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1.
Arch Biochem Biophys ; 679: 108220, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31812669

RESUMO

Quiescent and contractile VSMC can switch to proliferative and migratory phenotype in response to growth factors and cytokines, an effect underscored by Nox family NADPH oxidases, particularly Nox1. We previously showed that quiescin/sulfhydryl oxidase 1 (QSOX1) has a role in neointima formation in balloon-injured rat carotid. Here, we investigated the intracellular redox mechanisms underlying these effects in primary VSMC. Our results show that exogenous incubation with wild type QSOX1b (wt QSOX), or with secreted QSOX1, but not with the inactive C452S QSOX 1b (C452S QSOX) or secreted inactive C455S QSOX1, induces VSMC migration and chemotaxis. PEG-catalase (PEG-CAT) prevented, while PEG-superoxide dismutase (PEG-SOD) increased migration induced by wt QSOX. Moreover, wt QSOX-induced migration was abrogated in NOX1-null VSMC. In contrast, both wt QSOX and C452S QSOX, and both secreted QSOX1 and C455S QSOX1, induce cell proliferation. Such effect was unaltered by PEG-CAT, while being inhibited by PEG-SOD. However, QSOX1-induced proliferation was not significantly affected in NOX1-null VSMC, compared with WT VSMC. These results indicate that hydrogen peroxide and superoxide mediate, respectively, migration and proliferation. However, Nox1 was required only for QSOX1-induced migration. In parallel, QSOX1-induced proliferation was independent of its redox activity, although mediated by intracellular superoxide.


Assuntos
Movimento Celular , Músculo Liso Vascular/citologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Animais , Proliferação de Células , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Espaço Intracelular/metabolismo , Camundongos , NADPH Oxidase 1/metabolismo , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo
2.
J Exp Med ; 217(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31658986

RESUMO

Plasmodium infection in Anopheles is influenced by mosquito-derived factors. We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9). Here, we show that female mosaic mosGILT mutant mosquitoes display defects in ovarian development and refractoriness to Plasmodium. Following infection by either Plasmodium berghei or Plasmodium falciparum, mutant mosquitoes have significantly reduced oocyst numbers as a result of increased thioester-containing protein 1 (TEP1)-dependent parasite killing. Expression of vitellogenin (Vg), the major yolk protein that can reduce the parasite-killing efficiency of TEP1, is severely impaired in mutant mosquitoes. MosGILT is a mosquito factor that is essential for ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity.


Assuntos
Anopheles/genética , Anopheles/parasitologia , Malária/parasitologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Culicidae/genética , Feminino , Masculino , Camundongos , Mutação/genética , Plasmodium berghei/patogenicidade , Plasmodium falciparum/patogenicidade , Proteínas de Ligação a RNA/genética , Vitelogeninas/genética
3.
Ecotoxicol Environ Saf ; 187: 109819, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31654864

RESUMO

Cadmium (Cd) is a dangerous environmental pollutant with high toxicity to plants. The adenosine 5'-phosphosulfate reductase 2 (APR2) is the dominant APRs in Arabidopsis and plays an important role in reductive sulfate assimilation pathway. However, whether the involvement of plant APRs in Cd stress response is largely unclear. Herein, we report that APR2 functions in Cd accumulation and tolerance in Arabidopsis. The transcript levels of APR2 were markedly induced by Cd exposure. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, exogenous GSH application effectively rescued Cd hypersensitivity in APR2-knockout plants. Further analysis showed that buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) treatment completely eliminated the enhanced Cd tolerance phenotypes of APR2-overexpressing plants, implying that APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesis-related genes under Cd stress. Taken together, our results indicated that APR2 regulated Cd accumulation and tolerance possibly through modulating GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. APR2 could be exploited for engineering heavy metal-tolerant plants in phytoremediation.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/efeitos dos fármacos , Cádmio/toxicidade , Glutationa/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais
4.
Biomed Res Int ; 2019: 3869825, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31815134

RESUMO

Flavoproteins and their interacting proteins play important roles in mitochondrial electron transport, fatty acid degradation, and redox regulation. However, their clinical significance and function in esophageal squamous cell carcinoma (ESCC) are little known. Here, using survival analysis and machine learning, we mined 179 patient expression profiles with ESCC in GSE53625 from the Gene Expression Omnibus (GEO) database and constructed a signature consisting of two flavoprotein genes (GPD2 and PYROXD2) and four flavoprotein interacting protein genes (CTTN, GGH, SRC, and SYNJ2BP). Kaplan-Meier analysis revealed the signature was significantly associated with the survival of ESCC patients (mean survival time: 26.77 months in the high-risk group vs. 54.97 months in the low-risk group, P < 0.001, n = 179), and time-dependent ROC analysis demonstrated that the six-gene signature had good predictive ability for six-year survival for ESCC (AUC = 0.86, 95% CI: 0.81-0.90). We then validated its prediction performance in an independent set by RT-PCR (mean survival: 15.73 months in the high-risk group vs. 21.1 months in the low-risk group, P=0.032, n = 121). Furthermore, RNAi-mediated knockdown of genes in the flavoprotein signature led to decreased proliferation and migration of ESCC cells. Taken together, CTTN, GGH, GPD2, PYROXD2, SRC, and SYNJ2BP have an important clinical significance for prognosis of ESCC patients, suggesting they are efficient prognostic markers and potential targets for ESCC therapy.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Algoritmos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sobrevida , Transcriptoma , Cicatrização
5.
Mol Cells ; 42(12): 893-905, 2019 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-31822044

RESUMO

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion processes that closely related to their function. Disruption of mitochondrial dynamics has been demonstrated in acute kidney injury (AKI), which could eventually result in cell injury and death. Previously, we reported that augmenter of liver regeneration (ALR) alleviates renal tubular epithelial cell injury. Here, we gained further insights into whether the renoprotective roles of ALR are associated with mitochondrial dynamics. Changes in mitochondrial dynamics were examined in experimental models of renal ischemia-reperfusion (IR). In a model of hypoxia-reoxygenation (HR) injury in vitro , dynamin-related protein 1 (Drp1) and mitochondrial fission process protein 1 (MTFP1), two key proteins of mitochondrial fission, were downregulated in the Lv-ALR + HR group. ALR overexpression additionally had an impact on phosphorylation of Drp1 Ser637 during AKI. The inner membrane fusion protein, Optic Atrophy 1 (OPA1), was significantly increased whereas levels of outer membrane fusion proteins Mitofusin-1 and -2 (Mfn1, Mfn2) were not affected in the Lv-ALR + HR group, compared with the control group. Furthermore, the mTOR/4E-BP1 signaling pathway was highly activated in the Lv-ALR + HR group. ALR overexpression led to suppression of HR-induced apoptosis. Our collective findings indicate that ALR gene transfection alleviates mitochondrial injury, possibly through inhibiting fission and promoting fusion of the mitochondrial inner membrane, both of which contribute to reduction of HK-2 cell apoptosis. Additionally, fission processes are potentially mediated by promoting tubular cell survival through activating the mTOR/4E-BP1 signaling pathway.


Assuntos
Túbulos Renais/patologia , Rim/lesões , Dinâmica Mitocondrial , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose , Linhagem Celular Transformada , Expressão Gênica , Humanos , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais
7.
Emerg Microbes Infect ; 8(1): 1511-1523, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31631785

RESUMO

Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Febre Lassa/imunologia , Vírus Lassa/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina L/genética , Catepsina L/imunologia , Linhagem Celular , Ebolavirus/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Febre Lassa/genética , Febre Lassa/virologia , Vírus Lassa/genética , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/virologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Replicação Viral
8.
Endocrinology ; 160(11): 2543-2555, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504396

RESUMO

A defining characteristic of the hypothalamus-pituitary-gonad reproductive endocrine axis is the episodic secretion of the pituitary gonadotropin hormones LH and FSH by the anterior pituitary gonadotropes. Hormone secretion is dictated by pulsatile stimulation, with GnRH released by hypothalamic neurons that bind and activate the G protein-coupled GnRH receptor expressed by gonadotropes. Hormone secretion and synthesis of gonadotropins are influenced by the amplitude and frequency of GnRH stimulation; variation in either affects the proportion of LH and FSH secreted and the differential regulation of hormone subunit gene expression. Therefore, proper decoding of GnRH signals is essential for appropriate gonadotropin synthesis and secretion. The GnRH receptor robustly activates downstream signaling cascades to facilitate exocytosis and stimulate gene expression and protein synthesis. It is necessary to rapidly quench signaling to preserve sensitivity and adaptability to changing pulse patterns. Reactive oxygen species (ROS) generated by receptor-activated oxidases fulfill the role of rapid signaling intermediates that facilitate robust and transient signaling. However, excess ROS can be detrimental and, unchecked, can confuse signal interpretation. We demonstrate that sulfiredoxin (SRXN1), an ATP-dependent reductase, is essential for normal responses to GnRH receptor signaling and plays a central role in resolution of ROS induced by GnRH stimulation. SRXN1 expression is mitogen-activated protein kinase dependent, and knockdown reduces Lhb and Fshb glycoprotein hormone subunit mRNA and promoter activity. Loss of SRXN1 leads to increased basal and GnRH-stimulated ROS levels. We conclude that SRXN1 is essential for normal responses to GnRH stimulation and plays an important role in ROS management.


Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peroxirredoxinas/metabolismo , Animais , Linhagem Celular , Sistema de Sinalização das MAP Quinases , Camundongos , NADPH Oxidases/metabolismo , Oxirredução
9.
J Mol Model ; 25(10): 308, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31502063

RESUMO

In the last years, H2S has been recognized as a signaling molecule in mammals, which can synthesize and catabolize (by oxidation) such species. The latter process is accelerated by a sulfide:quinone oxidoreductase (SQR, E.C. 1.8.5.4), a flavin-dependent sulfide oxidase (FDSO). FDSOs catalyze electron transfer from H2S to an acceptor in catalytic cycles involving two phases: (I) reduction of FAD by H2S (SH-) and (II) electron transfer from FADH- to the electron acceptor. The first step of FAD reduction consists on the reaction of SH- with a catalytic disulfide at the active site of the enzyme, to yield a thiolate and a persulfide in the protein. This step is ca. 106 times faster than the analogous reaction with low-molecular-weight disulfides (LMWDs) and the causes of such extraordinary acceleration remain unknown. Using the IEF-PCM(ε ≈ 10)/M06-2X-D3/6-31+G(d,p) level of theory, we have modeled the reaction of SH- with a disulfide as located in a representative model of the active site extracted from a prokaryotic SQR, assessing the effects of partial covalent interactions (PCIs) between the leaving sulfur atom and flavin ring on the activation Gibbs free-energy barrier at 298 K (∆‡G298K). To also evaluate the importance of entropic penalties on the first step, we have modeled at the same level of theory the reaction of (bis)hydroxyethyl disulfide in aqueous solution, a LMWD for which experimental data is available. Our results show that PCIs between the leaving sulfur atom and the flavin group only have a minor effect (∆‡G298K reduced by 1.6 kcal mol-1) while compensating entropic penalties could have a much larger effect (up to 8.3 kcal mol-1). Finally, we also present here a first model of some of further steps in the phase I of the catalytic cycle as in mammalian FDSOs, providing some light about their detailed mechanism. Graphical abstract .


Assuntos
Teoria da Densidade Funcional , Flavinas/metabolismo , Sulfeto de Hidrogênio/metabolismo , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Acidithiobacillus/enzimologia , Biocatálise , Domínio Catalítico , Dissulfetos/metabolismo , Entropia , Oxirredução
10.
PLoS Comput Biol ; 15(8): e1007207, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31442220

RESUMO

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.


Assuntos
Afinidade de Anticorpos , Desenho de Fármacos , Região Variável de Imunoglobulina/genética , Engenharia de Proteínas/métodos , Animais , Afinidade de Anticorpos/genética , Biologia Computacional , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/estatística & dados numéricos , Estabilidade Proteica , Software , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia
11.
ACS Chem Biol ; 14(9): 1981-1989, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31449382

RESUMO

The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the ß- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the ß-carbon of Asp (S-Cß). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Biossíntese Peptídica/fisiologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Paenibacillus polymyxa/enzimologia , Peptídeos/química , Especificidade por Substrato
12.
Cells ; 8(8)2019 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-31382676

RESUMO

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.


Assuntos
Acetilcisteína/farmacologia , Neoplasias do Colo/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Sulfurtransferases/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Depuradores de Radicais Livres/farmacologia , Humanos
13.
Methods Mol Biol ; 2033: 131-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332752

RESUMO

As a critical feature of the next generation of antibody-drug conjugates (ADCs), site-specific bioconjugation approaches can help to optimize stability, pharmacokinetics, efficacy, and safety as well as improve manufacturing consistency. The SMARTag® technology platform offers a practical and efficient chemoenzymatic solution for site-specific protein modifications. A bioorthogonal aldehyde handle is introduced through the oxidation of a cysteine residue, embedded in a specific peptide sequence (CxPxR), to the aldehyde-bearing formylglycine (fGly). This enzymatic modification is carried out by the formylglycine-generating enzyme (FGE). The broad recognition of this short sequence by FGE within the context of heterologous proteins allows for the introduction of fGly residues at chosen sites in proteins expressed in prokaryotic and eukaryotic systems. The protocol presented here describes the methods for expressing fGly-containing antibodies in eukaryotic cells and subsequent site-specific conjugation with a payload-linker using aldehyde-specific Hydrazino-Iso-Pictet-Spengler (HIPS) chemistry.


Assuntos
Imunoconjugados/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Engenharia de Proteínas/métodos , Proteínas/química , Aldeídos/química , Glicina/análogos & derivados , Humanos , Imunoconjugados/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Peptídeos/química , Peptídeos/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/genética
14.
Biochem J ; 476(13): 1955-1956, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308158

RESUMO

Recently, Guenter Schwarz and colleagues published an elegant study in the Biochemical Journal (2019) 476, 1805-1815 which combines kinetic and spectroscopic studies with protein engineering to provide a mechanism for sulfite oxidase (SO)-catalyzed nitrite reduction that yields nitric oxide (NO). This work is noteworthy as it demonstrates that (i) for NO generation, both sulfite and nitrite must bind to the same molybdenum (Mo) center; (ii) upon sulfite reduction, Mo is reduced from +6 (MoVI) to +4 (MoIV) and MoIV reduces nitrite to NO yielding MoV; (iii) the heme moiety, linked to the Mo-center by an 11 amino acid residue tether, gets reduced by intramolecular electron transfer (IET) resulting in MoV being oxidized to MoVI; (iv) the reduced heme transfers its electron to a second nitrite molecule converting it to NO; (v) the authors demonstrate steady-state NO production in the presence of the natural electron acceptor cytochrome c; (vi) Finally, the authors use protein engineering to shorten the heme tether to reduce the heme-Mo-center distance with the aim of increasing NO production. Consequently, the rate of IET to cytochrome c is decreased but the enzymatic turnover rate for NO production is increased by ∼10-fold. This paper is unique as it provides strong evidence for a novel mechanism for steady-state NO production for human mitochondrial SO and serves as a potential template for studying NO production mechanisms in other enzymes by integrating the information gained from enzyme kinetics with EPR and UV/vis spectroscopy and protein engineering.


Assuntos
Sulfito Oxidase , Catálise , Humanos , Cinética , Molibdênio , Óxido Nítrico , Nitritos , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre
15.
Molecules ; 24(11)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167508

RESUMO

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of ß2-microglobulin (ß2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of ß2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove ß2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with ß2-m. In this study, we modified the anti-ß2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of ß2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed ß2-m from blood, therefore opening a new avenue for efficient hemodialysis.


Assuntos
Imunoadsorventes , Anticorpos de Domínio Único , Microglobulina beta-2 , Adsorção , Catálise , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoadsorventes/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Diálise Renal/métodos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Microglobulina beta-2/imunologia
16.
Biochem J ; 476(12): 1805-1815, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31167903

RESUMO

In addition to nitric oxide (NO) synthases, molybdenum-dependent enzymes have been reported to reduce nitrite to produce NO. Here, we report the stoichiometric reduction in nitrite to NO by human sulfite oxidase (SO), a mitochondrial intermembrane space enzyme primarily involved in cysteine catabolism. Kinetic and spectroscopic studies provide evidence for direct nitrite coordination at the molybdenum center followed by an inner shell electron transfer mechanism. In the presence of the physiological electron acceptor cytochrome c, we were able to close the catalytic cycle of sulfite-dependent nitrite reduction thus leading to steady-state NO synthesis, a finding that strongly supports a physiological relevance of SO-dependent NO formation. By engineering SO variants with reduced intramolecular electron transfer rate, we were able to increase NO generation efficacy by one order of magnitude, providing a mechanistic tool to tune NO synthesis by SO.


Assuntos
Proteínas Mitocondriais/química , Óxido Nítrico/química , Nitritos/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico/genética , Nitritos/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo
17.
Methods Mol Biol ; 2012: 63-81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31161504

RESUMO

Use of the formylglycine generating enzyme (FGE)-a copper-dependent posttranslational protein modifier-represents a particularly elegant method taken directly from nature of introducing a unique amino acid into the larger context of a protein. Formylglycine (fGly) is a crucial component of the active site of sulfatases, where it directly participates in the breakdown of sulfate ester substrates. In the context of bioconjugation this aldehyde containing amino acid can be an invaluable reactive handle for the chemical conjugation of molecules. Here we describe a detailed method for generating formylglycine-containing proteins in a mammalian system developed specifically for the production of antibody-drug conjugates (ADCs) but applicable to a wide range of proteins.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Proteínas/química , Coloração e Rotulagem , Sequência de Aminoácidos , Aminoácidos/química , Sequência Consenso , Humanos , Imunoconjugados/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade
18.
Mitochondrion ; 47: 114-124, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31170524

RESUMO

Pyridine Nucleotide-Disulphide Oxidoreductase Domain 2 (PYROXD2), a Hepatitis B virus X protein (HBx)-interacting protein, is significantly down-regulated in hepatocellular carcinoma (HCC), however its exact biological function remains unclear. The aim of this study is to investigate the subcellular localization and biological function of PYROXD2 in hepatic cells. The results showed that PYROXD2 was imported to the mitochondrial inner membrane/matrix by Tom40 and Tim23, but not Mia40. PYROXD2 151-230aa might be the mitochondrial targeting sequence. PYROXD2 interacted with complex IV subunit COX5B. Knockout of PYROXD2 decreased MMP, intracellular ROS, complex IV activity, cell proliferation, ATP content and mtDNA copy number, but increased mtROS levels and the number of immature mitochondria. In summary, our data illustrated that PYROXD2 localizes to the mitochondrial inner membrane/matrix, and it plays important roles in regulating mitochondrial function.


Assuntos
Mitocôndrias Hepáticas/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Hep G2 , Humanos , Mitocôndrias Hepáticas/genética , Proteínas Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Espécies Reativas de Oxigênio/metabolismo
19.
PLoS One ; 14(6): e0214534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166951

RESUMO

OBJECTIVE: To study the role of miRNA-181a and augmenter of liver regeneration in TGF-ß-induced fibrosis in hepatic stellate cells. METHODS: LX2 cells were treated with 20 ng/ml TGF-ß for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-ß receptor II (TGFß-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and ß-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA. RESULTS: TGF-ß induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFß-RII and increased expression E-cadherin. CONCLUSION: TGF-ß induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-ß action by decreasing the expression of TGFß-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.


Assuntos
Células Estreladas do Fígado/citologia , Cirrose Hepática/genética , MicroRNAs/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fator de Crescimento Transformador beta/farmacologia , Actinas/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Colágeno Tipo I/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
20.
Diabetes Care ; 42(8): 1414-1421, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31152121

RESUMO

OBJECTIVE: Type 1 diabetes (T1D) is a highly heritable disease with much lower incidence but more adult-onset cases in the Chinese population. Although genome-wide association studies (GWAS) have identified >60 T1D loci in Caucasians, less is known in Asians. RESEARCH DESIGN AND METHODS: We performed the first two-stage GWAS of T1D using 2,596 autoantibody-positive T1D case subjects and 5,082 control subjects in a Chinese Han population and evaluated the associations between the identified T1D risk loci and age and fasting C-peptide levels at T1D diagnosis. RESULTS: We observed a high genetic correlation between children/adolescents and adult T1D case subjects (r g = 0.87), as well as subgroups of autoantibody status (r g ≥ 0.90). We identified four T1D risk loci reaching genome-wide significance in the Chinese Han population, including two novel loci, rs4320356 near BTN3A1 (odds ratio [OR] 1.26, P = 2.70 × 10-8) and rs3802604 in GATA3 (OR 1.24, P = 2.06 × 10-8), and two previously reported loci, rs1770 in MHC (OR 4.28, P = 2.25 × 10-232) and rs705699 in SUOX (OR 1.46, P = 7.48 × 10-20). Further fine mapping in the MHC region revealed five independent variants, including another novel locus, HLA-C position 275 (omnibus P = 9.78 × 10-12), specific to the Chinese population. Based on the identified eight variants, we achieved an area under the curve value of 0.86 (95% CI 0.85-0.88). By building a genetic risk score (GRS) with these variants, we observed that the higher GRS were associated with an earlier age of T1D diagnosis (P = 9.08 × 10-11) and lower fasting C-peptide levels (P = 7.19 × 10-3) in individuals newly diagnosed with T1D. CONCLUSIONS: Our results extend current knowledge on genetic contributions to T1D risk. Further investigations in different populations are needed for genetic heterogeneity and subsequent precision medicine.


Assuntos
Fatores Etários , Antígenos CD/genética , Butirofilinas/genética , Diabetes Mellitus Tipo 1/genética , Fator de Transcrição GATA3/genética , Loci Gênicos/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Autoanticorpos/sangue , Peptídeo C/sangue , Criança , China , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Jejum/sangue , Feminino , Estudo de Associação Genômica Ampla , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Masculino , Razão de Chances , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fatores de Risco
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