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1.
Food Chem ; 370: 131271, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34788952

RESUMO

5-Hydroxymethylfurfural (HMF) and acrylamide (AA) are neoformed food contaminants. In this study, the simultaneous inhibition of HMF and AA by histidine (His) were investigated. In the asparagine (Asn)/glucose (Glc) model system, the inhibition ratios of HMF and AA were in the range of 28-58% and 0-71% when 20 mmol/L His was added. In cookies, His also exhibited excellent inhibition effects on both HMF and AA. At the His concentration of 2% (w/w), the inhibition ratios of HMF and AA reached 90% and 65%. Additionally, the sensory quality of cookies was not affected significantly. Qualitative results suggested that His inhibited the formation of AA by the competitive reaction between His and Asn for Glc, as well as directly eliminated the formed HMF and AA via the carbonyl-amine reaction and the Michael addition, respectively. This study revealed that His could be applied for the inhibition of HMF and AA in heated food.


Assuntos
Acrilamida , Histidina , Asparagina , Furaldeído/análogos & derivados
2.
Elife ; 102021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34647520

RESUMO

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here, we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.


Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Asparagina/biossíntese , Desenvolvimento Ósseo/genética , Glutamina/biossíntese , Antígenos de Histocompatibilidade Menor/genética , Osteoblastos/metabolismo , Osteogênese , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Feminino , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo
3.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34551980

RESUMO

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.


Assuntos
Asparagina/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribonuclease Pancreático/metabolismo , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Bovinos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Polissacarídeos/química , Polissacarídeos/genética , Conformação Proteica , Engenharia de Proteínas , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
4.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576056

RESUMO

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0-6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.


Assuntos
Archaea/enzimologia , Asparaginase/isolamento & purificação , Biotecnologia/tendências , Thermococcus/enzimologia , Sequência de Aminoácidos/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Asparaginase/química , Asparaginase/genética , Asparagina/metabolismo , Estabilidade Enzimática/genética , Escherichia coli/efeitos dos fármacos , Cinética , Especificidade por Substrato/genética
5.
Biochem Biophys Res Commun ; 573: 93-99, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34403810

RESUMO

ATF4 is a crucial transcription factor in the integrated stress response, a major adaptive signaling pathway activated by tumor microenvironment and therapeutic stresses. BRAF inhibitors, such as vemurafenib, induce ATF4 in BRAF-mutated melanoma cells, but the mechanisms of ATF4 induction are not fully elucidated. Here, we show that ATF4 expression can be upregulated by eukaryotic initiation factor 4B (eIF4B) in BRAF-mutated A375 cells. Indeed, eIF4B knockout (KO) prevented ATF4 induction and activation of the uORF-mediated ATF4 translation mechanism during vemurafenib treatment, which were effectively recovered by the rescue of eIF4B. Transcriptome analysis revealed that eIF4B KO selectively influenced ATF4-target gene expression among the overall gene expression changed by vemurafenib. Interestingly, eIF4B supported cellular proliferation under asparagine-limited conditions, possibly through the eIF4B-ATF4 pathway. Our findings indicate that eIF4B can regulate ATF4 expression, thereby contributing to cellular stress adaptation, which could be targeted as a therapeutic approach against malignancies, including melanoma.


Assuntos
Fator 4 Ativador da Transcrição/genética , Asparagina/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fator 4 Ativador da Transcrição/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Fatores de Iniciação em Eucariotos/deficiência , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas , Vemurafenib/farmacologia
6.
Sci Transl Med ; 13(605)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349034

RESUMO

Group A streptococcus (GAS) is among the top 10 causes of mortality from an infectious disease, producing mild to invasive life-threatening manifestations. Necrotizing fasciitis (NF) is characterized by a rapid GAS spread into fascial planes followed by extensive tissue destruction. Despite prompt treatments of antibiotic administration and tissue debridement, mortality from NF is still high. Moreover, there is no effective vaccine against GAS, and early diagnosis of NF is problematic because its clinical presentations are not specific. Thus, there is a genuine need for effective treatments against GAS NF. Previously, we reported that GAS induces endoplasmic reticulum (ER) stress to gain asparagine from the host. Here, we demonstrate that GAS-mediated asparagine induction and release occur through the PERK-eIF2α-ATF4 branch of the unfolded protein response. Inhibitors of PERK or integrated stress response (ISR) blocked the formation and release of asparagine by infected mammalian cells, and exogenously added asparagine overcame this inhibition. Moreover, in a murine model of NF, we show that the inhibitors minimized mortality when mice were challenged with a lethal dose of GAS and reduced bacterial counts and lesion size when mice were challenged with a sublethal dose. Immunohistopathology studies demonstrated that PERK/ISR inhibitors protected mice by enabling neutrophil infiltration into GAS-infected fascia and reducing the pro-inflammatory response that causes tissue damage. Inhibitor treatment was also effective in mice when started at 12 hours after infection. We conclude that host metabolic alteration induced by PERK or ISR inhibitors is a promising therapeutic strategy to treat highly invasive GAS infections.


Assuntos
Fasciite Necrosante , Infecções Estreptocócicas , Animais , Asparagina , Fasciite Necrosante/tratamento farmacológico , Camundongos , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus pyogenes , Resposta a Proteínas não Dobradas
7.
Vet Microbiol ; 261: 109215, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34455356

RESUMO

Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.


Assuntos
Infecções por Flavivirus/veterinária , Flavivirus/genética , Doenças das Aves Domésticas/virologia , Proteínas não Estruturais Virais/metabolismo , Viremia/veterinária , Virulência/genética , Replicação Viral/genética , Animais , Asparagina/genética , Asparagina/metabolismo , Patos , Infecções por Flavivirus/virologia , Glicosilação , Viremia/genética , Viremia/virologia
8.
Angew Chem Int Ed Engl ; 60(41): 22207-22211, 2021 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-34396662

RESUMO

Peptidyl asparaginyl ligases (PALs) are powerful tools for peptide macrocyclization. Herein, we report that a derivative of Asn, namely Nγ -hydroxyasparagine or Asn(OH), is an unnatural P1 substrate of PALs. By Asn(OH)-mediated cyclization, we prepared cyclic peptides as new matrix metalloproteinase 2 (MMP2) inhibitors displaying the hydroxamic acid moiety of Asn(OH) as the key pharmacophore. The most potent cyclic peptide (Ki =2.8±0.5 nM) was built on the hyperstable tetracyclic scaffold of rhesus theta defensin-1. The Asn(OH) residue in the cyclized peptides can also be readily oxidized to Asp. By this approach, we synthesized several bioactive Asp-containing cyclic peptides (MCoTI-II, kB2, SFTI, and integrin-targeting RGD peptides) that are otherwise difficult targets for PAL-catalyzed cyclization owing to unfavorable kinetics of the P1-Asp substrates. This study demonstrates that substrate engineering is a useful strategy to expand the application of PAL ligation in the synthesis of therapeutic cyclic peptides.


Assuntos
Aminoácidos/farmacologia , Asparagina/farmacologia , Inibidores Enzimáticos/farmacologia , Peptídeo Sintases/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Aminoácidos/química , Asparagina/química , Inibidores Enzimáticos/química , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/química , Especificidade por Substrato
9.
J Agric Food Chem ; 69(32): 9419-9433, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34374283

RESUMO

Usage of sprouted grains is an increasing trend in thermally processed foods. Sprouting alters the composition of sugars and amino acids, which are Maillard reaction precursors. Free asparagine, total free amino acids, and sugars were monitored during sprouting and yeast and sourdough fermentations. Acrylamide and 5-hydroxymethylfurfural (HMF) were analyzed in heated samples. The asparagine concentration decreased up to 40% after 24-36 h of sprouting, except for buckwheat, and then increased to the initial concentration after 48 h and several folds after 72 h. The increased amount of reducing sugars after sprouting caused higher acrylamide and HMF formation even if the asparagine concentration was lower. Acrylamide and HMF formation decreased after fermentation of sprouted wholemeal because sugars and asparagine were consumed by yeast. A pH drop of 3 units by sourdough fermentation decreased acrylamide formation but increased HMF formation. Results indicated that sprouted cereal products should be produced under controlled conditions to be used in heated foods.


Assuntos
Fagopyrum , Hordeum , Acrilamida , Asparagina , Avena , Fermentação , Furaldeído/análogos & derivados , Calefação , Temperatura Alta , Reação de Maillard , Secale , Açúcares , Triticum
10.
Nature ; 596(7871): 301-305, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321660

RESUMO

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.


Assuntos
Microscopia Crioeletrônica , Ketamina/química , Ketamina/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/ultraestrutura , Antidepressivos/química , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/química , Glicina/metabolismo , Glicina/farmacologia , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ketamina/metabolismo , Leucina/química , Leucina/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo
11.
Cell Death Dis ; 12(7): 693, 2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34247201

RESUMO

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.


Assuntos
Antineoplásicos/farmacologia , Asparagina/biossíntese , Aspartato-Amônia Ligase/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
BMC Plant Biol ; 21(1): 302, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34187359

RESUMO

BACKGROUND: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. RESULTS: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. CONCLUSIONS: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.


Assuntos
Asparagina/metabolismo , Aspartato-Amônia Ligase/genética , Deleção de Genes , Triticum/genética , Aspartato-Amônia Ligase/metabolismo , Qualidade dos Alimentos , Genes de Plantas/genética , Estudos de Associação Genética , Variação Genética , Triticum/enzimologia , Triticum/metabolismo
13.
Biochemistry ; 60(26): 2064-2070, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34137579

RESUMO

Here we show that an NH-π interaction between a highly conserved Asn and a nearby Trp stabilizes the WW domain of the human protein Pin1. The strength of this NH-π interaction depends on the structure of the arene, with NH-π interactions involving Trp or naphthylalanine being substantially more stabilizing than those involving Tyr or Phe. Calculations suggest arene size and polarizability are key structural determinants of NH-π interaction strength. Methylation or PEGylation of the Asn side-chain amide nitrogen each strengthens the associated NH-π interaction, though likely for different reasons. We hypothesize that methylation introduces steric clashes that destabilize conformations in which the NH-π interaction is not possible, whereas PEGylation strengthens the NH-π interaction via localized desolvation of the protein surface.


Assuntos
Asparagina/química , Ligação de Hidrogênio/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA/química , Polietilenoglicóis/química , Triptofano/química , Domínios WW/efeitos dos fármacos , Sequência de Aminoácidos , Humanos , Metilação , Modelos Moleculares , Mutação , Peptidilprolil Isomerase de Interação com NIMA/genética , Conformação Proteica , Termodinâmica , Domínios WW/genética
14.
Cancer Chemother Pharmacol ; 88(4): 655-664, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34170389

RESUMO

PURPOSE: We evaluated effects of asparaginase dosage, schedule, and formulation on CSF asparagine in children with acute lymphoblastic leukemia (ALL). METHODS: We evaluated CSF asparagine (2114 samples) and serum asparaginase (5007 samples) in 482 children with ALL treated on the Total XVI study (NCT00549848). Patients received one or two 3000 IU/m2 IV pegaspargase doses during induction and were then randomized in continuation to receive 2500 IU/m2 or 3500 IU/m2 IV intermittently (four doses) on the low-risk (LR) or continuously (15 doses) on the standard/high risk (SHR) arms. A pharmacokinetic-pharmacodynamic model was used to estimate the duration of CSF asparagine depletion below 1 uM. RESULTS: During induction, CSF asparagine depletion after two doses of pegaspargase was twice as long as one dose (median 30.7 vs 15.3 days, p < 0.001). During continuation, the higher dose increased the CSF asparagine depletion duration by only 9% on the LR and 1% in the SHR arm, consistent with the nonlinear pharmacokinetics of serum asparaginase. Pegaspargase caused a longer CSF asparagine depletion duration (1.3-5.3-fold) compared to those who were switched to erwinase (p < 0.001). The median (quartile range) serum asparaginase activity needed to maintain CSF asparagine below 1 µM was 0.44 (0.20, 0.99) IU/mL. Although rare, CNS relapse was higher with decreased CSF asparagine depletion (p = 0.0486); there was no association with relapse at any site (p = 0.3). CONCLUSIONS: The number of pegaspargase doses has a stronger influence on CSF asparagine depletion than did dosage, pegaspargase depleted CSF asparagine longer than erwinase, and CSF asparagine depletion may prevent CNS relapses.


Assuntos
Antineoplásicos/administração & dosagem , Asparaginase/administração & dosagem , Asparagina/líquido cefalorraquidiano , Polietilenoglicóis/administração & dosagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antineoplásicos/farmacocinética , Asparaginase/farmacocinética , Criança , Relação Dose-Resposta a Droga , Esquema de Medicação , Humanos , Modelos Biológicos , Polietilenoglicóis/farmacocinética , Estudos Prospectivos
15.
Biosci Biotechnol Biochem ; 85(8): 1853-1860, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34077498

RESUMO

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type (WT) XynR and S92E. The optimal pH values of T315N and S92E/T315N were 6.5-9.5 while those of WT and S92E were 6.5-8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0-8.5 for T315N and S92E/T315N, but 6.0-10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance.


Assuntos
Álcalis/metabolismo , Asparagina/química , Treonina/química , Xilosidases/metabolismo , Substituição de Aminoácidos , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Temperatura , Xilosidases/química , Xilosidases/genética
16.
Appl Microbiol Biotechnol ; 105(11): 4515-4534, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34059941

RESUMO

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.


Assuntos
Antineoplásicos/metabolismo , Asparaginase/biossíntese , Asparagina , Biotecnologia , Dickeya chrysanthemi , Escherichia coli , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/biossíntese
17.
Yakugaku Zasshi ; 141(5): 635-639, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33952744

RESUMO

One of the current critical issues in nucleic acid delivery is the efficient mRNA delivery into target cells, directed toward clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genome editing. To this end, we have developed a variety of cationic polyaspartamide derivatives with varying side chain structures because they can form nanocomplexes, termed polyplexes, with mRNA through electrostatic interactions. Interestingly, the delivery functions were highly affected by the chemical structures of the polyaspartamide side chains. Therefore, we review our previous research and provide a rationale for designing polypeptides for mRNA delivery.


Assuntos
Sistemas de Liberação de Medicamentos , Edição de Genes , Ácidos Nucleicos/metabolismo , Peptídeos , RNA Mensageiro/metabolismo , Animais , Asparagina/química , Proteínas Associadas a CRISPR , Cátions , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenho de Fármacos , Humanos , Camundongos , Nanocompostos , Eletricidade Estática
18.
Biochemistry ; 60(21): 1708-1721, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33983715

RESUMO

Endoplasmic reticulum (ER) stress has been reported in a variety of diseases. Although ER stress can be detected using specific markers, it is still difficult to quantitatively evaluate the degree of stress and to identify the cause of the stress. The ER is the primary site for folding of secretory or transmembrane proteins as well as the site where glycosylation is initiated. This study therefore postulates that tracing the biosynthetic pathway of asparagine-linked glycans (N-glycans) would be a reporter for reflecting the state of the ER and serve as a quantitative descriptor of ER stress. Glycoblotting-assisted mass spectrometric analysis of the HeLa cell line enabled quantitative determination of the changes in the structures of N-glycans and degraded free oligosaccharides (fOSs) in response to tunicamycin- or thapsigargin-induced ER stress. The integrated analysis of neutral and sialylated N-glycans and fOSs showed the potential to elucidate the cause of ER stress, which cannot be readily done by protein markers alone. Changes in the total amount of glycans, increase in the ratio of high-mannose type N-glycans, increase in fOSs, and changes in the ratio of sialylated N-glycans in response to ER stress were shown to be potential descriptors of ER stress. Additionally, drastic clearance of accumulated N-glycans was observed in thapsigargin-treated cells, which may suggest the observation of ER stress-mediated autophagy or ER-phagy in terms of glycomics. Quantitative analysis of N-glycoforms composed of N-glycans and fOSs provides the dynamic indicators reflecting the ER status and the promising strategies for quantitative evaluation of ER stress.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/patologia , Asparagina/metabolismo , Biomarcadores , Glicosilação , Células HeLa , Humanos , Manose/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Relação Estrutura-Atividade , Tunicamicina/farmacologia
19.
Biochem Biophys Res Commun ; 562: 9-14, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030043

RESUMO

Although the PVR/TIGIT immune checkpoint axis has been suggested as a promising target for cancer immunotherapy and multiple TIGIT-targeting therapies are undergoing clinical trials, the underlying regulatory mechanisms of PVR/TIGIT interaction remain inconclusive. Here we show that TIGIT N-glycosylations are critical for maintaining the interaction between TIGIT and PVR. TIGIT has two N-glycosylation residues, N32 and N101. N-glycosylation on N101 of TIGIT and, to less extent, on N32, play potent roles in PVR binding. Taken together, these findings suggest that the N-glycosylation sites on TIGIT, especially residue N101, may be potential targets for PVR/TIGIT immune checkpoint blockade.


Assuntos
Asparagina/metabolismo , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Glicosilação , Células HEK293 , Humanos , Ligação Proteica , Receptores Imunológicos/química
20.
J Med Chem ; 64(11): 7189-7209, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34029087

RESUMO

Factor inhibiting hypoxia-inducible factor (FIH) is a JmjC domain 2-oxogluarate and Fe(II)-dependent oxygenase that catalyzes hydroxylation of specific asparagines in the C-terminal transcriptional activation domain of hypoxia-inducible factor alpha (HIF-α) isoforms. This modification suppresses the transcriptional activity of HIF by reducing its interaction with the transcriptional coactivators p300/CBP. By contrast with inhibition of the HIF prolyl hydroxylases (PHDs), inhibitors of FIH, which accepts multiple non-HIF substrates, are less studied; they are of interest due to their potential ability to alter metabolism (either in a HIF-dependent and/or -independent manner) and, provided HIF is upregulated, to modulate the course of the HIF-mediated hypoxic response. Here we review studies on the mechanism and inhibition of FIH. We discuss proposed biological roles of FIH including its regulation of HIF activity and potential roles of FIH-catalyzed oxidation of non-HIF substrates. We highlight potential therapeutic applications of FIH inhibitors.


Assuntos
Oxigenases de Função Mista/metabolismo , Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Asparagina/metabolismo , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Proteínas I-kappa B/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Oxigênio/química , Proteínas Repressoras/antagonistas & inibidores , Especificidade por Substrato , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismo
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