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1.
Nat Commun ; 12(1): 4687, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344901

RESUMO

Lipoproteins are important for bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE recognizes its substrate by interacting with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Acilação , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutação , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico
2.
J Immunol ; 207(3): 966-973, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290104

RESUMO

Neutrophils, polymorphonuclear leukocytes (PMN), play a critical role in the innate immune response to Staphylococcus aureus, a pathogen that continues to be associated with significant morbidity and mortality. Neutrophil extracellular trap (NET) formation is involved in ensnaring and killing of S. aureus, but this host-pathogen interaction also leads to host tissue damage. Importantly, NET components including neutrophil proteases are under consideration as therapeutic targets in a variety of disease processes. Although S. aureus lipoproteins are recognized to activate cells via TLRs, specific mechanisms of interaction with neutrophils are poorly delineated. We hypothesized that a lipoprotein-containing cell membrane preparation from methicillin-resistant S. aureus (MRSA-CMP) would elicit PMN activation, including NET formation. We investigated MRSA-CMP-elicited NET formation, regulated elastase release, and IL-8 production in human neutrophils. We studied PMN from healthy donors with or without a common single-nucleotide polymorphism in TLR1, previously demonstrated to impact TLR2/1 signaling, and used cell membrane preparation from both wild-type methicillin-resistant S. aureus and a mutant lacking palmitoylated lipoproteins (lgt). MRSA-CMP elicited NET formation, elastase release, and IL-8 production in a lipoprotein-dependent manner. TLR2/1 signaling was involved in NET formation and IL-8 production, but not elastase release, suggesting that MRSA-CMP-elicited elastase release is not mediated by triacylated lipoproteins. MRSA-CMP also primed neutrophils for enhanced NET formation in response to a subsequent stimulus. MRSA-CMP-elicited NET formation did not require Nox2-derived reactive oxygen species and was partially dependent on the activity of peptidyl arginine deiminase (PAD). In conclusion, lipoproteins from S. aureus mediate NET formation via TLR2/1 with clear implications for patients with sepsis.


Assuntos
Membrana Celular/metabolismo , Armadilhas Extracelulares/metabolismo , Lipoproteínas/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Neutrófilos/imunologia , Proteína-Arginina Desiminase do Tipo 1/metabolismo , Infecções Estafilocócicas/imunologia , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Lipoproteínas/genética , Lipoilação , Staphylococcus aureus Resistente à Meticilina/genética , Mutação/genética , Elastase Pancreática/metabolismo , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/genética , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo
3.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298929

RESUMO

Dyslipidemia is characterized by increasing plasma levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides (TGs) and TG-rich lipoproteins (TGRLs) and is a major risk factor for the development of atherosclerotic cardiovascular disorders (ASCVDs). It is important to understand the metabolic mechanisms underlying dyslipidemia to develop effective strategies against ASCVDs. Angiopoietin-like 3 (ANGPTL3), a member of the angiopoietin-like protein family exclusively synthesized in the liver, has been demonstrated to be a critical regulator of lipoprotein metabolism to inhibit lipoprotein lipase (LPL) activity. Genetic, biochemical, and clinical studies in animals and humans have shown that loss of function, inactivation, or downregulated expression of ANGPTL3 is associated with an obvious reduction in plasma levels of TGs, LDL-C, and high-density lipoprotein-cholesterol (HDL-C), atherosclerotic lesions, and the risk of cardiovascular events. Therefore, ANGPTL3 is considered an alternative target for lipid-lowering therapy. Emerging studies have focused on ANGPTL3 inhibition via antisense oligonucleotides (ASOs) and monoclonal antibody-based therapies, which have been carried out in mouse or monkey models and in human clinical studies for the management of dyslipidemia and ASCVDs. This review will summarize the current literature on the important role of ANGPTL3 in controlling lipoprotein metabolism and dyslipidemia, with an emphasis on anti-ANGPTL3 therapies as a potential strategy for the treatment of dyslipidemia and ASCVDs.


Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , Dislipidemias/metabolismo , Lipoproteínas/metabolismo , Animais , Aterosclerose/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol , Humanos , Triglicerídeos/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203861

RESUMO

Circulating hemopexin is the primary protein responsible for the clearance of heme; therefore, it is a systemic combatant against deleterious inflammation and oxidative stress induced by the presence of free heme. This role of hemopexin is critical in hemolytic pathophysiology. In this review, we outline the current research regarding how the dynamic activity of hemopexin is implicated in sickle cell disease, which is characterized by a pathological aggregation of red blood cells and excessive hemolysis. This pathophysiology leads to symptoms such as acute kidney injury, vaso-occlusion, ischemic stroke, pain crises, and pulmonary hypertension exacerbated by the presence of free heme and hemoglobin. This review includes in vivo studies in mouse, rat, and guinea pig models of sickle cell disease, as well as studies in human samples. In summary, the current research indicates that hemopexin is likely protective against these symptoms and that rectifying depleted hemopexin in patients with sickle cell disease could improve or prevent the symptoms. The data compiled in this review suggest that further preclinical and clinical research should be conducted to uncover pathways of hemopexin in pathological states to evaluate its potential clinical function as both a biomarker and therapy for sickle cell disease and related hemoglobinopathies.


Assuntos
Anemia Falciforme/patologia , Anemia Falciforme/fisiopatologia , Citoproteção , Hemopexina/metabolismo , Animais , Humanos , Imunomodulação , Lipoproteínas/metabolismo , Microvasos/patologia
5.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298933

RESUMO

In photodynamic therapy (PDT) for neoplasms, photosensitizers selectively accumulate in cancer tissue. Upon excitation with light of an optimal wavelength, the photosensitizer and surrounding molecules generate reactive oxygen species, resulting in cancer cell-specific cytotoxicity. Porphylipoprotein (PLP) has a porphyrin-based nanostructure. The porphyrin moiety of PLP is quenched because of its structure. When PLP is disrupted, the stacked porphyrins are separated into single molecules and act as photosensitizers. Unless PLP is disrupted, there is no photosensitive disorder in normal tissues. PLP can attenuate the photosensitive disorder compared with other photosensitizers and is ideal for use as a photosensitizer. However, the efficacy of PLP has not yet been evaluated. In this study, the mechanism of cancer cell-specific accumulation of PLP and its cytotoxic effect on cholangiocarcinoma cells were evaluated. The effects were investigated on normal and cancer-like mutant cells. The cytotoxicity effect of PLP PDT in cancer cells was significantly stronger than in normal cells. In addition, reactive oxygen species regulated intracellular PLP accumulation. The cytotoxic effects were also investigated using a cholangiocarcinoma cell line. The cytotoxicity of PLP PDT was significantly higher than that of laserphyrin-based PDT, a conventional type of PDT. PLP PDT could also inhibit tumor growth in vivo.


Assuntos
Antineoplásicos/farmacologia , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Lipoproteínas/metabolismo , Porfirinas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanoestruturas/química , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo
6.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281154

RESUMO

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria-bacteria and bacteria-host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.


Assuntos
Vesículas Extracelulares/imunologia , Lipoproteínas/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Inflamação/imunologia , Lipoproteínas/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Receptor 2 Toll-Like/metabolismo
7.
Commun Biol ; 4(1): 526, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953337

RESUMO

The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.


Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Microscopia Crioeletrônica/métodos , Lipoproteínas/química , Lipoproteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica
8.
Microvasc Res ; 137: 104188, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34022205

RESUMO

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been led to a pandemic emergency. So far, different pathological pathways for SARS-CoV-2 infection have been introduced in which the excess release of pro-inflammatory cytokines (such as interleukin 1 ß [IL-1ß], IL-6, and tumor necrosis factor α [TNFα]) has earned most of the attentions. However, recent studies have identified new pathways with at least the same level of importance as cytokine storm in which endothelial cell (EC) dysfunction is one of them. In COVID-19, two main pathologic phenomena have been seen as a result of EC dysfunction: hyper-coagulation state and pathologic angiogenesis. The EC dysfunction-induced hypercoagulation state seems to be caused by alteration in the levels of different factors such as plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF) antigen, soluble thrombomodulin, and tissue factor pathway inhibitor (TFPI). As data have shown, these thromboembolic events are associated with severity of disease severity or even death in COVID-19 patients. Other than thromboembolic events, pathologic angiogenesis is among the recent findings. Furthermore, over-expression/higher levels of different proangiogenic factors such as vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1 α (HIF-1α), IL-6, TNF receptor super family 1A and 12, and angiotensin-converting enzyme 2 (ACE2) have been found in the lung biopsies/sera of both survived and non-survived COVID-19 patients. Also, there are some hypotheses regarding the role of nitric oxide in EC dysfunction and acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection. It has been demonstrated that different pathways involved in inflammation are generally common with EC dysfunction and angiogenesis. Altogether, considering the common possible upstream pathways in cytokine storm, pathologic angiogenesis, and EC dysfunction, it seems that targeting these molecules (such as nuclear factor κB) could be more effective in the management of patients with COVID-19.


Assuntos
COVID-19/sangue , COVID-19/fisiopatologia , Células Endoteliais/metabolismo , Neovascularização Patológica , Enzima de Conversão de Angiotensina 2/metabolismo , Biomarcadores/metabolismo , Coagulação Sanguínea , Síndrome da Liberação de Citocina , Humanos , Inflamação , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipoproteínas/metabolismo , Óxido Nítrico/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , SARS-CoV-2 , Fator de Necrose Tumoral alfa/metabolismo , Fator de von Willebrand/metabolismo
9.
Med Sci Monit ; 27: e928784, 2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958572

RESUMO

BACKGROUND A small proportion of familial hypercholesterolemia (FH) patients can adequately control this condition, although achieving the recommended targets for low-density lipoprotein cholesterol (LDL-c) levels remains a challenge. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are new and potent lipid-lowering drugs. However, there is scarce literature on real-world data about their use in patients with FH. MATERIAL AND METHODS We examined the reduction in LDL-c levels from the baseline, after PCSK9i initiation in heterozygous familial hypercholesterolemia patients referred for lipoprotein apheresis in our regional lipid clinic. The study was conducted from March 2018 to September 2019, the period immediately after PCSK9i reimbursement was available in France. PCSK9i was added on top of the patients' maximal tolerated lipid-lowering regimens. RESULTS The study had 123 patients with heterozygous FH. The mean age of the patients was 59±11 years. The mean baseline LDL-c for all the participants was 277±78 mg/dl. It was 283±81 mg/dl in the PCSK9i monotherapy group (n=83), 247±68 mg/dl in the PCSK9i plus ezetimibe group (n=12), and 264±78 mg/dl in the PCSK9i plus statin and ezetimibe group (n=28). The mean decrease observed in the LDL-c level from baseline was 136±70 mg/dl (n=123), 125±60 mg/dl (n=83), 103±77 mg/dl (n=12), and 175±70 mg/dl (n=28), respectively. CONCLUSIONS An overall reduction of 49.1% from the baseline LDL-c was observed in the heterozygous FH population after PCSK9i initiation in a real-world experience. The group treated with PCSK9i ezetimibe plus statin showed further reduction of their LDL-c levels with a better responder rate, achieving the target 50% reduction in LDL-c from the baseline.


Assuntos
Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Pró-Proteína Convertase 9/antagonistas & inibidores , Subtilisinas/uso terapêutico , Remoção de Componentes Sanguíneos/métodos , LDL-Colesterol/metabolismo , Estudos de Coortes , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipoproteinemia Tipo II/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos , Lipoproteínas/metabolismo , Masculino , Pessoa de Meia-Idade
10.
Elife ; 102021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33847565

RESUMO

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the ß-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Membrana Externa Bacteriana/fisiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lipoproteínas/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Aptidão Genética , Lipoproteínas/metabolismo , Dobramento de Proteína
11.
J Bacteriol ; 203(13): e0014921, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33875545

RESUMO

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.


Assuntos
Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transferases/metabolismo , Animais , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Escherichia coli/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos , Peptidoglicano/metabolismo , Transferases/química , Transferases/genética , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo
13.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807969

RESUMO

Sitosterolemia is a lipid disorder characterized by the accumulation of dietary xenosterols in plasma and tissues caused by mutations in either ABCG5 or ABCG8. ABCG5 ABCG8 encodes a pair of ABC half transporters that form a heterodimer (G5G8), which then traffics to the surface of hepatocytes and enterocytes and promotes the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. We review the literature from the initial description of the disease, the discovery of its genetic basis, current therapy, and what has been learned from animal, cellular, and molecular investigations of the transporter in the twenty years since its discovery. The genomic era has revealed that there are far more carriers of loss of function mutations and likely pathogenic variants of ABCG5 ABCG8 than previously thought. The impact of these variants on G5G8 structure and activity are largely unknown. We propose a classification system for ABCG5 ABCG8 mutants based on previously published systems for diseases caused by defects in ABC transporters. This system establishes a framework for the comprehensive analysis of disease-associated variants and their impact on G5G8 structure-function.


Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Colesterol/metabolismo , Hipercolesterolemia , Enteropatias , Erros Inatos do Metabolismo Lipídico , Lipoproteínas , Mutação , Fitosteróis/efeitos adversos , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/história , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/história , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Enterócitos/metabolismo , Enterócitos/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , História do Século XXI , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/história , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Enteropatias/genética , Enteropatias/história , Enteropatias/metabolismo , Enteropatias/patologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/história , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Lipoproteínas/genética , Lipoproteínas/história , Lipoproteínas/metabolismo , Fitosteróis/genética , Fitosteróis/história , Fitosteróis/metabolismo
14.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673180

RESUMO

Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32-/- mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32-/- mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32-/- neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32-/- mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocinas/metabolismo , Lipoproteínas/metabolismo , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Quimiocinas/genética , Lipoproteínas/genética , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
15.
Nutrients ; 13(2)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671529

RESUMO

The number of nutrigenetic studies dedicated to the identification of single nucleotide polymorphisms (SNPs) modulating blood lipid profiles in response to dietary interventions has increased considerably over the last decade. However, the robustness of the evidence-based science supporting the area remains to be evaluated. The objective of this review was to present recent findings concerning the effects of interactions between SNPs in genes involved in cholesterol metabolism and transport, and dietary intakes or interventions on circulating cholesterol concentrations, which are causally involved in cardiovascular diseases and established biomarkers of cardiovascular health. We identified recent studies (2014-2020) that reported significant SNP-diet interactions in 14 cholesterol-related genes (NPC1L1, ABCA1, ABCG5, ABCG8, APOA1, APOA2, APOA5, APOB, APOE, CETP, CYP7A1, DHCR7, LPL, and LIPC), and which replicated associations observed in previous studies. Some studies have also shown that combinations of SNPs could explain a higher proportion of variability in response to dietary interventions. Although some findings still need replication, including in larger and more diverse study populations, there is good evidence that some SNPs are consistently associated with differing circulating cholesterol concentrations in response to dietary interventions. These results could help clinicians provide patients with more personalized dietary recommendations, in order to lower their risk for cardiovascular disease.


Assuntos
Colesterol na Dieta/sangue , Colesterol/sangue , Metabolismo dos Lipídeos/genética , Polimorfismo de Nucleotídeo Único , Colesterol na Dieta/metabolismo , Regulação da Expressão Gênica , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo
16.
Microb Cell Fact ; 20(1): 33, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531008

RESUMO

BACKGROUND: The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins. RESULTS: In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp'OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp'OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy. CONCLUSIONS: To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Bactérias/ultraestrutura , Membrana Celular/metabolismo , Escherichia coli/ultraestrutura , Engenharia Genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Cell Metab ; 33(2): 231-233, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33535097

RESUMO

Chylomicrons and very-low-density lipoproteins (VLDLs) are large, complex cargos that may require specific chaperones for efficient transport from the ER to Golgi. In this issue of Cell Metabolism, Wang et al. (2020) identify SURF4, in coordination with SAR1B, as an essential player in COPII transport of VLDLs from ER to Golgi, suggesting that SURF4 may be a target for approaches aimed at reducing secretion of triglyceride-rich, atherogenic lipoproteins from the liver.


Assuntos
Apolipoproteínas B , Proteínas Monoméricas de Ligação ao GTP , Animais , Apolipoproteínas B/metabolismo , Quilomícrons/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Humanos , Lipoproteínas/metabolismo , Proteínas de Membrana , Camundongos
18.
Sci Rep ; 11(1): 967, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441737

RESUMO

Colony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.


Assuntos
Flavobacterium/metabolismo , Flavobacterium/fisiologia , Movimento/fisiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flavobacterium/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Mutação/genética
19.
Clin Chem ; 67(1): 183-196, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33409533

RESUMO

BACKGROUND: Triglycerides, cholesterol, and their metabolism are linked due to shared packaging and transport within circulating lipoprotein particles. While a case for a causal role of cholesterol-carrying low-density lipoproteins (LDLs) in atherosclerosis is well made, the body of scientific evidence for a causal role of triglyceride-rich lipoproteins (TRLs) is rapidly growing, with multiple lines of evidence (old and new) providing robust support. CONTENT: This review will discuss current perspectives and accumulated evidence that an overabundance of remnant lipoproteins stemming from intravascular remodeling of nascent TRLs-chylomicrons and very low-density lipoproteins (VLDL)-results in a proatherogenic milieu that augments cardiovascular risk. Basic mechanisms of TRL metabolism and clearance will be summarized, assay methods reviewed, and pivotal clinical studies highlighted. SUMMARY: Remnant lipoproteins are rendered highly atherogenic by their high cholesterol content, altered apolipoprotein composition, and physicochemical properties. The aggregate findings from multiple lines of evidence suggest that TRL remnants play a central role in residual cardiovascular risk.


Assuntos
Aterosclerose/etiologia , Lipoproteínas/metabolismo , Triglicerídeos/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/epidemiologia , Aterosclerose/genética , Remanescentes de Quilomícrons/metabolismo , Ensaios Clínicos como Assunto , Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Fíbricos/uso terapêutico , Humanos , Hipertrigliceridemia/tratamento farmacológico , Hipertrigliceridemia/etiologia , Lipoproteínas/análise , Lipoproteínas VLDL/metabolismo , Fatores de Risco , Triglicerídeos/análise
20.
Biomed Res Int ; 2021: 8239135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33490276

RESUMO

The study is aimed at investigating the role and mechanism of LpqH of Mycobacterium tuberculosis in the activation of NLRP3 inflammasome in mouse Ana-1 macrophages. ExPASy-ProtParam, PHYRE2, ABCpred, and SYFPEITHI were used to predict and analyze the physicochemical properties, protein structure, and B cell/T cell-associated epitopes of LpqH protein. The recombinant LpqH protein was purified, and its immunoreactivity was analyzed with western blot. The LPS-treated mouse Ana-1 macrophages were incubated with purified LpqH protein directly. The expression of NLRP3, ASC, and caspase-1 protein was detected by western blot. The secretion of IL-1ß was detected by ELISA, and LDH was detected by a kit. Cell death was detected by flow cytometry. LpqH consisted of 159 amino acids and was a hydrophobic protein with stable properties. Its secondary structure contained 47% random coils, 53% ß-sheets, and 3% α-helix. The tertiary structure showed a relatively loose spatial conformation. Additionally, it had 8 B cell epitopes (score > 0.8) and 10 CTL cell epitopes (score ≥ 20). The recombinant LpqH, which had strong immunoreactivity, significantly increased the levels of NLRP3, ASC, and caspase-1 p20 (P < 0.01) and promoted the secretion of IL-1ß by the cells (P < 0.01). In addition, high concentration of KCl significantly inhibited the effect of LpqH on mouse Ana-1 macrophages and reduced the expression of NLRP3, ASC, and caspase-1 p20 (P < 0.01). However, there was no significant change in LDH (P > 0.05). Meanwhile, LpqH protein did not cause additional cell death (P > 0.05). LpqH protein has good immunogenicity and can activate the NLRP3 inflammasome through the potassium efflux pathway without causing cell death.


Assuntos
Proteínas de Bactérias/metabolismo , Inflamassomos/metabolismo , Lipoproteínas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Linhagem Celular , Biologia Computacional , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos
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