Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.     

磷脂组成对脱脂蛋白模型多肽与脂质体相互作用的影响

根据脱脂蛋白的脂结合序列合成了两个两亲性多肽Ａｍｐ１和Ａｍｐ２,在Ａｍｐ２在缬氨酸残基取代了Ａｍｐ１第４位的赖氨酸残基。用内源荧光谱发射峰的蓝移,包埋的钙氯黄素在脂质体中的渗漏,丙烯酰胺对多肽色氨酸残基的淬灭等手段比较了Ａｍｐ１与Ａｍｐ２与具有不同磷脂组成的脂质体的相互作用,并研究了温度的影响。     

Lysozyme Mutants Accumulate in Cells while Associated at their N-terminal Alpha-domain with the Endoplasmic Reticulum Chaperone GRP78/BiP

Amyloidogenic human lysozyme variants deposit in cells and cause systemic amyloidosis. We recently observed that such lysozymes accumulate in the endoplasmic reticulum (ER) with the ER chaperone GRP78/BiP, accompanying the ER stress response. Here we investigated the region of lysozyme that is critical to its association with GRP78/BiP. In addition to the above-mentioned variants of lysozyme, we constructed lysozyme truncation or substitution mutants. These were co-expressed with GRP78/BiP (tagged with FLAG) in cultured human embryonic kidney cells, which were analyzed by western blotting and immunocytochemistry using anti-lysozyme and anti-FLAG antibodies. The amyloidogenic variants were confirmed to be strongly associated with GRP78/BiP as revealed by the co-immunoprecipitation assay, whereas N-terminal mutants pruned of 1-41 or 1-51 residues were found not to be associated with the chaperone. Single amino acid substitutions for the leucine array along the α-helices in the N-terminal region resulted in wild-type lysozyme remaining attached to GRP78/BiP. These mutations also tended to show lowered secretion ability. We conclude that the N-terminal α-helices region of the lysozyme is pivotal for its strong adhesion to GRP78/BiP. We suspect that wild-type lysozyme interacts with the GRP at this region as a step in the proper folding monitored by the ER chaperone.     

Involvement of the N-terminal B-box Domain of Arabidopsis BBX32 Protein in Interaction with Soybean BBX62 Protein

Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.     

Compromised Catalysis and Potential Folding Defects in in Vitro Studies of Missense Mutants Associated with Hereditary Phosphoglucomutase 1 Deficiency

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with k_cat/K_m often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency.     

Structural Basis for Ca2+-mediated Interaction of the Perforin C2 Domain with Lipid Membranes

Natural killer cells and cytotoxic T-lymphocytes deploy perforin and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca²⁺ and lipid binding to the C2 domain by NMR spectroscopy and x-ray crystallography. Calcium titrations, together with dodecylphosphocholine micelle experiments, confirmed that multiple Ca²⁺ ions bind within the calcium-binding regions, activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and have shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca²⁺ binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.     

Fatty Acids Bind Tightly to the N-terminal Domain of Angiopoietin-like Protein 4 and Modulate Its Interaction with Lipoprotein Lipase

Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.     

Maintaining Connexin43 Gap Junctional Communication in v-Src Cells Does Not Alter Growth Properties Associated with the Transformed Phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine “normal” cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Alteration of the Rugose Phenotype in waaG and ddhC Mutants of Salmonella enterica Serovar Typhimurium DT104 Is Associated with Inverse Production of Curli and Cellulose

The rugose (also known as wrinkled or rdar) phenotype in Salmonella enterica serovar Typhimurium DT104 Rv has been associated with cell aggregation and the ability, at low temperature under low-osmolarity conditions, to form pellicles and biofilms. Two Tn5 insertion mutations in genes that are involved in lipopolysaccharide (LPS) synthesis, ddhC (A1-8) and waaG (A1-9), of Rv resulted in diminished expression of colony rugosity. Scanning electron micrographs revealed that the ddhC mutant showed reduced amounts of extracellular matrix, while there was relatively more, profuse matrix production in the waaG mutant, compared to Rv. Both mutants appeared to produce decreased levels of curli, as judged by Western blot assays probed with anti-AgfA (curli) antibodies but, surprisingly, were observed to have increased amounts of cellulose relative to Rv. Comparison with a non-curli-producing mutant suggested that the alteration in curli production may have engendered the increased presence of cellulose. While both mutants had impaired biofilm formation when grown in rich medium with low osmolarity, they constitutively formed larger amounts of biofilms when the growth medium was supplemented with either glucose or a combination of glucose and NaCl. These observations indicated that LPS alterations may have opposing effects on biofilm formation in these mutants, depending upon either the presence or the absence of these osmolytes. The phenotypes of the waaG mutant were further confirmed in a constructed, nonpolar deletion mutant of S. enterica serovar Typhimurium LT2, where restoration to the wild-type phenotypes was accomplished by complementation. These results highlight the importance of an integral LPS, at both the O-antigen and core polysaccharide levels, in the modulation of curli protein and cellulose production, as well as in biofilm formation, thereby adding another potential component to the complex regulatory system which governs multicellular behaviors in S. enterica serovar Typhimurium.     

Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases

Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1.     

DnaK Chaperone-dependent Disaggregation by Caseinolytic Peptidase B (ClpB) Mutants Reveals Functional Overlap in the N-terminal Domain and Nucleotide-binding Domain-1 Pore Tyrosine

Protein disaggregation in Escherichia coli is carried out by ClpB, an AAA(+) (ATPases associated with various cellular activities) molecular chaperone, together with the DnaK chaperone system. Conformational changes in ClpB driven by ATP binding and hydrolysis promote substrate binding, unfolding, and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and -2 (NBD-2), which reside in flexible loops extending into the central pore of the ClpB hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding, and like the NBD-1 pore loop tyrosine, it is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and a Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant.     

Inability of Pseudomonas stutzeri Denitrification Mutants with the Phenotype of Pseudomonas aeruginosa to Grow in Nitrous Oxide

[This corrects the article on p. 1301 in vol. 50.].     

Drug-Carrier and Stability Properties of the Long-Lived Lipid Vesicles. Cryptosomes,in Vitro and in Vivo

Abstract

Lipid vesicles composed of phosphatidylcholine and suitable polyoxyethylene-derivat-ives of phosphatidylethanolamine (cryptosomes) remain in circulation 8–10 times longer than standard liposomes after an i.v. administration in mice. In contrast to previous belief, this longevity is not destroyed by the net charges on the lipid vesicle surface and is not a direct consequence of the high surface hydrophilicity; also bilayer fluidity is not an obstacle for the attainment of long circulation times. All these three factors, however, can affect the effectiveness of the drug encapsulation into lipid vesicles and the stability of the resulting carrier suspensions. Terminal head-group modifications, moreover, can affect the final carrier and drug distribution after vesicle applications in vivo and lead to accumulation in certain body subsites, such as the gut.     

Polymorphism of the Apolipoprotein B Gene and Association with Plasma Lipid and Lipoprotein Levels in the Mongolian Buryat

Allele frequencies at six RFLP sites (Ins/Del, ApaLI, AluI, XbaI, MspI, and EcoRI) of the apolipoprotein B gene (APOB) and the relationship of genotypes with plasma lipid and lipoprotein levels in the Mongolian Buryat were investigated. Common alleles at these sites in 110 Buryat subjects were I, G, A-, X-, M+, and E+; the frequencies of 0.809-0.991 differed strikingly from those of a few Asians and most Europeans. Five unambiguous haplotypes of all sites were revealed at 74%; haplotype IGA-X-M+E+ (000000) was the most frequent (67%), followed by IGA+X-M+E+ (001000) (19%). The frequency constitution differed significantly from the Chinese, Malaysians, and Caucasians but resembled the Indians. No APOB polymorphisms were associated with cholesterol levels (total, HDL and LDL). Significant associations of genotypes were shown with the triglyceride level only at the AluI and XbaI sites. The lipid level of A-A+ females or X-X+ males was higher than that of A-A- females or X-X- males, respectively.     

Circulating Irisin Concentrations Are Associated with a Favourable Lipid Profile in the General Population

Background/aimsIrisin is a myokine, which is mainly inversely associated with the risk for non-communicable diseases. Irisin improves cellular energy metabolism by uncoupling the mitochondrial respiratory chain resulting in increased energy expenditure using lipids. To date potential associations between irisin concentration and lipid profile are poorly understood. Therefore, this investigation aimed to evaluate potential associations between irisin and lipid levels in the general population.MethodsData of 430 men and 537 women from the population-based Study of Health in Pomerania (SHIP-TREND) with available irisin and lipid concentrations were used. Analyses of variance, linear and logistic regression models adjusted for age, HBA1c, waist circumference, physical activity, smoking, alcohol consumption, systolic blood pressure, ALAT were calculated.ResultsWe detected significantly inverse associations between irisin and circulating levels of total [beta coefficient 0.21 (standard error 0.08), p = 0.01], low-density cholesterol [-0.16 (0.07), p = 0.03] and triglycerides [-0.17 (0.08), p = 0.02] for men. Females without lipid lowering medication had an inverse association between irisin and total cholesterol [-0.12 (0.06), p = 0.05]. Further, male subjects with irisin concentrations in the third tertile had an increased odds for elevated low-density cholesterol [odds ratio 1.96 (95% confidence interval 1.07–3.48), p = 0.03) and triglyceride [1.95 (1.09–3.47), p = 0.02] levels, even after exclusion of subjects with lipid lowering medication. In addition, our data revealed an annual rhythm of serum irisin levels with peak levels arise in winter and summer months.ConclusionThis is the first investigation to report a significant association between circulating irisin and a favourable lipid profile in the general population. This may infer that higher irisin concentrations are associated with a reduced risk for non-communicable diseases.