The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.     

Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus

Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.     

Effect of tumor-associated mutant E-cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E-cadherin is frequently down-regulated or mutated in tumors. In addition to down-regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E-cadherin and matrix metalloproteinase 3 (MMP-3), we examined expression of MMP-3 in human MDA-MB-435S cells transfected with wild-type (wt) or three different tumor-associated mutant E-cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E-cadherin, the MMP-3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down-regulation of MMP-3 was not found in MDA-MB-435S transfectants expressing mutant E-cadherin variants which indicates that E-cadherin mutations interfere with the MMP-3 suppressing function of E-cadherin. The mechanism of regulation of MMP-3 by E-cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E-cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP-3 NNGH and small interfering RNA (siRNA) directed against MMP-3 reduce mutant E-cadherin-enhanced cell motility. Taken together, our results point to a functional link between MMP-3 and E-cadherin. MMP-3 is differentially regulated by expression of wt or mutant E-cadherin. On the other hand, MMP-3 plays a role in the enhancement of cell motility by mutant E-cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

The solution structure of the SODD BAG domain reveals additional electrostatic interactions in the HSP70 complexes of SODD subfamily BAG domains

The solution structure of an N-terminally extended construct of the SODD BAG domain was determined by nuclear magnetic resonance spectroscopy. A homology model of the SODD-BAG/HSP70 complex reveals additional possible interactions that are specific for the SODD subfamily of BAG domains while the overall geometry of the complex remains the same. Relaxation rate measurements show that amino acids N358-S375 of SODD which were previously assigned to its BAG domain are not structured in our construct. The SODD BAG domain is thus indeed smaller than the homologous domain in Bag1 defining a new subfamily of BAG domains.     

Design of chimeric receptor mimics with different TcRVbeta isoforms. Type-specific inhibition of superantigen pathogenesis

The Staphylococcus aureus enterotoxins (S.E.) A-I, and toxic-shock syndrome toxin TSST-1 act as superantigens to cause overstimulation of the host immune system, leading to the onset of various diseases including food poisoning and toxic shock syndrome. SAgs bind as intact proteins to the DRalpha1 domain of the MHC class II receptor and the TcRVbeta domain from the T cell receptor and cause excessive release of cytokines such as IL-2, TNF-alpha, and IFN-gamma, and hyperproliferation of T cells. In addition, different SAgs bind and activate different TcRVbeta isoforms during pathogenesis of human immune cells. These two properties of SAgs prompted us to design several chimeric DRalpha1-linker-TcRVbeta proteins using different TcRVbeta isoforms to create chimeras that would specifically inhibit the pathogenesis of SAgs against which they were designed. In this study, we compare the design, interaction, and inhibitory properties of three different DRalpha1-linker-TcRVbeta chimeras targeted against three different SAgs, SEB, SEC3, and TSST-1. The inhibitory properties of the chimeras were tested by monitoring IL-2 release and T cell proliferation using a primary human cell model. We demonstrate that the three chimeras specifically inhibit the pathogenesis of their target superantigen. We performed molecular modeling to analyze the structural basis of the type specificity exhibited by different chimeras designed against their target SAgs, examine the role of the linker in determining binding and specificity, and suggest site-specific mutations in the chimera to enhance binding affinity. The fact that our strategy works equally well for SEB and TSST-1, two widely different phylogenic variants, suggests that the DRalpha1-linker-TcRVbeta chimeras may be developed as a general therapy against a broad spectrum of superantigens released during Staphylococcal infection.     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase

Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8A resolution to R and Rfree values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three alpha helices. The homotetramer buries a large internal cavity of roughly 15,000 A3, from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.     

Influence of a Fusarium culmorum inoculation of wheat on the progression of mycotoxin accumulation, ingredient concentrations and ruminal in sacco dry matter degradation of wheat residues

The Fusarium head blight (FHB)-susceptible winter wheat cv. Ritmo was inoculated with spores of Fusarium culmorum at the beginning of full blossom. Samples of whole wheat plants were taken once weekly from anthesis until harvest and subsequently fractionated into straw, glumes and spindles, which were examined for deoxynivalenol (DON) and zearalenone (ZON). Additionally, the content of crude protein (CP) and non-starch polysaccharides (NSP) was scrutinized. Synthesis of the Fusarium toxins DON and ZON generally differed in terms of date of formation and concentration. Final mean DON concentrations of 37.5, 28.1 and 5.0 mg/kg DM were measured in glumes, spindles and straw, respectively, at the time of harvest. At this time, maximal mean ZON concentrations of 587, 396 and 275 microg/kg DM in spindles, glumes and straw, respectively, were determined. Moreover, Fusarium infected wheat residues contained higher CP but lower NSP contents at the last three sampling dates. In addition, collective samples of wheat straw and chaff were taken to investigate the effect of the Fusarium contamination on their in sacco DM degradation in dairy cows. Samples were analysed for mycotoxins and selected quality parameters. The dried and milled collective samples of straw and chaff were weighed into nylon bags and subjected to ruminal incubation for 4, 8, 16, 24, 48, 72, 96 and 120 h in two dairy cows equipped with a permanent rumen cannula. Marked differences in level of mycotoxin contamination as well as in ingredient composition between the variants of straw and chaff were detected. Moreover, after 120 h rumen incubation the in sacco DM degradation of inoculated straw and chaff were lower compared to the accordant controls. The soluble fraction was increased in inoculated samples, whereas a diminishment in the potentially degradable but insoluble fraction was more pronounced. Thereby, a decrease in the potential degradability was obtained for inoculated straw and even if less pronounced for chaff compared to the non inoculated corresponding controls. In conclusion, infection with F. culmorum of wheat involves an increased risk of mycotoxin contamination in straw. Also, a Fusarium infection may have an impact on chemical composition and may result in Fusarium growth-related modifications of host cell wall components.