Lipid posttranslational modifications. Farnesyl transferase inhibitors

Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the C terminus. Protein isoprenylation promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include small GTPases, tyrosine phosphatases, nuclear lamina, cochaperones, and centromere-associated proteins. Prenylation is required for the transforming activity of Ras. Because of the high frequency of Ras mutations in cancer, farnesyl transferase inhibitors (FTIs) were investigated as a means to antagonize Ras function. Evaluation of FTIs led to the finding that both K- and N-Ras are alternatively modified by geranylgeranyl prenyltransferase-1 in FTI-treated cells. Geranylgeranylated forms of Ras retain the ability to associate with the plasma membrane and activate substrates. Despite this, FTIs are effective at inhibiting the growth of human tumor cells in vitro, suggesting that activity is dependent on blocking the farnesylation of other proteins. FTIs also inhibit the in vivo growth of human tumor xenografts and sensitize these models to chemotherapeutics, most notably taxanes. Several FTIs have entered clinical trials for various cancer indications. In some clinical settings, primarily hematologic malignancies, FTIs have displayed evidence of single-agent activity. Clinical studies in progress are exploring the antitumor activity of FTIs as single agents and in combination. This review will summarize the basic biology of FTIs, their antitumor activity in preclinical models, and the current status of clinical studies with these agents.     

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

Manipulation of the oxytocin system alters social behavior and attraction in pair-bonding primates, Callithrix penicillata

The establishment and maintenance of stable, long-term male-female relationships, or pair-bonds, are marked by high levels of mutual attraction, selective preference for the partner, and high rates of sociosexual behavior. Central oxytocin (OT) affects social preference and partner-directed social behavior in rodents, but the role of this neuropeptide has yet to be studied in heterosexual primate relationships. The present study evaluated whether the OT system plays a role in the dynamics of social behavior and partner preference during the first 3 weeks of cohabitation in male and female marmosets, Callithrix penicillata. OT activity was stimulated by intranasal administration of OT, and inhibited by oral administration of a non-peptide OT-receptor antagonist (L-368,899; Merck). Social behavior throughout the pairing varied as a function of OT treatment. Compared to controls, marmosets initiated huddling with their social partner more often after OT treatments but reduced proximity and huddling after OT antagonist treatments. OT antagonist treatment also eliminated food sharing between partners. During the 24-h preference test, all marmosets interacted more with an opposite-sex stranger than with the partner. By the third-week preference test, marmosets interacted with the partner and stranger equally with the exception that intranasal-OT treatments facilitated initial partner-seeking behavior over initial contact with the stranger. Our findings demonstrate that pharmacological manipulations of OT activity alter partner-directed social behavior during pair interactions, suggesting that central OT may facilitate the process of pair-bond formation and social relationships in marmoset monkeys.     

Characterization of membrane properties of inositol phosphorylceramide

Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature (T_m) of P-IPC was 48 °C. Addition of 5 mM CaCl₂ during vesicle preparation markedly increased the T_m (65 °C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes.     

非肽类胰岛素类似物的研究

糖尿病是二十一世纪人类健康的主要威胁之一。而胰岛素又是糖尿病患终生依赖的药物。如果能筛选出既有良好药效,又具有安全、无毒特性,且能口服的非肽类胰岛素类似物,无疑将给糖尿病患带来福音。本对有希望成为治疗药物的L-83,28l、钒化合物以及中草药中可能存在的非肽类胰岛素类似物的研究进展作一概述。     

Agonist-induced activation releases peroxisome proliferator-activated receptor β/δ from its inhibition by palmitate-induced nuclear factor-κB in skeletal muscle cells

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood, but there is a strong correlation between insulin resistance and intramyocellular lipid accumulation in skeletal muscle. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The aim of this work was to study whether the exposure of skeletal muscle cells to palmitate affected peroxisome proliferator-activated receptor (PPAR) β/δ activity. Here, we report that exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate reduced (74%, P<0.01) the mRNA levels of the PPARβ/δ-target gene pyruvatedehydrogenase kinase 4 (PDK-4), which is involved in fatty acid utilization. This reduction was not observed in the presence of the PPARβ/δ agonist L-165041. This drug prevented palmitate-induced nuclear factor (NF)-κB activation. Increased NF-κB activity after palmitate exposure was associated with enhanced protein–protein interaction between PPARβ/δ and p65. Interestingly, treatment with the PPARβ/δ agonist L-165041 completely abolished this interaction. These results indicate that palmitate may reduce fatty acid utilization in skeletal muscle cells by reducing PPARβ/δ signaling through increased NF-κB activity.     

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodoacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.     

Structural organisation of band 3 in Melanesian ovalocytes

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normacytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.     

Irreversible binding of DOPA and dopamine metabolites to protein by rat liver microsomes.

Rat liver microsomes catalyze NADPH-dependent irreversible binding of metabolites of DOPA and DOPAmine to microsomal protein and to BSA. Binding is inhibited by cysteine and the singlet oxygen quencher 1,4-diaza-bicyclo(2.2.2)octane. Irreversible binding to BSA is also catalyzed by mushroom tyrosinase, xanthine oxidase, and NADPH-cytochrome c reductase. The results suggest that in the microsomal system the participation of the hemoprotein, cytochrome P-450, is not an absolute requirement for the irreversible binding of metabolites of DOPA and DOPAmine to proteins.