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1.
Life Sci Alliance ; 5(1)2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34764206

RESUMO

Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed.


Assuntos
COVID-19/metabolismo , Modelos Animais de Doenças , Esfingolipídeos/metabolismo , Replicação Viral/fisiologia , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Cromatografia Líquida/métodos , Dioxanos/farmacologia , Gangliosídeos/sangue , Gangliosídeos/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Humanos , Espectrometria de Massas/métodos , Camundongos Transgênicos , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Esfingolipídeos/sangue , Esfingosina/análogos & derivados , Esfingosina/sangue , Esfingosina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
2.
Enzyme Microb Technol ; 151: 109919, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34649690

RESUMO

Improving enzyme stability is very important for enzyme applications. Structural modification is a reliable and effective method to improve the characteristics of protein. By artificially extending the C-terminus, 6 domain modification variants of different sizes were constructed, and a new enzyme species with high stability was obtained. Experimental results affirmed that high stability can be achieved by decreasing the degree of domain freedom. The optimum temperatures of domain modification variants were improved by 10 °C compared with the original enzyme. Specifically, compared with the original enzyme, the half-life of the variant dexYG-fdx (D-F) was increased to 280% under 35 °C and 200% under 45 °C, and the pH tolerance range was wider. Further structural simulations and molecular docking studies provided a reasonable explanation (The increased domain reduced the degree of freedom of the enzyme terminal to some extent) for this variant to increase stability and produce dextran. This study can provide valuable information for increasing the characteristics of recombinant dextransucrase.


Assuntos
Glucosiltransferases , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Simulação de Acoplamento Molecular , Temperatura
3.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638879

RESUMO

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.


Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo , Dioxanos/farmacologia , Glicoesfingolipídeos , Pirrolidinas/farmacologia , Esferoides Celulares , Animais , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/genética , Células HCT116 , Humanos , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
4.
Cell Mol Life Sci ; 78(21-22): 7025-7041, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34626204

RESUMO

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.


Assuntos
Biomarcadores Tumorais/metabolismo , Metabolismo Energético/fisiologia , Glucosilceramidas/metabolismo , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Resistência a Múltiplos Medicamentos/fisiologia , Glucosiltransferases/metabolismo , Glicólise/fisiologia , Glicoesfingolipídeos/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologia
5.
Appl Microbiol Biotechnol ; 105(19): 7309-7319, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34542685

RESUMO

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. KEY POINTS: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants.


Assuntos
Glucosídeos/metabolismo , Glucosiltransferases/metabolismo , Estabilidade Enzimática
6.
Nat Commun ; 12(1): 4713, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354054

RESUMO

Maize (Zea mays L.) is a cold-sensitive species that often faces chilling stress, which adversely affects growth and reproduction. However, the genetic basis of low-temperature adaptation in maize remains unclear. Here, we demonstrate that natural variation in the type-A Response Regulator 1 (ZmRR1) gene leads to differences in chilling tolerance among maize inbred lines. Association analysis reveals that InDel-35 of ZmRR1, encoding a protein harboring a mitogen-activated protein kinase (MPK) phosphorylation residue, is strongly associated with chilling tolerance. ZmMPK8, a negative regulator of chilling tolerance, interacts with and phosphorylates ZmRR1 at Ser15. The deletion of a 45-bp region of ZmRR1 harboring Ser15 inhibits its degradation via the 26 S proteasome pathway by preventing its phosphorylation by ZmMPK8. Transcriptome analysis indicates that ZmRR1 positively regulates the expression of ZmDREB1 and Cellulose synthase (CesA) genes to enhance chilling tolerance. Our findings thus provide a potential genetic resource for improving chilling tolerance in maize.


Assuntos
Zea mays/genética , Zea mays/fisiologia , Alelos , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética
7.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298903

RESUMO

Sucrose content is a crucial indicator of quality and flavor in peanut seed, and there is a lack of clarity on the molecular basis of sucrose metabolism in peanut seed. In this context, we performed a comprehensive comparative transcriptome study on the samples collected at seven seed development stages between a high-sucrose content variety (ICG 12625) and a low-sucrose content variety (Zhonghua 10). The transcriptome analysis identified a total of 8334 genes exhibiting significantly different abundances between the high- and low-sucrose varieties. We identified 28 differentially expressed genes (DEGs) involved in sucrose metabolism in peanut and 12 of these encoded sugars will eventually be exported transporters (SWEETs). The remaining 16 genes encoded enzymes, such as cell wall invertase (CWIN), vacuolar invertase (VIN), cytoplasmic invertase (CIN), cytosolic fructose-bisphosphate aldolase (FBA), cytosolic fructose-1,6-bisphosphate phosphatase (FBP), sucrose synthase (SUS), cytosolic phosphoglucose isomerase (PGI), hexokinase (HK), and sucrose-phosphate phosphatase (SPP). The weighted gene co-expression network analysis (WGCNA) identified seven genes encoding key enzymes (CIN, FBA, FBP, HK, and SPP), three SWEET genes, and 90 transcription factors (TFs) showing a high correlation with sucrose content. Furthermore, upon validation, six of these genes were successfully verified as exhibiting higher expression in high-sucrose recombinant inbred lines (RILs). Our study suggested the key roles of the high expression of SWEETs and enzymes in sucrose synthesis making the genotype ICG 12625 sucrose-rich. This study also provided insights into the molecular basis of sucrose metabolism during seed development and facilitated exploring key candidate genes and molecular breeding for sucrose content in peanuts.


Assuntos
Arachis/genética , Arachis/metabolismo , Sacarose/metabolismo , Transcriptoma/genética , Metabolismo dos Carboidratos/genética , Parede Celular/genética , Parede Celular/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Sementes/genética , Sementes/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209164

RESUMO

For many years, the biology of glycosphingolipids was elucidated with the help of glucosylceramide synthase (GCS) inhibitors such as 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Additionally, PDMP gained interest because of its chemosensitizing effects. Several studies have successfully combined PDMP and anti-cancer drugs in the context of cancer therapy. However, the mechanism of action of PDMP is not fully understood and seems to go beyond glycolipid inhibition. Here, we used a functionalized sphingosine analogue (pacSph) to investigate the acute effects of PDMP on cellular sphingolipid distribution and found that PDMP, but not other GCS inhibitors, such as ND-DNJ (also called Miglustat), induced sphingolipid accumulation in lysosomes. This effect could be connected to defective export from lysosome, as monitored by the prolonged lysosomal staining of sphingolipids as well as by a delay in the metabolic conversion of the pacSph precursor. Additionally, other lipids such as lysobisphosphatidic acid (LBPA) and cholesterol were enriched in lysosomes upon PDMP treatment in a time-dependent manner. We could further correlate early LBPA enrichment with dissociation of the mechanistic target of rapamycin (mTOR) from lysosomes followed by nuclear translocation of its downtream target, transcription factor EB (TFEB). Altogether, we report here a timeline of lysosomal lipid accumulation events and mTOR inactivation arising from PDMP treatment.


Assuntos
Glucosiltransferases/antagonistas & inibidores , Metabolismo dos Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Morfolinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Glucosiltransferases/metabolismo , Células HeLa , Humanos
9.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1858-1868, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227281

RESUMO

Sucrose is a natural product occurs widely in nature. In living organisms such as plants, sucrose phosphate synthase (SPS) is the key rate-limiting enzyme for sucrose synthesis. SPS catalyzes the synthesis of sucrose-6-phosphate, which is further hydrolyzed by sucrose phosphatase to form sucrose. Researches on SPS in recent decades have been focused on the determination of enzymatic activity of SPS, the identification of the inhibitors and activators of SPS, the covalent modification of SPS, the carbohydrate distribution in plants regulated by SPS, the mechanism for promoting plant growth by SPS, the sweetness of fruit controlled by SPS, and many others. A systematic review of these aspects as well as the crystal structure and catalytic mechanism of SPS are presented.


Assuntos
Glucosiltransferases , Sacarose , Metabolismo dos Carboidratos , Glucosiltransferases/metabolismo , Plantas/metabolismo
10.
Molecules ; 26(14)2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34299608

RESUMO

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.


Assuntos
Glucosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Domínio Catalítico , Glucosiltransferases/química , Glucosiltransferases/genética , Modelos Moleculares , Mutação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/química , Plantas/genética
11.
Plant Cell Rep ; 40(10): 1875-1888, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34272585

RESUMO

KEY MESSAGE: GmFULa improved soybean yield by enhancing carbon assimilation. Meanwhile, different from known yield-related genes, it did not alter flowering time or maturity. Soybean (Glycine max (L.) Merr.) is highly demanded by a continuously growing human population. However, increasing soybean yield is a major challenge. FRUITFULL (FUL), a MADS-box transcription factor, plays important roles in multiple developmental processes, especially fruit and pod development, which are crucial for soybean yield formation. However, the functions of its homologs in soybean are not clear. Here, through haplotype analysis, we found that one haplotype of the soybean homolog GmFULa (GmFULa-H02) is dominant in cultivated soybeans, suggesting that GmFULa-H02 was highly selected during domestication and varietal improvement of soybean. Interestingly, transgenic overexpression of GmFULa enhanced vegetative growth with more biomass accumulated and ultimately increased the yield but without affecting the plant height or changing the flowering time and maturity, indicating that it enhances the efficiency of dry matter accumulation. It also promoted the yield factors like branch number, pod number and 100-seed weight, which ultimately increased the yield. It increased the palisade tissue cell number and the chlorophyll content to promote photosynthesis and increase the soluble sugar content in leaves and fresh seeds. Furthermore, GmFULa were found to be sublocalized in the nucleus and positively regulate sucrose synthases (SUSs) and sucrose transporters (SUTs) by binding with the conserved CArG boxes in their promoters. Overall, these results showed GmFULa promotes the capacity of assimilation and the transport of the resultant assimilates to increase yield, and provided insights into the link between GmFULa and sucrose synthesis with transport-related molecular pathways that control seed yield.


Assuntos
Carbono/metabolismo , Flores/fisiologia , Proteínas de Plantas/genética , Soja/genética , Clorofila/metabolismo , Flores/genética , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Haplótipos , Fotossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/genética , Sementes/metabolismo , Soja/fisiologia
12.
Chem Biodivers ; 18(8): e2100455, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34185351

RESUMO

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U-87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates.


Assuntos
Modelos Biológicos , Triterpenos Pentacíclicos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Senna (Planta)/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Apoptose/efeitos dos fármacos , Reatores Biológicos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Senna (Planta)/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
13.
Commun Biol ; 4(1): 678, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083706

RESUMO

Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO2 nanoparticles (TiO2NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO2NPs. TiO2NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO2NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO2NPs, thereby mitigating TiO2NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.


Assuntos
Vesículas Citoplasmáticas/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Pressão Osmótica/efeitos dos fármacos , Titânio/toxicidade , Vesículas Citoplasmáticas/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Microscopia de Força Atômica/métodos , Mutação
14.
Plant Physiol Biochem ; 166: 495-504, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34166976

RESUMO

Flavonoid glycosides are important plant secondary metabolites with broad pharmacological activities. Flavonoid glycosides are generated from aglycones, in reactions catalyzed by typical uridine diphosphate-dependent glycosyltransferases (UGTs). Liverworts produce various types of flavonoid glycosides; however, only two UGTs have been characterized from liverworts to date. Here, we isolated three genes encoding UGTs (MeUGT1, MeUGT2, and MpalUGT1) from the liverwort species Marchantia emarginata and Marchantia paleacea through transcriptome sequencing. Recombinant MeUGT1, MeUGT2, and MpalUGT1 proteins heterologously produced in Escherichia coli exhibited catalytic activity towards multiple flavonoids. MeUGT1 and MpalUGT1 catalyzed the glycosylation of flavonols into the corresponding 3-O-glucosides with UDP-glucose as the sugar donor, while MeUGT2 exhibited a wider substrate specificity that included flavonols, flavones, and flavanones. When MeUGT2 was expressed in E. coli, the yield of flavonol 3-O-glucosides reached to 40-60% with feeding of the substrates kaempferol or quercetin under optimal conditions. Furthermore, heterologous expression of MeUGT1 in Arabidopsis thaliana increased the flavonol glycoside contents in the plants. Therefore, the UGTs characterized in this study could provide new data that will be useful for examining flavonoid biosynthesis in liverworts.


Assuntos
Glucosiltransferases , Marchantia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Marchantia/genética , Marchantia/metabolismo , Especificidade por Substrato
15.
EMBO J ; 40(15): e107240, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34152647

RESUMO

Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.


Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Lisossomos/metabolismo , Polissacarídeos/metabolismo , Animais , Calnexina/genética , Calnexina/metabolismo , Fibroblastos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Oligossacarídeos/metabolismo , Pró-Colágeno/genética , Pró-Colágeno/metabolismo , Dobramento de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
16.
Int J Biol Macromol ; 183: 1248-1256, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-33965495

RESUMO

Potato starch with high viscosity and digestibility cannot be added into some foods. To address this issue, a novel starch-acting enzyme 4,6-α-glucosyltransferase from Streptococcus thermophilus (StGtfB) was used. StGtfB decreased the iodine affinity and the molecular weight, but increased the degree of branching of starch at a mode quite different from glycogen 1,4-α-glucan branching enzyme (GBE). StGtfB at 5 U/g substrate mainly introduced DP 1-7 into amylose (AMY) or DP 1-12 branches into amylopectin (AMP), and increased the ratio of short- to long-branches from 0.32 to 2.22 or from 0.41 to 2.50. The DP 3 branch chain was the most abundant in both StGtfB-modified AMY and StGtfB-modified AMP. The DP < 6 branch chain contents in StGtfB-modified AMY were 42.68%, much higher than those of GBE-modified AMY. StGtfB significantly decreased viscoelasticity but still kept pseudoplasticity of starch. The modifications also slowed down the glucose generation rate of products at the mammalian mucosal α-glucosidase level. The slowly digestible fraction in potato starch increased from 34.29% to 53.22% using StGtfB of 5 U/g starch. This low viscoelastic and slowly digestible potato starch had great potential with respect to low and stable postprandial blood glucose.


Assuntos
Glucosiltransferases/metabolismo , Solanum tuberosum/química , Amido/química , Streptococcus thermophilus/enzimologia , Amilopectina/metabolismo , Amilose/metabolismo , Proteínas de Bactérias/metabolismo , Elasticidade , Hidrólise , Iodo/química , Peso Molecular , Viscosidade
17.
Phytomedicine ; 88: 153556, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33958276

RESUMO

BACKGROUND: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as ß(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of ß(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. PURPOSE: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. METHODS: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms "natural antifungals" and "plant extracts" with "fungal cell wall". RESULTS: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. CONCLUSION: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Produtos Biológicos/farmacologia , Parede Celular/efeitos dos fármacos , Fungos/citologia , Caspofungina/farmacologia , Parede Celular/química , Parede Celular/metabolismo , Quitina/biossíntese , Equinocandinas/farmacocinética , Fungos/efeitos dos fármacos , Glucanos/biossíntese , Glucosiltransferases/metabolismo , Humanos , Micoses/tratamento farmacológico
18.
Enzyme Microb Technol ; 147: 109788, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33992410

RESUMO

Isomaltooligosaccharide (IMO), considered to be a prebiotic, reportedly has health effects, particularly in terms of digestion; however, the prebiotic effects of IMOs depend largely on the degree of polymerization. Currently, IMOs are commercially produced using transglucosidase (TG) derived from Aspergillus niger. Here, we report a novel Thermoanaerobacter thermocopriae-derived TG (TtTG) that can produce long-chain IMOs (L-IMOs) using maltodextrin as the main substrate. A putative carbohydrate-binding gene comprising carbohydrate-binding module 35 and glycoside hydrolase family 15 domain was cloned and successfully overexpressed in Escherichia coli BL21 (DE3) cells. The resulting purified recombinant enzyme (TtTG) had a molecular mass of 94 kDa. TtTG displayed an optimal pH of 4.0 (higher than that of commercial TG) and an optimal temperature of 60 °C (same as that of commercial TG). TtTG also enabled the synthesis of oligosaccharides using various saccharides, such as palatinose, kojibiose, sophorose, maltose, cellobiose, isomaltose, gentiobiose, and trehalose, which acted as specific acceptors. TtTG could also produce a medium-sized L-IMO, different from that by dextran-dextrinase and TG, from maltodextrin, as the sole substrate. Thus, the novel combination of maltodextrin and TtTG shows potential as an effective method for commercially producing L-IMOs with improved prebiotic effects.


Assuntos
Glucosiltransferases , Thermoanaerobacter , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oligossacarídeos , Polissacarídeos , Especificidade por Substrato , Thermoanaerobacter/genética
19.
J Biol Chem ; 297(1): 100843, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34058199

RESUMO

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding ß1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.


Assuntos
Fenda Labial/enzimologia , Fenda Labial/genética , Córnea/anormalidades , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Transtornos do Crescimento/enzimologia , Transtornos do Crescimento/genética , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Mutação/genética , Proteínas ADAMTS/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Córnea/enzimologia , Estabilidade Enzimática , Fucose/metabolismo , Galactosiltransferases/química , Glucose/metabolismo , Glucosiltransferases/química , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Sequências Repetitivas de Aminoácidos , Homologia Estrutural de Proteína
20.
J Biol Chem ; 296: 100772, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989636

RESUMO

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel ß-sheets of seven and six strands, arranged as a distorted ß-sandwich. Furthermore, two long loops partially cover the concave face of the ß-sandwich defined by the ß-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.


Assuntos
Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Domínio B30.2-SPRY , Sítios de Ligação , Cristalografia por Raios X , Glucosiltransferases/química , Glicoproteínas/química , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química
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