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1.
Nature ; 577(7788): 109-114, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31827280

RESUMO

Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.

2.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1607-1618, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559743

RESUMO

With the rapid development of modern biotechnology, fermentation process is increasingly important in industrial production. To guarantee the stability of products, fermentation process should be elaborately monitored and controlled. Biomass is an important parameter for on-line monitoring in bioprocesses because biomass can reflect cell growth in a bioreactor directly. In-situ microscope, a non-invasive and image-analysis based technology, can real-time monitor cells in biological process. This review summarizes the development and application of in-situ microscopy in biomass monitoring.


Assuntos
Biomassa , Microscopia , Reatores Biológicos , Biotecnologia , Fermentação
3.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(7): 843-849, 2019 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-31340919

RESUMO

OBJECTIVE: To evaluate the application of femtosecond laser technology in the management of subluxated lens. METHODS: We retrospectively analyzed the data of the patients with subluxated lens undergoing femtosecond laser- assisted surgery at the Cataract Center of Guangzhou Aier Eye Hospital between March, 2017 and May, 2019. The LenSx femtosecond laser-assisted cataract surgery system was used to perform capsulotomy and lens fragmentation. According to the patients' eye condition, anterior vitrectomy was performed and capsular retractors was used. After phacoemulsification, I/A and insertion of the tension rings, the intraocular lens (IOL) was implanted into the capsular bag. The perioperative data, complications, visual acuity and intraocular pressure after the operation were recorded, and the stability of the capsular bag and IOLs were assessed. RESULTS: We analyzed the data of 25 cases (29 eyes) of subluxated lens, including 16 (16 eyes; 55.17%) as the result of traumatic lens subluxation, 5 (9 eyes; 31.03%) of Mafan syndromes, 1 case (1 eye; 3.45%) of high myopia and 3 cases (3 eyes; 10.34%) of unknown causes. Thirteen 13 eyes (44.83%) showed mild subluxation, 7 (24.14%) had moderate subluxation, and 9 (31.03%) had severe subluxation. Femtosecond laser- assisted capsulorhexis, lens fragmentation and phacoemulsification were successfully completed for 29 eyes, of which 28 eyes (96.55%) retained the complete capsular bag and with successful implantation of the capsular tension devices and IOLs. Nine eyes (31.03%) were treated with anterior segment vitrectomy; iris hooks were used for 2 eyes (6.90%) and capsular bag hooks for 9 eyes (31.03%). The best corrected visual acuity was significantly improved in 29 eyes after operation (P < 0.05). At 1 month after the surgery, 26 eyes (89.66%) showed stably centered IOLs, 2 eyes (6.90%) showed slight tilt of the IOLs, and 3 eyes (10.34%) had anterior capsular contraction. The intraoperative complications included subconjunctival hemorrhage (75.87%), incomplete capsulotomy (17.24%) and contracted pupils (13.79%). CONCLUSIONS: The application of femtosecond laser assisted technology enhances the surgical safety and effectiveness for subluxated lens, facilitates the choice of individualized surgical options, and promotes maximum recovery of the patients' visual function.


Assuntos
Terapia a Laser , Subluxação do Cristalino , Lentes Intraoculares , Humanos , Implante de Lente Intraocular , Subluxação do Cristalino/cirurgia , Facoemulsificação , Estudos Retrospectivos
4.
Int J Ophthalmol ; 12(7): 1122-1126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341802

RESUMO

AIM: To observe the changes in ocular surface and the dry eye symptoms following femtosecond laser-assisted cataract surgery (FLACS). METHODS: Patients with no eye signs or symptoms in Guangzhou Aier Eye Hospital between October 2017 and September 2018, who underwent FLACS and intraocular lens (IOL) implantation for age-related cataract were enrolled. Tear film stability assessed with OCULUS Keratograph 5M, Schirmer's I test (SIT), and corneal fluorescein staining (CFS) were evaluated before and after surgery at 1d, 1wk, 1, and 3mo in order. Ocular Surface Disease Index scores (OSDI) and Subjective Symptom Questionnaires (SSQs) were recorded at the same time point. RESULTS: Thirty-eight eyes of 38 patients were enrolled. The noninvasive tear film break-up time (first break-up time and average break-up time) decreased in a peak at the 1wk visit, and then increased to basic levels at 1mo. The tear meniscus height (TMH) increased transiently at 1d, and declined in the following 3mo visits. The SIT had a transient increase at 1d (P=0.357) and a decrease at 1wk and 1mo (both P<0.05) but returned to the preoperative levels at 3mo after surgery (P=0.062). CFS scores were significantly improved compared with those before surgery, and had a statistical difference (P<0.05). OSDI scores and SSQs after surgery were obviously higher, and had a statistical difference (P<0.001) but didn't return to the basic level by 3mo. CONCLUSION: Dry eye signs and symptoms can occur immediately following FLACS and have a peak severity on day 7 postoperatively. Most signs of dry eye can return to preoperative basic levels within 3mo postoperatively. However, all cases can not recover from CFS and dry eye symptoms at 3mo postoperatively.

5.
Appl Microbiol Biotechnol ; 103(14): 5617-5626, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104100

RESUMO

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L-1. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and eep of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor.


Assuntos
Amidoidrolases/metabolismo , Aminoidrolases/metabolismo , Arabis/enzimologia , Pregabalina/metabolismo , Aminoidrolases/genética , Arabis/genética , Biotransformação , Catálise , Enzimas , Escherichia coli/genética , Hidrólise , Cinética , Mutação , Pantoea/enzimologia , Especificidade por Substrato
6.
Proc Natl Acad Sci U S A ; 116(8): 2996-3005, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718432

RESUMO

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.


Assuntos
Autofagia/genética , Glutationa Peroxidase/genética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Chaperonas Moleculares/genética , Necrose/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Caspases/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Ferro/metabolismo , Ligantes , Peróxidos Lipídicos/genética , Peróxidos Lipídicos/metabolismo , Chaperonas Moleculares/metabolismo , Piperazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética
7.
Bioresour Technol ; 274: 371-378, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30544042

RESUMO

To develop a highly efficient method for aprepitant chiral intermediate (S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu2+-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.2% activity recovery. The immobilized amidase exhibited excellent thermal stability with the longest half-life of 1456.8 h at 40 °C ever reported. (S)-4-fluorophenylglycine was continuously produced using the reaction system with 49.9% conversion, 99.9% ee, and an outstanding space-time yield of 5.29 kg L-1 d-1. Moreover, the efficient reaction system exhibited a high operational stability and retained 86.3% catalytic activity after 25-day continuous operation. This efficient continuous bioprocess presents great industrial potential for large-scale production of (S)-4-fluorophenylglycine.


Assuntos
Amidoidrolases/metabolismo , Aprepitanto/metabolismo , Reatores Biológicos , Enzimas Imobilizadas/metabolismo
8.
Appl Environ Microbiol ; 85(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578259

RESUMO

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. (Pa-Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with k cat/Km values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 gproduct liter-1 day-1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships.IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa-Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa-Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa-Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Niacinamida/análogos & derivados , Niacinamida/biossíntese , Pantoea/enzimologia , Engenharia de Proteínas , Amidoidrolases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotransformação , Catálise , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação
10.
BMC Ophthalmol ; 18(1): 81, 2018 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-29566650

RESUMO

BACKGROUND: To evaluate the changes of choroidal vascular structures in patients after phacoemulsification surgery. METHODS: A self-control study was conducted on 36 eyes of 36 patients who had uneventful phacoemulsification. Choroidal images were acquired preoperatively, 7 days (D7), 1 month (M1), and 3 months (M3) after surgery from enhanced depth imaging (EDI) optical coherence tomography (OCT) scans. Choroidal vascularity index (CVI) was used to assess vascular status of the choroid using image binarization by the Niblack method. The postoperative values of mean CVI were compared with baseline by paired t-test. Univariate and multiple linear regression analyses were performed to determine the associations between CVI and other factors. RESULTS: The mean age of the recruited patients was 63.1 ± 6.9 years. The mean CVI at baseline was 60.1 ± 5.5%. After surgery, the CVI significantly increased to 61.7 ± 5.3% at D7, 63.6 ± 4.4% at M1 and 64.8 ± 4.0% at M3 (p = 0.035, 0.0006, < 0.0001, respectively). Univariate and multiple regression analysis revealed a positive association between CVI and subfoveal choroidal thickness (SFCT) at pre-operation and no significant association with age, axial length (AL), intraocular pressure (IOP) and gender at all timepoints. CONCLUSIONS: Phacoemulsification induced increased CVI in patients diagnosed with cataract. Evaluation of the long-term change of CVI following surgery may provide valuable information for studying the relationship between phacoemulsification and disorders of the choroid.


Assuntos
Corioide/irrigação sanguínea , Facoemulsificação/efeitos adversos , Vasos Retinianos/diagnóstico por imagem , Idoso , Comprimento Axial do Olho , Feminino , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Tomografia de Coerência Óptica/métodos
11.
J Biotechnol ; 266: 20-26, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29217400

RESUMO

As the important chiral building block of levetiracetam, the synthesis of (S)-2-aminobutanamide has attracted a great deal of attention. The d-aminopeptidase catalyzed kinetic resolution of 2-aminobutanamide was demonstrated as an effective strategy for (S)-2-aminobutanamide production. In this study, a novel d-aminopeptidase from Brucella sp. (Bs-Dap) was screened and systematically characterized. The enzyme exhibited maximum activity at 45°C, pH 8.0 and it showed relatively low Km value toward 2-aminobutanamide, indicating its high affinity to the substrate. Kinetic resolution of 300g/L 2-aminobutanamide by recombinant Escherichia coli whole cells (4g/L wet cell weight) resulted in 50% conversion and >99% e.e. within 80min. The catalytic properties of Bs-Dap demonstrated its great potential for industrial production of (S)-2-aminobutanamide.


Assuntos
Amidas/síntese química , Aminopeptidases/química , Proteínas de Bactérias/química , Brucella/enzimologia , Catálise
12.
Bioorg Chem ; 76: 81-87, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29153589

RESUMO

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Niacinamida/análogos & derivados , Ácidos Nicotínicos/síntese química , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Biocatálise , Domínio Catalítico , Técnicas de Química Sintética , Ensaios Enzimáticos , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Niacinamida/química , Pantoea/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
Genes Dev ; 31(11): 1162-1176, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701375

RESUMO

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.


Assuntos
Proteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/genética , Animais , Apoptose/genética , Células Cultivadas , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases/genética , Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/genética
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 37(7): 933-937, 2017 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-28736371

RESUMO

OBJECTIVE: To evaluate the changes in retinal functions using multifocal electroretinography (mfERG) following intravitreal injection of Lucentis for treatment of wet age-related macular degeneration. METHODS: This prospective study was conducted in 14 patients (9 men and 5 women, 14 eyes) with wet age-related macular degeneration receiving treatment with intravitreal injections of ranibizumab (Lucentis) in our hospital between October, 2014 and January, 2016. All the patients received the treatment following a 1+PRN protocol and after the initial injection, the patients were followed up monthly for 6 months to decide if additional injections were needed. The corrected visual acuity and mfERG findings of the patients were assessed before and at l, 3 and 6 months after the initial injection. RESULTS: At the last follow-up, the patients received injections for a mean of 2.86∓1.58 times. The best corrected visual acuity (BCVA) at 1 month after the initial treatment was not significantly different from that before treatment (P=0.07), but showed significant improvements at 3 and 6 months (P<0.05). In mfERG, the implicit time of the 6 rings showed no significant decrease after the treatment, but the amplitude density of P1 and N1 in rings 1 and 2 improved significantly at 1, 3, and 6 months after the initial injection (P<0.05). CONCLUSION: Multifocal electroretinography can serve as a useful modality for evaluating visual function changes in patients receiving intravitreal injection of Lucentis for wet age-related macular degeneration.

15.
Protein Expr Purif ; 129: 60-68, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27640050

RESUMO

Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli BL21, purified and functionally characterized. The optimal pH and temperature for Pl-Ami were 9.5 and 45 °C, respectively. Pl-Ami preferred long chain aliphatic amides as substrates, while no activity was detected towards aromatic, heterocyclic and other amides. The highest enzyme activity of 128 U/mg was obtained when hexanoamide was used as substrate. Kinetic analysis indicated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover number (kcat) was higher with long chain aliphatic amides as substrates. The obtained results provided a distinct understanding of substrate specificity of AS family amidases.


Assuntos
Alphaproteobacteria/genética , Amidoidrolases , Proteínas de Bactérias , Clonagem Molecular , Alphaproteobacteria/enzimologia , Amidoidrolases/biossíntese , Amidoidrolases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
16.
Appl Microbiol Biotechnol ; 101(5): 1953-1964, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27832306

RESUMO

Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM-1 s-1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.


Assuntos
Amidoidrolases/genética , Amidoidrolases/metabolismo , Burkholderia/enzimologia , Hidroxibutiratos/metabolismo , Proteínas Recombinantes/metabolismo , Biocatálise , Burkholderia/genética , Burkholderia/metabolismo , Clonagem Molecular , Cobalto/metabolismo , Cinética , Proteínas Recombinantes/genética , Especificidade por Substrato
17.
Enzyme Microb Technol ; 86: 93-102, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26992798

RESUMO

Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates. Dt-Ami 2 showed maximum activity near a quite alkaline pH (11.0) and exhibited opposite enantioselectivity to Dt-Ami 6. Furthermore, a novel bioprocess for hydrolysis of 1-cyanocyclohexaneacetamide was developed using Dt-Ami 6 as biocatalyst, resulting in >99% conversion within 1.5h at a substrate loading of 100g/L by 0.5g/L of Escherichia coli cells. On the other hand, nitrilase superfamily amidase only hydrolyzed aliphatic amides. The Km values of Dt-Ami 7 were considerably increased with the extension of chain length of aliphatic amides. The characterized enzymes from different families showed distinct biochemical characteristics and catalytic properties, leading to a better understanding of the two super amidase family members.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Delftia/enzimologia , Amidas/química , Amidas/metabolismo , Amidoidrolases/classificação , Amidoidrolases/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Clonagem Molecular , Delftia/genética , Estabilidade Enzimática , Genes Bacterianos , Cinética , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato
18.
Autophagy ; 11(4): 617-28, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25906440

RESUMO

ISG15 (ISG15 ubiquitin-like modifier), a ubiquitin-like protein, is one of the major type I IFN (interferon) effector systems. ISG15 can be conjugated to target proteins (ISGylation) via the stepwise action of E1, E2, and E3 enzymes. Conjugated ISG15 can be removed (deISGylated) from target proteins by USP18 (ubiquitin-specific peptidase 18). Here we investigated the role of deISGylation by USP18 in regulating autophagy and EGFR degradation in cells treated with type I IFNs. We show that type I IFN induced expression of ISG15 leads to ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 which competes with Lys63 ubiquitination of BECN1. We demonstrate that ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 serve an important role in negative regulation of intracellular processes including autophagy and EGFR degradation that are critically dependent upon the activity of class III PtdIns 3-kinase. Our studies provide fundamental new mechanistic insights into the innate immunity response implemented by type I IFNs.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Citocinas/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitinas/metabolismo , Proteína Beclina-1 , Humanos , Imunidade Inata , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Ubiquitinação/fisiologia
19.
Protein Expr Purif ; 101: 1-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24859676

RESUMO

A novel esterase encoding gene, tle, was cloned from the thermophilic fungus Thermomyces lanuginosus DSM 10635. The tle had an open reading frame of 945bp encoding TLE of 314 amino acids with a theoretical molecular mass of 34.5kDa. The putative catalytic triad of TLE was consisted of Ser151, His279, and Asp249. TLE was heterologously expressed in Escherichia coli in biologically active form and purified to homogeneity. Several biochemical properties of TLE were studied: Among the tested p-nitrophenol esters, TLE showed the highest hydrolytic activity with p-nitrophenyl butyrate (C4) and exhibited the maximum activity at 60°C and pH 8.5. The enzyme was stable at temperatures below 60°C and retained 53% of the maximum activity after treatment at 70°C for 60min. Esterase activity was notably enhanced by addition of Ca(2+) and Ba(2+), respectively. Furthermore, TLE showed high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE), which produce a valuable chiral intermediate-(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid for Pregabalin. These unique properties of the esterase indicate that TLE is a potential candidate for industrial application.


Assuntos
Butiratos/metabolismo , Esterases/genética , Eurotiales/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Bário/química , Sequência de Bases , Cálcio/química , Caproatos/química , Domínio Catalítico/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
20.
Eye Sci ; 27(1): 25-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22447548

RESUMO

PURPOSE: To observe the efficacy of vitrectomy with internal limiting membrane (ILM) peeling combined with phacoemulsification with intraocular lens (IOL) implantation in the treatment of cataract with co-existing macular diseases. METHODS: A total of 28 cataract patients (28 eyes) with co-existing macular diseases were admitted to Aier Eye Hospital between May 2008 and May 2011. The clinical characteristics were analyzed in this study. Subjects included 6 men and 22 women, aged from 56 to 77 years (mean 64 years), with duration of disease ranging from 2 to 36 months (mean 9.3 months). All patients underwent phacoemulsification with implantation of a hydrophobic acrylic IOL into the capsular bag and pars plana vitrectomy with ILM peeling. RESULTS: Postoperatively, patients underwent 3- to 18-months of follow-up (mean 7.2 months). Only one eye had macular hole failing to close. Normal macular structure was restored in the other 27 eyes. The presenting visual acuity and best corrected visual acuity (BCVA) did not differ significantly (t=-1.724, P=0.096), with the BCVA in 27 eyes (96.4%) improving by 2 lines or more. The improvement in minimum angle of resolution (MAR) was > 0.3 in 21 eyes, ≥ 0.1 in 6 eyes and < 0.1 in 1 eye. The mean spherical equivalent (SE) was -4.67±5.98D preoperatively and -0.38±0.69D postoperatively (t=4.157, P<0.005). CONCLUSION: Combined phacovitrectomy surgery is a reliable and safe procedure in the treatment of cataract complicated by macular disease.


Assuntos
Membrana Basal/cirurgia , Macula Lutea/cirurgia , Facoemulsificação/métodos , Doenças Retinianas/cirurgia , Vitrectomia/métodos , Idoso , Catarata/complicações , Terapia Combinada/métodos , Feminino , Humanos , Implante de Lente Intraocular/métodos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Doenças Retinianas/complicações , Perfurações Retinianas , Resultado do Tratamento , Acuidade Visual
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