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1.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1869(4): 159467, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38382574

RESUMO

Gram-negative bacteria possess an asymmetric outer membrane (OM) primarily composed of lipopolysaccharides (LPS) on the outer leaflet and phospholipids on the inner leaflet. The outer membrane functions as an effective permeability barrier to compounds such as antibiotics. Studying LPS biosynthesis is therefore helpful to explore novel strategies for new antibiotic development. Metabolic glycan labeling of the bacterial surface has emerged as a powerful method to investigate LPS biosynthesis. However, the previously reported methods of labeling LPS are based on radioactivity or difficult-to-produce analogs of bacterial sugars. In this study, we report on the incorporation of azido galactose into the LPS of the Gram-negative bacteria Escherichia coli and Salmonella typhi via metabolic labeling. As a common sugar analog, azido galactose successfully labeled both O-antigen and core of Salmonella LPS, but not E. coli LPS. This labeling of Salmonella LPS, as shown by SDS-PAGE analysis and fluorescence microscopy, differs from the previously reported labeling of either O-antigen or core of LPS. Our findings are useful for studying LPS biogenesis pathways in Gram-negative bacteria like Salmonella. In addition, our approach is helpful for screening for agents that target LPS biosynthesis as it allows for the detection of newly synthesized LPS that appears in the OM. Furthermore, this approach may also aid in isolating chemically modified LPS for vaccine development or immunotherapy.


Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Galactose/metabolismo , Antígenos O/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Antibacterianos
2.
iScience ; 25(8): 104753, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35942089

RESUMO

N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.

3.
EMBO J ; 41(2): e106837, 2022 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34873731

RESUMO

Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.


Assuntos
Mitocôndrias/metabolismo , Fosfolipídeos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Transporte Biológico , Carboxiliases/genética , Carboxiliases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Eur Biophys J ; 46(1): 91-101, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27815573

RESUMO

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.


Assuntos
Bicamadas Lipídicas/química , Maleatos/química , Poliestirenos/química , Membrana Celular/química , Solubilidade
5.
J Biomed Nanotechnol ; 13(2): 204-20, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29377650

RESUMO

The efficacy of photodynamic therapy (PDT) in some solid tumors is limited by the poor biodistributive properties of conventional photosensitizers and a natural predisposition of tumor cells to survive hypoxia and oxidative stress. This study investigated the therapeutic potential of a third-generation photosensitizer, liposomal zinc phthalocyanine (ZnPC), in combination with the hypoxic cytotoxin tirapazamine (TPZ). TPZ induces DNA double strand breaks (DSBs) under hypoxic conditions and subsequent apoptosis via p53 signaling. Experiments were performed in tumor cells with functional p53 (Sk-Cha1) and dysfunctional p53 (A431). The combination therapy of TPZ and PDT induced DNA DSBs and cell cycle stalling and enhanced the cytotoxicity of PDT by exacerbating apopotic and non-apoptotic tumor cell death. These phenomena occurred regardless of oxygen tension and the mechanism of cell death differed per cell line. Liposomes containing both ZnPC and TPZ exhibited no dark toxicity but were more lethal to both cell types after PDT compared to ZnPC-liposomes lacking TPZ­an effect that was more pronounced under hypoxic conditions. In conclusion, TPZ is a suitable pharmaceutical compound to increase PDT efficacy by exploiting the post-PDT tumor hypoxia. The inclusion of TPZ and ZnPC into a single liposomal delivery system was feasible. The PDT strategy described in this study may be valuable for the treatment of PDT-recalcitrant tumors.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Indóis/farmacologia , Lipossomos/farmacologia , Compostos Organometálicos/farmacologia , Fotoquimioterapia/métodos , Triazinas/farmacologia , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indóis/química , Isoindóis , Lipossomos/química , Neoplasias/metabolismo , Compostos Organometálicos/química , Estresse Oxidativo/efeitos dos fármacos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio , Tirapazamina , Triazinas/química , Compostos de Zinco
6.
Food Funct ; 4(8): 1209-15, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23719714

RESUMO

Treatment of rat brain C6 astroglioma cells with furan fatty acid F6 prior to exposure to hydrogen peroxide shows a strong protective effect of F6 against cell death resulting from oxidative stress. This protective effect is obtained only for F6 administered as a free fatty acid and with an intact furan ring. It is proposed that brain cells are rescued by F6 scavenging radicals elicited by lipid peroxidation within the cell membrane. Oxidative processes outside the cell membrane, such as protein carbonylation, are not affected by F6. Furan fatty acids such as those present in fish oils and marine organisms are likely beneficial for consumption in reducing the risk of diseases that have been implicated to arise from oxidative stress, such as Alzheimer's disease.


Assuntos
Apoptose , Encéfalo/citologia , Encéfalo/metabolismo , Ácidos Graxos/metabolismo , Furanos/metabolismo , Peróxido de Hidrogênio/líquido cefalorraquidiano , Estresse Oxidativo , Substâncias Protetoras/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ácidos Graxos/química , Furanos/química , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/química , Ratos
7.
EMBO Rep ; 14(5): 434-40, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23519169

RESUMO

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilcolinas/biossíntese , Saccharomyces cerevisiae/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidato Fosfatase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Biochim Biophys Acta ; 1831(6): 1167-76, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23501167

RESUMO

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.


Assuntos
Proteínas de Transporte/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Lisofosfolipase/metabolismo , Proteínas de Membrana/metabolismo , Palmitatos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipases A2/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray
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