The amyloid fibrils of the constant domain of immunoglobulin light chain

Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β₂-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.     

High density lipoprotein inhibits the activation of sterol regulatory element-binding protein-1 in cultured cells

A link between cellular uptake of high density lipoprotein (HDL) and regulation of sterol regulatory element-binding protein-1 (SREBP-1) was investigated in vitro. HDL decreased nuclear SREBP-1 levels as well as SREBP-1 target gene expression in HepG2 and HEK293 cells. However, HDL did not repress an exogenously expressed, constitutively active form of SREBP-1. HDL increased cellular cholesterol levels, and cellular cholesterol depletion by methyl-β-cyclodextrin abolished the effects of HDL. These results suggest that HDL inhibits the activation of SREBP-1 through a cholesterol-dependent mechanism, which may play an important role in regulating lipid synthetic pathways mediated by SREBP-1.     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.     

Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine

The alkaloid drug berberine (BBR) was recently described to decrease plasma cholesterol and triglycerides (TGs) in hypercholesterolemic patients by increasing expression of the hepatic low density lipoprotein receptor (LDLR). Using HepG2 human hepatoma cells, we found that BBR inhibits cholesterol and TG synthesis in a similar manner to the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Significant increases in AMPK phosphorylation and AMPK activity were observed when the cells were incubated with BBR. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK, correlated with a subsequent increase in fatty acid oxidation. All of these effects were abolished by the mitogen-activated protein kinase kinase inhibitor PD98059. Treatment of hyperlipidemic hamsters with BBR decreased plasma LDL cholesterol and strongly reduced fat storage in the liver. These findings indicate that BBR, in addition to upregulating the LDLR, inhibits lipid synthesis in human hepatocytes through the activation of AMPK. These effects could account for the strong reduction of plasma TGs observed with this drug in clinical trials.     

Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria

Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate-limiting step for the "acidic" pathway of bile acid synthesis. This work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol in beta-cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time-, proteinase K-, and substrate concentration-dependent. Proteinase K treatment decreased the apparent K(m) of CYP27A1 for cholesterol from 400 to 150 microM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity compared with mitochondria freshly isolated from rat liver tissue.     

Docosahexaenoic acid facilitates cell maturation and beta-adrenergic transmission in astrocytes

The effects of docosahexaenoic acid (DHA; 22:6 n-3), a major omega-3 PUFA in the mammalian brain, on the structure and function of astrocytes were studied using primary cultures from rat cerebra. Gas-liquid chromatography of methyl esters of FAs isolated from cultures exposed to individual FAs, namely, stearic acid, linoleic acid, arachidonic acid, and DHA, showed alterations in the lipid profiles of the membranes, with a preferential incorporation of the FA to which the cells were exposed. Immunofluorescence studies demonstrated that unlike treatment with other FAs, after which the astrocytes remained as immature radial forms, DHA-treated astrocytes showed distinct differentiation, having morphology comparable to those grown in normal serum-containing medium. Receptor binding studies to determine the concentration of various neurotransmitter receptors showed that DHA selectively increased the number of beta-adrenergic receptors (beta-ARs) compared with FA-untreated controls, suggesting a greater role of DHA on beta-AR expression in membranes. This was also reflected by an increase in downstream events of the beta-AR pathways, such as the induction of protein kinase A and glycogen turnover by isoproterenol (ISP), a beta-AR agonist in DHA-treated cells. Moreover, ISP completely transformed DHA-treated cells into mature astrocytes bearing long processes, as in cells grown under normal conditions. Together, our observations suggest that DHA plays a unique role in facilitating some of the vital functions of astrocytes in the developing brain.     

Immune responses of locusts to challenge with the pathogenic fungus Metarhizium or high doses of laminarin

Two isolates of Metarhizium anisopliae var acridum were tested for their effects on the locust immune system and for comparison with the effects of challenge by injection with laminarin. Isolate IMI 330189 (referred to hereafter as Met 189) is highly pathogenic whether applied topically as conidia or injected as blastospores. However, isolate ARSEF 728 (referred to hereafter as Met 728) is pathogenic only when injected as blastospores, suggesting that the lack of pathogenicity of topically applied conidia from this isolate is due to a failure to penetrate the insect cuticle and gain access to the haemocoel. After topical application of conidia from Met 189, no activation of prophenoloxidase is detected, but injection of blastospores from Met 189 brings about a transient increase in phenoloxidase activity in the haemolymph in both adult locusts and 5th instar nymphs, although this does not prevent fungal-induced mortality. Co-injection of adipokinetic hormone-I (AKH-I) with blastospores prolongs the activation of prophenoloxidase in the haemolymph of adult locusts, and enhances it in nymphs. It is argued that the lack of activation of prophenoloxidase in nymphs shown previously (Mullen, L., Goldsworthy, G., 2003. Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens. Insect Biochemistry and Molecular Biology 33, 661-670), reflects differences in the sensitivity of the immune system between adults and nymphs rather than distinct qualitative differences, and this is confirmed in this study by the demonstration that doses of laminarin higher than those used previously (>or=100 microg) do activate the prophenoloxidase cascade in 5th instar nymphs. Nodules are formed in locusts of all ages in response to fungal infection or injection of laminarin, although there is wide variation in the number, size and distribution of nodules formed. During the examination of 5th instar nymphs for nodule formation, a previously unknown phenomenon was observed in which the salivary glands melanise in response to injections of blastospores or high doses of laminarin. In c. 85% of such nymphs, this reaction is so strong that the whole salivary gland is intensely black. Such a response is not observed in the salivary glands of mature adult locusts.     

Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.     

Sequence motifs and antimotifs in beta-barrel membrane proteins from a genome-wide analysis: the Ala-Tyr dichotomy and chaperone binding motifs

Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. Although sequence motifs have been studied in alpha-helical membrane proteins and have been shown to play important roles in their assembly, it is not clear whether over-represented motifs and under-represented anti-motifs exist in beta-barrel membrane proteins. We have developed probabilistic models to identify sequence motifs of residue pairs on the same strand separated by an arbitrary number of residues. A rigorous statistical model is essential for this study because of the difficulty associated with the short length of the strands and the small amount of structural data. By comparing to the null model of exhaustive permutation of residues within the same beta-strand, propensity values of sequence patterns of two residues and p-values measuring statistical significance are calculated exactly by several analytical formulae we have developed or by enumeration. We find that there are characteristic sequence motifs and antimotifs in transmembrane (TM) beta-strands. The amino acid Tyr plays an important role in several such motifs. We find a general dichotomy consisting of favorable Aliphatic-Tyr sequence motifs and unfavorable Tyr-Aliphatic antimotifs. Tyr is also part of a terminal motif, YxF, which is likely to be important for chaperone binding. Our results also suggest several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of beta-barrel membrane proteins.