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1.
J Agric Food Chem ; 67(42): 11703-11709, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31578056

RESUMO

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Engenharia Metabólica , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/biossíntese
2.
Microbiol Res ; 229: 126329, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31518853

RESUMO

The genus Serratia is a predominantly unexplored source of antimicrobial secondary metabolites. The aim of the current study was thus to isolate and evaluate the antimicrobial properties of biosurfactants produced by Serratia species. Forty-nine (n = 34 pigmented; n = 15 non-pigmented) biosurfactant producing Serratia strains were isolated from environmental sources and selected isolates (n = 11 pigmented; n = 11 non-pigmented) were identified as Serratia marcescens using molecular typing. The swrW gene (serrawettin W1 synthetase) was detected in all the screened pigmented strains and one non-pigmented strain and primers were designed for the detection of the swrA gene (non-ribosomal serrawettin W2 synthetase), which was detected in nine non-pigmented strains. Crude extracts obtained from S. marcescens P1, NP1 and NP2 were chemically characterised using ultra-performance liquid chromatography coupled to electrospray ionisation mass spectrometry (UPLC-ESI-MS), which revealed that P1 produced serrawettin W1 homologues and prodigiosin, while NP1 produced serrawettin W1 homologues and glucosamine derivative A. In contrast, serrawettin W2 analogues were predominantly identified in the crude extract obtained from S. marcescens NP2. Both P1 and NP1 crude extracts displayed broad-spectrum antimicrobial activity against clinical, food and environmental pathogens, such as multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Cryptococcus neoformans. In contrast, the NP2 crude extract displayed antibacterial activity against a limited range of pathogenic and opportunistic pathogens. The serrawettin W1 homologues, in combination with prodigiosin and glucosamine derivatives, produced by pigmented and non-pigmented S. marcescens strains, could thus potentially be employed as broad-spectrum therapeutic agents against multidrug-resistant bacterial and fungal pathogens.


Assuntos
Antibacterianos/farmacologia , Depsipeptídeos/farmacologia , Lipoproteínas/farmacologia , Peptídeos Cíclicos/farmacologia , Prodigiosina/farmacologia , Serratia marcescens/química , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Depsipeptídeos/química , Depsipeptídeos/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Prodigiosina/química , Prodigiosina/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Metabolismo Secundário , Serratia marcescens/metabolismo , Tensoativos/química , Tensoativos/metabolismo , Tensoativos/farmacologia
3.
J Insect Sci ; 19(4)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31346627

RESUMO

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.


Assuntos
Complexos de ATP Sintetase/metabolismo , Corpo Adiposo/citologia , Proteínas de Insetos/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Panstrongylus/citologia , Animais , Corpo Adiposo/enzimologia , Ninfa/citologia , Ninfa/enzimologia , Panstrongylus/enzimologia , Panstrongylus/crescimento & desenvolvimento
4.
Subcell Biochem ; 92: 39-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214984

RESUMO

The Lpp lipoprotein of Escherichia coli is the first identified protein with a covalently linked lipid. It is chemically bound by its C-terminus to murein (peptidoglycan) and inserts by the lipid at the N-terminus into the outer membrane. As the most abundant protein in E. coli (106 molecules per cell) it plays an important role for the integrity of the cell envelope. Lpp represents the type protein of a large variety of lipoproteins found in Gram-negative and Gram-positive bacteria and in archaea that have in common the lipid structure for anchoring the proteins to membranes but otherwise strongly vary in sequence, structure, and function. Predicted lipoproteins in known prokaryotic genomes comprise 2.7% of all proteins. Lipoproteins are modified by a unique phospholipid pathway and transferred from the cytoplasmic membrane into the outer membrane by a special system. They are involved in protein incorporation into the outer membrane, protein secretion across the cytoplasmic membrane, periplasm and outer membrane, signal transduction, conjugation, cell wall metabolism, antibiotic resistance, biofilm formation, and adhesion to host tissues. They are only found in bacteria and function as signal molecules for the innate immune system of vertebrates, where they cause inflammation and elicit innate and adaptive immune response through Toll-like receptors. This review discusses various aspects of Lpp and other lipoproteins of Gram-negative and Gram-positive bacteria and archaea.


Assuntos
Archaea , Bactérias , Lipoproteínas/química , Lipoproteínas/metabolismo , Animais , Archaea/química , Archaea/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/biossíntese , Peptidoglicano/química , Peptidoglicano/metabolismo
5.
Plant Sci ; 285: 230-238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203888

RESUMO

In higher plants, Fibrillins (FBNs) constitute a conserved plastid-lipid-associated (PAPs) protein family and modulate the metabolite transport and lipid metabolism in plastids of dicot species. However, FBNs have not functionally characterized in monocot species. In this study, the function of rice fibrillin 1 (OsFBN1) was investigated. The subcellular localization assay showed that the N-terminal chloroplast transport peptide (CTP) could facilitate the import of OsFBN1 into chloroplast. OsFBN1 specifically bound C18- and C20- fatty acids in vitro. Overexpressing OsFBN1 increased the tiller number but decreased the panicle length, grain-filling percent and JA levels compared to the wild type and RNAi silencing lines under heat stress. In addition, the overexpressing lines had more plastoglobules (PGs) than the wild type and RNAi silencing lines under both normal and heat stress conditions. Moreover, overexpressing OsFBN1 affected the transcription levels of OsAOS2 in JA synthesis, OsTHF1, OsABC1K7 and OsPsaE in thylakoid stability and photosynthesis, OsABC1-4 and OsSPS2 in ubiquinone-metabolism, OsHDR, OsDXR, and OsFPPS in isoprenoid metabolism. Collectively, these findings suggest the essential role of rice OsFBN1 in PG formation and lipid metabolism in chloroplasts, which coordinately regulate the growth and grain filling of the overexpressing lines under heat stress.


Assuntos
Cloroplastos/metabolismo , Ciclopentanos/metabolismo , Grão Comestível/metabolismo , Oryza/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Cloroplastos/ultraestrutura , Grão Comestível/crescimento & desenvolvimento , Resposta ao Choque Térmico , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Microscopia Eletrônica de Transmissão , Oryza/genética , Oryza/fisiologia , Oryza/ultraestrutura , Fotossíntese , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Tilacoides/metabolismo
6.
Nat Commun ; 10(1): 2635, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201302

RESUMO

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/ultraestrutura , Microscopia Intravital/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptidoglicano/metabolismo , Periplasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
8.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 368-376, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045566

RESUMO

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cloretos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Haemophilus influenzae/química , Lipoproteínas/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína
9.
Adv Exp Med Biol ; 1127: 67-82, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31140172

RESUMO

The liver plays a capital role in the control of whole body energy homeostasis through the metabolization of dietary carbohydrates and lipids. However, under excess macronutrient uptake, those pathways overcharge nucleus-to-endoplasmic reticulum (ER) traffic pathways, leading to luminal overload of unfolded proteins which activates a series of adaptive signaling pathways known as unfolded protein response (UPR). The UPR is a central network mechanism for cellular stress adaptation, however far from a global nonspecific all-or-nothing response. Such a complex signaling network is able to display considerable specificity of responses, with activation of specific signaling branches trimmed for distinct types of stimuli. This makes the UPR a fundamental mechanism underlying metabolic processes and diseases, especially those related to lipid and carbohydrate metabolism. Thus, for a better understanding of the role of UPR on the physiopathology of lipid metabolism disorders, the concepts discussed along this chapter will demonstrate how several metabolic derangements activate UPR components and, in turn, how UPR triggers several metabolic adaptations through its component signaling proteins. This dual role of UPR on lipid metabolism will certainly foment the pursuit of an answer for the question: is UPR cause or consequence of lipid and lipoprotein metabolism disturbances?


Assuntos
Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas
10.
PLoS Pathog ; 15(5): e1007731, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083688

RESUMO

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Secretina/química , Sistemas de Secreção Tipo II/química , Vibrio vulnificus/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Lipoproteínas/química , Modelos Moleculares , Conformação Proteica , Secretina/metabolismo , Homologia de Sequência , Sistemas de Secreção Tipo II/metabolismo , Vibrio vulnificus/crescimento & desenvolvimento
11.
Curr Med Chem ; 26(9): 1525-1543, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31096892

RESUMO

BACKGROUND: Dyslipidaemias is a recognized risk factor for atherosclerosis, however, new evidence brought to light by trials investigating therapies to enhance HDLcholesterol have suggested an increased atherosclerotic risk when HDL-C is high. RESULTS: Several studies highlight the central role in atherosclerotic disease of dysfunctional lipoproteins; oxidised LDL-cholesterol is an important feature, according to "oxidation hypothesis", of atherosclerotic lesion, however, there is today a growing interest for dysfunctional HDL-cholesterol. The target of our paper is to review the functions of modified and dysfunctional lipoproteins in atherogenesis. CONCLUSION: Taking into account the central role recognized to dysfunctional lipoproteins, measurements of functional features of lipoproteins, instead of conventional routine serum evaluation of lipoproteins, could offer a valid contribution in experimental studies as in clinical practice to stratify atherosclerotic risk.


Assuntos
Aterosclerose/metabolismo , Lipoproteínas/metabolismo , Animais , Humanos
12.
PLoS Pathog ; 15(4): e1007723, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002736

RESUMO

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to ß-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.


Assuntos
Proteínas de Bactérias/química , Parede Celular/metabolismo , Lipoproteínas/química , Staphylococcus aureus/enzimologia , Ácidos Teicoicos/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Lipoproteínas/metabolismo , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína
13.
MBio ; 10(2)2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992347

RESUMO

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Lipoproteínas/metabolismo , Transporte Proteico , Pseudomonas aeruginosa/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Aspártico/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Pseudomonas aeruginosa/genética
14.
Nat Commun ; 10(1): 1606, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962435

RESUMO

Vascular endothelial growth factor (VEGF) regulates vasculogenesis by using its tyrosine kinase receptors. However, little is known about whether Sec14-like phosphatidylinositol transfer proteins (PTP) are involved in this process. Here, we show that zebrafish sec14l3, one of the family members, specifically participates in artery and vein formation via regulating angioblasts and subsequent venous progenitors' migration during vasculogenesis. Vascular defects caused by sec14l3 depletion are partially rescued by restoration of VEGFR2 signaling at the receptor or downstream effector level. Biochemical analyses show that Sec14l3/SEC14L2 physically bind to VEGFR2 and prevent it from dephosphorylation specifically at the Y1175 site by peri-membrane tyrosine phosphatase PTP1B, therefore potentiating VEGFR2 signaling activation. Meanwhile, Sec14l3 and SEC14L2 interact with RAB5A/4A and facilitate the formation of their GTP-bound states, which might be critical for VEGFR2 endocytic trafficking. Thus, we conclude that Sec14l3 controls vasculogenesis in zebrafish via the regulation of VEGFR2 activation.


Assuntos
Neovascularização Fisiológica/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
15.
BMC Complement Altern Med ; 19(1): 88, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023287

RESUMO

BACKGROUND: Stevia rebaudiana Bertoni has various pharmacological actions, which includes antidiabetic, antioxidant, anti-inflammatory activities. The superoxide and consequently NADPH oxidase (Nox) are relevant targets involved in biological effects of Stevia. The presence of NADPH-containing superoxide-producing lipoprotein (suprol) in Stevia leaves has not yet been tested. The mechanism of producing superoxide radicals (O2-) by suprol was determined in vitro, which is associated with the electron transfer from NADPH in the composition of suprol by traces of transition metal ions (Fe3+ or Cu2+) to molecular oxygen, turning it into O2-. It is expected that the therapeutic efficacy of Stevia leaves is caused by specific activity of superoxide-producing lipoprotein fraction. METHODS: For the first time, from the dry leaves of Stevia the NADPH-containing superoxide-producing lipoprotein was isolated and purified. The specific content of suprol (milligrams in 1 g of Stevia leaves- mg/g) was determined after desalination of suprol and lyophilization. RESULTS: According to the method provided, the specific content of the isolated suprol from Stevia's leaves was up to 4.5 ± 0.2 mg / g (yields up to 68.5 ± 4.7%, p < 0.05, n = 6). Nox forms a stable complex with suprol. The optical absorption spectrum of the Nox-suprol complex represents the overlapping suprol and Nox spectra, with a certain background increase and characteristic features of optical absorption for Nox. Due to O2- producing capacity suprol-Nox complex discolors KMnO4 solutions, Coomassie brilliant blue, restores nitrotetrazolium blue to formazan and oxidizes epinephrine to adrenochrome. The oxidation activity of adrenaline is 50.3 ± 5.1 U / mg / ml (p < 0.05, n = 6). CONCLUSION: Superoxide-producing lipoprotein fraction-Nox complex from Stevia leaves (membranes) can modulate redox regulated signaling pathways and may play a positive role in type-2 diabetes by means of adrenaline oxidation mechanism.


Assuntos
Lipoproteínas , NADP , Proteínas de Plantas , Stevia/química , Superóxidos , Lipoproteínas/química , Lipoproteínas/metabolismo , NADP/química , NADP/metabolismo , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Oxirredução , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Superóxidos/química , Superóxidos/metabolismo
16.
Methods Mol Biol ; 1951: 111-133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825148

RESUMO

Measuring cholesterol efflux involves the tracking of cholesterol movement out of cells. Cholesterol efflux is an essential mechanism to maintain cellular cholesterol homeostasis, and this process is largely regulated via the LXR transcription factors and their regulated genes, the ATP-binding cassette (ABC) cholesterol transporters ABCA1 and ABCG1. Typically, efflux assays are performed utilizing radiolabeled cholesterol tracers to label intracellular cholesterol pools, and these assays may be tailored to quantify the efflux of exogenously delivered cholesterol or alternatively the efflux of newly synthesized (endogenous) cholesterol, in different cell types (macrophages, hepatocytes). Cholesterol efflux may also be customized to quantify cholesterol flux out of the cell to various exogenous cholesterol acceptors, such as apolipoprotein A-I, high-density lipoprotein, or methyl-beta-cyclodextrin, depending on the purpose of the experiment. Here, we provide comprehensive protocols to quantify the net flux of cholesterol out of cells and recommendations on how this assay may be tailored as a function of the experimental question at hand.


Assuntos
Colesterol/metabolismo , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Animais , Transporte Biológico , HDL-Colesterol , LDL-Colesterol/metabolismo , Humanos , Espaço Intracelular/metabolismo , Lipoproteínas/sangue , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos
17.
EcoSal Plus ; 8(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30900542

RESUMO

Lipoproteins are produced by both Gram-positive and Gram-negative bacteria. Once secreted, lipoproteins are quickly acylated, anchoring them into the plasma membrane. Recent work has shown that Gram-positive bacteria are able to generate considerable diversity in the acylation of their lipoproteins, though the mechanisms involved are only just beginning to emerge. In Gram-negative organisms, most lipoproteins are subsequently trafficked to the outer membrane (OM). Lipoprotein trafficking is an essential pathway in these bacteria. At least one OM lipoprotein component is required by each of the essential machines that assemble the OM (such as the Bam and Lpt machines) and build the peptidoglycan cell wall (Lpo-penicillin-binding protein complexes). The Lol pathway has been the paradigm for OM lipoprotein trafficking: a complex of LolCDE extracts lipoproteins from the plasma membrane, LolA shuttles them through the periplasmic space, and LolB anchors them into the OM. The peptide signals responsible for OM-targeting via LolCDE have long been known for Escherichia coli. Remarkably, production of novel lipoprotein acyl forms in E. coli has reinforced the idea that lipid signals also contribute to OM targeting via LolCDE. Moreover, recent work has shown that lipoprotein trafficking can occur in E. coli without either LolA or LolB. Therefore, current evidence suggests that at least one additional, LolAB-independent route for OM lipoprotein trafficking exists. This chapter reviews the posttranslocation modifications of all lipoproteins, with a focus on the trafficking of lipoproteins to the OM of Gram-negative bacteria.


Assuntos
Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Transporte Proteico , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Peptidoglicano/metabolismo , Periplasma/metabolismo
18.
Appl Microbiol Biotechnol ; 103(10): 4003-4015, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30923871

RESUMO

Poly-γ-glutamic acid (γ-PGA) is an extracellularly produced biodegradable polymer, which has been widely used as agricultural fertilizer, mineral fortifier, cosmetic moisturizer, and drug carrier. This study firstly discovered that lichenysin, as a biosurfactant, showed the capability to enhance γ-PGA production in Bacillus licheniformis. The exogenous addition of lichenysin improved the γ-PGA yield up to 17.9% and 21.9%, respectively, in the native strain B. licheniformis WX-02 and the lichenysin-deficient strain B. licheniformis WX02-ΔlchAC. The capability of intracellular biosynthesis of lichenysin was positively correlated with γ-PGA production. The yield of γ-PGA increased by 25.1% in the lichenysin-enhanced strain B. licheniformis WX02-Psrflch and decreased by 12.2% in the lichenysin-deficient strain WX02-ΔlchAC. Analysis of key enzyme activities and gene expression in the TCA cycle, precursor glutamate synthesis, and γ-PGA synthesis pathway revealed that the existence of lichenysin led to increased γ-PGA via shifting the carbon flux in the TCA cycle towards glutamate and γ-PGA biosynthetic pathways, minimizing by-product formation, and facilitating the uptake of extracellular substrates and the polymerization of glutamate to γ-PGA. Insight into the mechanisms of enhanced production of γ-PGA by lichenysin would define the essential parameters involved in γ-PGA biosynthesis and provide the basis for large-scale production of γ-PGA.


Assuntos
Bacillus licheniformis/efeitos dos fármacos , Bacillus licheniformis/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Lipoproteínas/metabolismo , Peptídeos Cíclicos/metabolismo , Ácido Poliglutâmico/análogos & derivados , Tensoativos/metabolismo , Carbono/metabolismo , Análise do Fluxo Metabólico , Ácido Poliglutâmico/biossíntese
19.
Oxid Med Cell Longev ; 2019: 3021785, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911344

RESUMO

Cardiovascular disease (CVD) events are the main causes of death in end-stage renal disease (ESRD) patients on dialysis. The number and severity of CVD events remain inappropriate and difficult to explain by considering only the classic CVD risk factors. Our aim was to clarify the changes and the relationship of lipoprotein subfractions with other CVD risk factors, namely, body mass index (BMI) and adipokines, inflammation and low-density lipoprotein (LDL) oxidation, and the burden of the most prevalent comorbidities, diabetes mellitus (DM) and hypertension (HT). We studied 194 ESRD patients on dialysis and 22 controls; lipid profile, including lipoprotein subpopulations and oxidized LDL (oxLDL), C-reactive protein (CRP), adiponectin, leptin, and paraoxonase 1 activity were evaluated. Compared to controls, patients presented significantly lower levels of cholesterol, high-density lipoprotein cholesterol (HDLc), LDLc, oxLDL, and intermediate and small HDL and higher triglycerides, CRP, adiponectin, large HDL, very-low-density lipoprotein (VLDL), and intermediate-density lipoprotein- (IDL) B. Adiponectin levels correlated positively with large HDL and negatively with intermediate and small HDL, oxLDL/LDLc, and BMI; patients with DM (n = 17) and with DM+HT (n = 70), as compared to patients without DM or HT (n = 69) or only with HT (n = 38), presented significantly higher oxLDL, oxLDL/LDLc, and leptin and lower adiponectin. Obese patients (n = 45), as compared to normoponderal patients (n = 81), showed lower HDLc, adiponectin, and large HDL and significantly higher leptin, VLDL, and intermediate and small HDL. In ESRD, the higher adiponectin seems to favor atheroprotective HDL modifications and protect LDL particles from oxidative atherogenic changes. However, in diabetic and obese patients, adiponectin presents the lowest values, oxLDL/LDLc present the highest ones, and the HDL profile is the more atherogenic. Our data suggest that the coexistence of DM and adiposity in ESRD patients on dialysis contributes to a higher CVD risk, as showed by their lipid and adipokine profiles.


Assuntos
Adiponectina/metabolismo , Índice de Massa Corporal , Diabetes Mellitus/patologia , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Lipoproteínas/metabolismo , Substâncias Protetoras/metabolismo , Arildialquilfosfatase/metabolismo , Proteína C-Reativa/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
20.
Proc Natl Acad Sci U S A ; 116(9): 3764-3773, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755523

RESUMO

Commensal and pathogenic bacteria hydrolyze host lipid substrates with secreted lipases and phospholipases for nutrient acquisition, colonization, and infection. Bacterial lipase activity on mammalian lipids and phospholipids can promote release of free fatty acids from lipid stores, detoxify antimicrobial lipids, and facilitate membrane dissolution. The gram-positive bacterium Staphylococcus aureus secretes at least two lipases, Sal1 and glycerol ester hydrolase (Geh), with specificities for short- and long-chain fatty acids, respectively, each with roles in the hydrolysis of environmental lipids. In a recent study from our group, we made the unexpected observation that Geh released by S. aureus inhibits activation of innate immune cells. Herein, we investigated the possibility that S. aureus lipases interface with the host immune system to blunt innate immune recognition of the microbe. We found that the Geh lipase, but not other S. aureus lipases, prevents activation of innate cells in culture. Mutation of geh leads to enhancement of proinflammatory cytokine production during infection, increased innate immune activity, and improved clearance of the bacterium in infected tissue. These in vitro and in vivo effects on innate immunity were not due to direct functions of the lipase on mammalian cells, but rather a result of inactivation of S. aureus lipoproteins, a major pathogen-associated molecular pattern (PAMP) of extracellular gram-positive bacteria, via ester hydrolysis. Altogether, these studies provide insight into an adaptive trait that masks microbial recognition by innate immune cells through targeted inactivation of a broadly conserved PAMP.


Assuntos
Hidrolases de Éster Carboxílico/genética , Imunidade Inata/genética , Lipase/genética , Infecções Estafilocócicas/enzimologia , Staphylococcus aureus/enzimologia , Animais , Hidrolases de Éster Carboxílico/imunologia , Interações Hospedeiro-Patógeno/genética , Ligantes , Lipase/imunologia , Lipólise/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutação , Pele/enzimologia , Pele/metabolismo , Pele/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade
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