Inhibitors of nonhousekeeping functions of the apicoplast defy delayed death in Plasmodium falciparum.
(57/220)
Targeting of apicoplast replication and protein synthesis in the apicomplexan Toxoplasma gondii has conventionally been associated with the typical "delayed death" phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in Plasmodium falciparum, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy. (+info)
The regulation of stearoyl-CoA desaturase gene expression is tissue specific in chickens.
(58/220)
Emerging evidence suggests a potential role of stearoyl-CoA desaturase (SCD)-1 in the control of body weight and energy homeostasis. The present study was conducted to investigate the effects of several energy balance-related factors (leptin, cerulenin, food deprivation, genotype, and gender) on SCD gene expression in chickens. In experiment 1, 6-week-old female and male broiler chickens were used. In experiment 2, two groups of 3-week-old broiler chickens were continuously infused with recombinant chicken leptin (8 micro g/kg/h) or vehicle for 6 h. In experiment 3, two groups of 2-week-old broiler chickens received i.v. injections of cerulenin (15 mg/kg) or vehicle. In experiment 4, two broiler chicken lines (fat and lean) were submitted to two nutritional states (food deprivation for 16 or 24 h and feeding ad libitum). At the end of each experiment, tissues were collected for analyzing SCD gene expression. Data from experiment 1 showed that SCD is ubiquitously expressed in chicken tissues with highest levels in the proventriculus followed by the ovary, hypothalamus, kidney, liver, and adipose tissue in female, and hypothalamus, leg muscle, pancreas, liver, and adipose tissue in male. Female chickens exhibited significantly higher SCD mRNA levels in kidney, breast muscle, proventriculus, and intestine than male chickens. However, hypothalamic SCD gene expression was higher in male than in female (P < 0.05). Leptin increased SCD gene expression in chicken liver (P < 0.05), whereas cerulenin decreased SCD mRNA levels in muscle. Both leptin and cerulenin significantly reduced food intake (P < 0.05). Food deprivation for either 16 or 24 h decreased the hepatic SCD gene expression in fat line and lean line chickens compared with their fed counterparts (P < 0.05). The hypothalamic SCD mRNA levels were decreased in both lines only after 24 h of food deprivation (P < 0.05). In conclusion, SCD is ubiquitously expressed in chickens and it is regulated by leptin, cerulenin, nutritional state, and gender in a tissue-specific manner. (+info)
Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase.
(59/220)
Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C(16) and C(18) products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C(6) and C(10)-C(12)) substrate preferences leading to the C(8) lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding. (+info)
The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K(+) (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH(2) and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH(2)-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5. (+info)
Myristoylation of proteins in the yeast secretory pathway.
(61/220)
Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the endoplasmic reticulum and Golgi (sec 18, 20), within the Golgi (sec 7), and between the Golgi and plasma membrane (sec 1, 3, 4, 5, 6, 8, 9). The accumulation of vesicles was paralleled by the enrichment of a defined subset of proteins modified either via ester or amide linkages to myristic acid: Myristoylated proteins of 21, 32, 49, 56, 75, and 136 kDa were enriched between the endoplasmic reticulum and Golgi; proteins of 21, 32, 45, 56, 75, 136 kDa were enriched by blocks within the Golgi; and proteins of 18, 21, 32, 36, 49, 68, and 136 kDa were trapped in a myristoylated form by blocks between the Golgi and plasma membrane. This enrichment of myristoylated proteins was reversed upon returning the cells to the permissive temperature for secretion. The fatty acid was linked to the 21-kDa protein via a hydroxylamine-resistant amide linkage (N-myristoylation) and to the proteins of 24, 32, 49, 56, 68, 136 kDa via hydroxylamine-labile ester linkage (E-myristoylation). In addition, myristoylated proteins of 21, 56, and 136 kDa were glycosylated via amino linkages to asparagine. This suggests they are exposed to the lumen of the secretory pathway. Three proteins (24, 32, and 56) were E-myristoylated in the presence of protein synthesis inhibitors, indicating this modification can occur posttranslationally. After using cycloheximide to clear protein passengers from the secretory pathway the 21-, 32-, and 56-kDa proteins continued to accumulate in a myristoylated form when vesicular transport was blocked between the Golgi and plasma membrane. These data suggest that myristoylation occurs on a component of the secretory machinery rather than on a passenger protein. (+info)
Links between de novo fatty acid synthesis and leptin secretion in bovine adipocytes.
(62/220)
Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants. (+info)
Mass spectrometry-based systems approach for identification of inhibitors of Plasmodium falciparum fatty acid synthase.
(63/220)
The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (beta-ketoacyl-ACP synthase), FabG (beta-ketoacyl-ACP reductase), FabZ (beta-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (-)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite. (+info)
The inhibitors of protein acylation, cerulenin and tunicamycin, increase voltage-dependent Ca(2+) currents in the insulin-secreting INS 832/13 cell.
(64/220)
As it has been suggested that protein acylation plays a role in nutrient stimulus-secretion coupling in the pancreatic beta-cell, we examined the insulin-secreting INS 832/13 beta-cell line for evidence that protein acylation was involved. The perforated whole-cell configuration was employed to voltage-clamp INS 832/13 cells. Voltage pulses were applied and Ca(2+) currents measured in the presence and absence of the protein acylation inhibitors cerulenin and tunicamycin. Both inhibitors enhanced the peak amplitude of I(Ca,L). Both increased the peak inward current in the range between -40 and +30mV and shifted the apparent maximum current by 10mV in the hyperpolarizing direction without affecting the activation threshold of -40mV. The two drugs had qualitatively and quantitatively similar effects. Steady-state activation curves revealed that cerulenin and tunicamycin shifted the activation curves in the hyperpolarization direction. Activation time constants were significantly reduced in the presence of both drugs. The Ca(2+) charge influx was increased by the drugs at all potentials tested. In contrast to these effects on the L-type Ca(2+) channel, the two inhibitors of protein acylation had no effect on the ATP-sensitive K(+) channel. The results suggest that protein acylation exerts a tonic inhibitory effect on L-type Ca(2+) channel function in the insulin-secreting beta-cell. (+info)