terminally acetylated form of α-synuclein exhibits stabilized helicity at its N terminus and increased affinity for lipid vesicles similar to synaptic vesicles, a binding target of the protein in vivo. Furthermore, the novel BOG-bound state of N-terminally acetylated α-synuclein may serve as a model of partly helical membrane-bound intermediates with a role in α-synuclein function and dysfunction.     

The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain IV. Rate constant determination

The expressions for the kinetic constants corresponding to the steady state model for hydrolysis of ATP catalyzed by (Na⁺ + K⁺)-ATPase proposed recently are analyzed with the object of determining the rate constants. The theoretical background for the necessary procedures is described. The results of this analysis are: (1) A small class (four) of rate constants are determined directly by the previously published values of the kinetic constants. (2) For a somewhat larger class of rate constants upper and lower bounds may be established. For several rate constants the upper and lower bounds differ by less than a factor 1.6 (for the ‘(Na⁺ + K⁺)-enzyme’, i.e. the enzyme activity with K⁺ and millimolar substrate concentration) and 1.2 (for the ‘Na⁺-enzyme’, i.e. the activity at micromolar substrate concentrations). (3) Experiments on inhibition by K⁺ of the Na⁺-enzyme at various Mg²⁺ concentrations are reported and analyzed. With the additional assumption that the rate constants governing the addition to ATP of Mg²⁺ is independent of whether or not ATP is bound to an enzyme molecule, a set of consistent values for all the 23 rate constants in the mechanism may be obtained. (4) The values of some rate constants lend further support to the contention discussed in a previous paper that the enzyme hydrolyzes ATP along two kinetically distinct pathways, depending on the presence of K⁺ and on the concentration of substrate, without the necessity of having more than one active substrate site per enzyme unit at any time. (5) The results show that while the two enzyme forms, the ‘Na⁺-enzyme’ E₁ and the “K⁺-enzyme” E₂K, add substrate with (second order) rate constants of the same order of magnitude (differing only by a factor of four in favor of the former), the rate constants for the reverse processes differ by a factor of 100, being largest for the K⁺-enzyme. This is the main reason for the large difference in the Michaelis constants for the two forms reported previously. (6) Compatibility of the model with the well-known rapid dephosphorylation of the phosphorylated enzyme in the presence of K⁺ requires the presence, at non-zero steady state concentration, of an enzyme-potassium-phosphate intermediate, which is acid labile and is therefore not detected as a phosphorylated enzyme using conventional methods.     

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Elongation factor eEF1B modulates functions of the release factors eRF1 and eRF3 and the efficiency of translation termination in yeast

Background  

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation.     

Effects of age and caloric restriction on glutathione redox state in mice

The main purpose of this study was to determine whether the aging process in the mouse is associated with a pro-oxidizing shift in the redox state of glutathione and whether restriction of caloric intake, which results in the extension of life span, retards such a shift. Amounts of reduced and oxidized forms of glutathione (GSH and GSSG, respectively) and protein-glutathione mixed disulfides (protein-SSG) were measured in homogenates and mitochondria of liver, kidney, heart, brain, eye, and testis of 4, 10, 22, and 26 month old ad libitum-fed (AL) mice and 22 month old mice fed a diet containing 40% fewer calories than the AL group from the age of 4 months. The concentrations of GSH, GSSG, and protein-SSG vary greatly (approximately 10-, 30-, and 9-fold, respectively) from one tissue to another. During aging, the ratios of GSH:GSSG in mitochondria and tissue homogenates decreased, primarily due to elevations in GSSG content, while the protein-SSG content increased significantly. Glutathione redox potential in mitochondria became less negative, i.e., more pro-oxidizing, as the animal aged. Caloric restriction (CR) lowered the GSSG and protein-SSG content. Results suggest that the aging process in the mouse is associated with a gradual pro-oxidizing shift in the glutathione redox state and that CR attenuates this shift.     

Widespread positive selection in synonymous sites of mammalian genes

Evolution of protein sequences is largely governed by purifying selection, with a small fraction of proteins evolving under positive selection. The evolution at synonymous positions in protein-coding genes is not nearly as well understood, with the extent and types of selection remaining, largely, unclear. A statistical test to identify purifying and positive selection at synonymous sites in protein-coding genes was developed. The method compares the rate of evolution at synonymous sites (Ks) to that in intron sequences of the same gene after sampling the aligned intron sequences to mimic the statistical properties of coding sequences. We detected purifying selection at synonymous sites in approximately 28% of the 1,562 analyzed orthologous genes from mouse and rat, and positive selection in approximately 12% of the genes. Thus, the fraction of genes with readily detectable positive selection at synonymous sites is much greater than the fraction of genes with comparable positive selection at nonsynonymous sites, i.e., at the level of the protein sequence. Unlike other genes, the genes with positive selection at synonymous sites showed no correlation between Ks and the rate of evolution in nonsynonymous sites (Ka), indicating that evolution of synonymous sites under positive selection is decoupled from protein evolution. The genes with purifying selection at synonymous sites showed significant anticorrelation between Ks and expression level and breadth, indicating that highly expressed genes evolve slowly. The genes with positive selection at synonymous sites showed the opposite trend, i.e., highly expressed genes had, on average, higher Ks. For the genes with positive selection at synonymous sites, a significantly lower mRNA stability is predicted compared to the genes with negative selection. Thus, mRNA destabilization could be an important factor driving positive selection in nonsynonymous sites, probably, through regulation of expression at the level of mRNA degradation and, possibly, also translation rate. So, unexpectedly, we found that positive selection at synonymous sites of mammalian genes is substantially more common than positive selection at the level of protein sequences. Positive selection at synonymous sites might act through mRNA destabilization affecting mRNA levels and translation.     

Multigene Phylogenetics Reveals Temporal Diversification of Major African Malaria Vectors

The major vectors of malaria in sub-Saharan Africa belong to subgenus Cellia. Yet, phylogenetic relationships and temporal diversification among African mosquito species have not been unambiguously determined. Knowledge about vector evolutionary history is crucial for correct interpretation of genetic changes identified through comparative genomics analyses. In this study, we estimated a molecular phylogeny using 49 gene sequences for the African malaria vectors An. gambiae, An. funestus, An. nili, the Asian malaria mosquito An. stephensi, and the outgroup species Culex quinquefasciatus and Aedes aegypti. To infer the phylogeny, we identified orthologous sequences uniformly distributed approximately every 5 Mb in the five chromosomal arms. The sequences were aligned and the phylogenetic trees were inferred using maximum likelihood and neighbor-joining methods. Bayesian molecular dating using a relaxed log normal model was used to infer divergence times. Trees from individual genes agreed with each other, placing An. nili as a basal clade that diversified from the studied malaria mosquito species 47.6 million years ago (mya). Other African malaria vectors originated more recently, and independently acquired traits related to vectorial capacity. The lineage leading to An. gambiae diverged 30.4 mya, while the African vector An. funestus and the Asian vector An. stephensi were the most closely related sister taxa that split 20.8 mya. These results were supported by consistently high bootstrap values in concatenated phylogenetic trees generated individually for each chromosomal arm. Genome-wide multigene phylogenetic analysis is a useful approach for discerning historic relationships among malaria vectors, providing a framework for the correct interpretation of genomic changes across species, and comprehending the evolutionary origins of this ubiquitous and deadly insect-borne disease.