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1.
Protein Expr Purif ; 197: 106110, 2022 09.
Article in English | MEDLINE | ID: mdl-35577182

ABSTRACT

Interferon alpha-2b (IFNα-2b) is an essential cytokine widely used in hepatitis and cancer treatment. This paper presents a novel protocol for purification and efficient refolding of recombinant interferon alpha-2b (IFNα-2b) expressed as insoluble inclusion bodies in Escherichia coli. Purification of IFNα-2b from solubilized inclusion bodies was performed by solvent extraction using 2-butanol. Refolding conditions were optimized using the response surface method (RSM). Under optimized conditions, refolding yield of solvent-extracted IFNα-2b was 1.5 fold higher than refolding yield of unpurified IFNα-2b. High-concentration protein refolding was carried out by pulse-fed method, and refolding yield of 75% was achieved at a protein concentration of 300 µg ml-1. Under optimized conditions, 259.16 mg of purified IFNα-2b with the biological activity of 2.4 × 108 IU mg-1 was achieved per liter of bacterial culture. The developed protocol provides an efficient production process of high-quality IFNα-2b suitable for research and pharmaceutical applications.


Subject(s)
Escherichia coli , Inclusion Bodies , Escherichia coli/genetics , Escherichia coli/metabolism , Inclusion Bodies/metabolism , Interferon alpha-2/metabolism , Protein Refolding , Recombinant Proteins , Solvents
2.
Biochem J ; 478(19): 3527-3537, 2021 10 15.
Article in English | MEDLINE | ID: mdl-34523671

ABSTRACT

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme.


Subject(s)
Human Growth Hormone/metabolism , Interferon alpha-2/metabolism , N-Acetylgalactosaminyltransferases/chemistry , N-Acetylgalactosaminyltransferases/metabolism , Protein Engineering/methods , Amino Acid Sequence , Catalytic Domain , Chromatography, High Pressure Liquid/methods , Chromatography, Reverse-Phase/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Glycosylation , Human Growth Hormone/genetics , Humans , Interferon alpha-2/genetics , Isoenzymes/metabolism , Kinetics , Mucins/metabolism , N-Acetylgalactosaminyltransferases/genetics , Polysaccharides/chemistry , Polysaccharides/metabolism , Recombinant Proteins/metabolism , Sequence Alignment , Serine/metabolism , Synthetic Biology/methods , Threonine/chemistry
3.
Front Endocrinol (Lausanne) ; 11: 522340, 2020.
Article in English | MEDLINE | ID: mdl-33329376

ABSTRACT

Background: Type 2 diabetes mellitus (T2DM) is a chronic, hyperglycemia-associated, metabolic disorder. Heart disease is a major complication of T2DM. The present study aimed to explore the effects of miR-216a-3p on cardiomyocyte proliferation, apoptosis, and inflammation in T2DM through the Toll-like receptor (TLR) pathway involving interferon-α2 (IFN-α2) mediation. Methods: T2DM was induced in rats by a high-fat diet, in combination with an intraperitoneal injection of low-dose streptozotocin. ELISAs were conducted to measure inflammatory-related factors in serum. Next, isolated cardiomyocytes were used in loss- and gain-of-function experiments, followed by MTT and flow cytometry assays, conducted to evaluate cell proliferation, cell cycle, and apoptosis. Results: Our results revealed an increase in the inflammatory response in T2DM rat models, accompanied by significantly increased expression of miR-216a-3p and TLR pathway-related genes. However, a decrease in the expression of IFN-α2 was observed. Moreover, the presence of an miR-216a-3p inhibitor and si-IFN-α2 increased the expression of TLR pathway-related genes and cell apoptosis, whereas cell proliferation was significantly decreased in the cardiomyocytes. Conclusion: We found that in T2DM, miR-216a-3p inhibited the proliferation and enhanced the apoptosis of cardiomyocytes and generated an inflammatory response through activation of the TLR pathway and targeting of IFN-α2.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Inflammation/metabolism , Interferon alpha-2/metabolism , MicroRNAs/metabolism , Myocytes, Cardiac/metabolism , Animals , Apoptosis/genetics , Cell Proliferation/genetics , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Diet, High-Fat , Female , Inflammation/genetics , Inflammation/pathology , Insulin Resistance/genetics , MicroRNAs/genetics , Rats , Signal Transduction/genetics , Toll-Like Receptors/metabolism
4.
Sci Adv ; 7(1)2021 01.
Article in English | MEDLINE | ID: mdl-33187978

ABSTRACT

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.


Subject(s)
Antiviral Agents/pharmacology , Azetidines/pharmacology , COVID-19/mortality , Enzyme Inhibitors/pharmacology , Janus Kinases/antagonists & inhibitors , Liver/virology , Purines/pharmacology , Pyrazoles/pharmacology , SARS-CoV-2/pathogenicity , Sulfonamides/pharmacology , Adult , Aged , Aged, 80 and over , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Cytokine Release Syndrome , Cytokines/metabolism , Drug Evaluation, Preclinical , Female , Gene Expression Profiling , Humans , Interferon alpha-2/metabolism , Italy , Janus Kinases/metabolism , Liver/drug effects , Male , Middle Aged , Patient Safety , Platelet Activation , Proportional Hazards Models , RNA-Seq , Spain , Virus Internalization/drug effects
5.
Science ; 369(6504): 718-724, 2020 08 07.
Article in English | MEDLINE | ID: mdl-32661059

ABSTRACT

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-ß and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Interferon alpha-2/metabolism , Interferon-alpha/metabolism , Interferon-beta/metabolism , Pneumonia, Viral/immunology , Adult , Aged , Betacoronavirus/physiology , COVID-19 , Coronavirus Infections/virology , Critical Illness , Cross-Sectional Studies , Female , Gene Expression Profiling , Humans , Immunity, Innate , Inflammation , Interleukin-6/metabolism , Male , Middle Aged , NF-kappa B/metabolism , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Signal Transduction , T-Lymphocyte Subsets/immunology , Tumor Necrosis Factor-alpha/metabolism , Viral Load
6.
Int J Med Sci ; 17(10): 1458-1463, 2020.
Article in English | MEDLINE | ID: mdl-32624702

ABSTRACT

Background: There are limited data regarding the efficacy of addition of entecavir (ETV) or tenofovir disoproxil fumarate (TDF) to Peg-IFNα-2b in HBeAg positive chronic hepatitis B (CHB) patients without early response to Peg-IFNα-2b. In this study, we aimed to evaluate the efficacy of ETV and TDF in HBeAg positive CHB patients who had a poor response to Peg-INFα-2b at the end of 12 weeks of monotherapy. Methods: A total of 40 HBeAg-positive CHB patients who were naive to antiviral therapy were recruited. The patients received a subcutaneous injection of Peg-IFNα-2b (180 µg) once a week for 12 weeks. However, the patients had a poor response to Peg-INFα-2b at the end of the 12-week-period monotherapy. The patients were then divided into two therapeutic protocol groups: (1) Group A: Patients received Peg-IFNα-2b (180 µg) subcutaneously weekly and ETV (0.5 mg) orally once daily for 48 weeks; (2) Group B: Patients received Peg-IFNα-2b (180 µg) subcutaneously weekly and TDF (300 mg) orally once daily for 48 weeks. The therapeutic efficacy was evaluated. Blood samples were collected at baseline and every 12 weeks. Routine biochemical tests including ALT, AST, etc. were measured by automated biochemical technique. HBV DNA was quantified using the TaqMan PCR assay. The levels of HBsAg, HBsAb, HBeAg, HBeAb and HBcAb were measured using a commercial chemiluminescent microparticle immunoassay. Results: The HBsAg level declined rapidly in both two treatment groups during the first 12 weeks and declined gradually in the next 36 weeks. At week 48, the mean ΔHBsAg level in Peg-IFNα-2b+TDF group was significantly higher than that in Peg-IFNα-2b +ETV group (-1.799 ± 0.3063 vs. -1.078 ± 0.2028, P=0.0491). The HBeAg loss rate was significantly higher in TDF add-on group than that in ETV add-on group at week 48 (40% vs. 10%, P=0.028). At week 48, the proportions of patients with undetectable HBV DNA (<500 IU/mL) were 80% (16 out of 20) and 95% (19 out of 20) in Peg-IFNα-2b+ETV group and Peg-IFNα-2b+TDF group, respectively. Conclusions: This real world study demonstrated that the efficacy of addition of TDF to Peg-IFNα-2b is superior to the efficacy of addition of ETV to Peg-IFNα-2b in HBeAg positive CHB patients with a poor response after 12 weeks of Peg-IFNα-2b treatment alone. However, this present study also requires a larger sample size study to verify in the future.


Subject(s)
Guanine/analogs & derivatives , Hepatitis B e Antigens/metabolism , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/metabolism , Interferon alpha-2/metabolism , Interferon-alpha/metabolism , Polyethylene Glycols/metabolism , Tenofovir/therapeutic use , Adult , Antiviral Agents/therapeutic use , Erythrocytes/metabolism , Female , Guanine/therapeutic use , Humans , Immunoassay , Lymphocytes/metabolism , Male , Neutrophils/metabolism , Recombinant Proteins/metabolism , Young Adult
7.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 750-762, 2020 Apr 25.
Article in Chinese | MEDLINE | ID: mdl-32347069

ABSTRACT

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.


Subject(s)
Antiviral Agents , Interferon-alpha , Polyethylene Glycols , Transglutaminases , Animals , Humans , Interferon alpha-2/metabolism , Interferon-alpha/biosynthesis , Interferon-alpha/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Protein Structure, Secondary , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/pharmacology , Reproducibility of Results , Transglutaminases/metabolism
8.
Biotechnol J ; 15(8): e1900385, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32277577

ABSTRACT

In this study, the effectiveness of PASylation in enhancing the potency and plasma half-life of pharmaceutical proteins has been accredited as an alternative technique to the conventional methods such as PEGylation. Proline, alanine, and serine (PAS) chain has shown some advantages including biodegradability improvement and plasma half-life enhancement while lacking immunogenicity or toxicity. Although some experimental studies have been performed to find the mechanism behind PASylation, the detailed mechanism of PAS effects on the pharmaceutical proteins has remained obscure, especially at the molecular level. In this study, the interaction of interferon α-2a (IFN) and PAS chain is investigated using molecular dynamics simulation method. Several important parameters including secondary structure, root-mean-square distance, and solvent accessible surface area to investigate the stability, bioavailability, and bioactivity of the PASylated protein are studied. The results demonstrate that IFN conformation is not affected critically through PASylation while it results in improvement of the protein stability and bioactivity. Therefore, PASylation can be considered as a proper biological alternative technique to increase the plasma half-life of the biopharmaceutical proteins through enlarging apparent volume. The proposed simulation represents a computational approach that would provide a basis for the study of PASylated pharmaceutical proteins for different future applications.


Subject(s)
Interferon alpha-2 , Molecular Dynamics Simulation , Pharmaceutical Preparations , Plasma , Animals , Chemistry, Pharmaceutical , Half-Life , Humans , Interferon alpha-2/chemistry , Interferon alpha-2/metabolism , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Plasma/metabolism
9.
Eur J Pharm Biopharm ; 149: 105-112, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32035237

ABSTRACT

We recently reported the discovery of a novel protein stabilizing dipeptide, glycyl-D-asparagine, through a structure-based approach. As the starting hypothesis leading to the discovery, we postulated a stabilizing effect achieved by binding of the dipeptide to an aggregation prone region on the protein's surface. Here we present a detailed study of the interaction mechanism between the dipeptide and Interferon-alpha-2A (IFN) through the construction of a Markov state model from molecular dynamics trajectories. We identify multiple binding sites and compare these to aggregation prone regions. Additionally, we calculate the lifetime of the protein-excipient complex. If the excipient remained bound to IFN after administration, it could alter the protein's therapeutic efficacy. We establish that the lifetime of the complex between IFN and glycyl-D-asparagine is extremely short. Under these circumstances, stabilization by stoichiometric binding is consequently no impediment for a safe use of an excipient.


Subject(s)
Dipeptides/metabolism , Excipients/chemistry , Interferon alpha-2/metabolism , Binding Sites , Markov Chains , Molecular Dynamics Simulation
10.
Enzyme Microb Technol ; 128: 49-58, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31186110

ABSTRACT

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.


Subject(s)
Gene Expression , Immunologic Factors/metabolism , Interferon alpha-2/metabolism , Metabolic Engineering/methods , Pichia/metabolism , Animals , Glycosylation , Half-Life , Hepacivirus/drug effects , Hepatitis E virus/drug effects , Humans , Immunologic Factors/chemistry , Immunologic Factors/pharmacokinetics , Immunologic Factors/pharmacology , Interferon alpha-2/chemistry , Interferon alpha-2/pharmacokinetics , Interferon alpha-2/pharmacology , Pichia/genetics , Plasma/chemistry , Polysaccharides/analysis , Rats, Wistar , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Virus Replication/drug effects
11.
J Appl Microbiol ; 126(5): 1438-1453, 2019 May.
Article in English | MEDLINE | ID: mdl-30776176

ABSTRACT

AIMS: The present study was aimed at design of experiments (DoE)- and artificial intelligence-based culture medium optimization for high level extracellular production of a novel recombinant human interferon alpha 2b (huIFNα2b) in glycoengineered Pichia pastoris and its characterization. METHODS AND RESULTS: The artificial neural network-genetic algorithm model exhibited improved huIFNα2b production and better predictability compared to response surface methodology. The optimized medium exhibited a fivefold increase in huIFNα2b titre compared to the complex medium. A maximum titre of huIFNα2b (436 mg l-1 ) was achieved using the optimized medium in the bioreactor. Real-time capacitance data from dielectric spectroscopy were utilized to model the growth kinetics with unstructured models. Biological characterization by antiproliferative assay proved that the purified recombinant huIFNα2b was biologically active, exhibiting growth inhibition on breast cancer cell line. CONCLUSIONS: Culture medium optimization resulted in enhanced production of huIFNα2b in glycoengineered P. pastoris at both shake flask and bioreactor level. The purified huIFNα2b was found to be N-glycosylated and biologically active. SIGNIFICANCE AND IMPACT OF THE STUDY: DoE-based medium optimization strategy significantly improved huIFNα2b production. The antiproliferative activity of huIFNα2b substantiates its potential scope for application in cancer therapy.


Subject(s)
Bioreactors/microbiology , Interferon alpha-2 , Pichia , Recombinant Proteins , Cell Count , Computer Simulation , Culture Media , Humans , Interferon alpha-2/analysis , Interferon alpha-2/genetics , Interferon alpha-2/isolation & purification , Interferon alpha-2/metabolism , Pichia/genetics , Pichia/metabolism , Protein Engineering , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
12.
Cell Chem Biol ; 26(2): 203-212.e5, 2019 02 21.
Article in English | MEDLINE | ID: mdl-30503285

ABSTRACT

We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a ß1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.


Subject(s)
Growth Hormone/metabolism , Interferon alpha-2/metabolism , Polysaccharides/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Escherichia coli/metabolism , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Glycosylation , Growth Hormone/genetics , Humans , Interferon alpha-2/genetics , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , UDPglucose 4-Epimerase/genetics , UDPglucose 4-Epimerase/metabolism
13.
Front Immunol ; 9: 2494, 2018.
Article in English | MEDLINE | ID: mdl-30483249

ABSTRACT

Events related to HCMV infection drive accumulation of functionally enhanced CD57posNKG2Cpos adapted NK cells. We investigated NK cell adaptation to HCMV along a proposed continuum progressing from acute activation through maturation and memory formation towards functional exhaustion. Acute exposure to conditioned medium collected 24 h after HCMV infection (HCMVsn) increased NK cell cytotoxicity for all HCMV-seronegative and seropositive donors tested, with mean 38 and 29% boosts in natural and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively. Increases in NK cell cytotoxicity were completely abrogated by blocking type I interferon (IFN) receptors and equivalent responses occurred with exposure to IFN-α2 alone at the same concentration present in HCMVsn. To study longer term effects of HCMV infection, we focused on three groups of human immunodeficiency virus (HIV)-infected subjects distinguished as HCMV-seronegative or HCMV-seropositive with either high (>20%) or low (<6%) fractions of their NK cells expressing NKG2C. The NK cells of all three HIV-infected groups responded to HCMVsn and IFN-α2 in a manner similar to the NK cells of either HCMV-seronegative or seropositive controls. Neither HCMV status, nor the extent of phenotypic evidence of adaptation to HCMV infection significantly affected mean levels of ADCC or CD16-mediated NK cell degranulation and IFN-γ production compared between the HIV-infected groups. Levels of IFN-γ production correlated significantly with the fraction of NK cells lacking FcεRIγ (FcRγ), but not with the fraction of NK cells expressing NKG2C. There was negligible expression of exhaustion markers Lag-3 and PD-1 on NK cells in any of the groups and no significant difference between groups in the fraction of NK cells expressing Tim-3. The fraction of NK cells expressing Tim-3 was unaffected by CD16 stimulation. Relative to the total NK cell population, responses of Tim-3-expressing cells to CD16 stimulation were variably compromised in HCMV seronegative and seropositive groups. In general, NK cell function in response to signaling through CD16 was well preserved in HIV infection and although HCMV had a clear effect on NK cell FcRγ and NKG2C expression, there was little evidence that the level of adaptation to HCMV infection affected CD16-dependent NK cell signaling in HIV infection.


Subject(s)
Cytomegalovirus Infections/immunology , Cytomegalovirus/physiology , HIV Infections/immunology , HIV-1/physiology , Killer Cells, Natural/immunology , Adult , Antibody-Dependent Cell Cytotoxicity , Cytomegalovirus Infections/complications , Cytotoxicity, Immunologic , Female , HIV Infections/complications , HIV Seropositivity , Humans , Interferon alpha-2/metabolism , K562 Cells , Lymphocyte Activation , Male , Middle Aged , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Phenotype , Receptors, IgG/metabolism , Signal Transduction
14.
Anim Biotechnol ; 30(4): 358-365, 2019 Oct.
Article in English | MEDLINE | ID: mdl-30179066

ABSTRACT

The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 ∼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated. The results showed that the designed shRNA1/2 fragments of SUV39H1 gene had an obvious inhibition effect on the expression of SUV39H1 gene, reached 53.07 and 31.28%, respectively by qRT-PCR analysis. Compared with the control group, the expression of IFNα-2b gene in transgenic Bcap-37 cells infected with shRNA1 and 2 viruses significantly increased by 96.25 and 121.08%, respectively (p < 0.05). In addition, the expression of DNMT1, HDAC1 and G9a gene in the shRNA infected cells reduced significantly, and the expression of the HAT1 gene increased significantly (p < 0.05). The above results indicated that the expression of exogenous transgenic gene could be promoted by suppressing SUV39H1 gene at the cell level.


Subject(s)
Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Interferon alpha-2/metabolism , RNA, Small Interfering , Transgenes , Animals , Buffaloes , Cattle , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Gene Expression , HEK293 Cells , Histone Acetyltransferases/metabolism , Histone Deacetylase 1/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans
15.
Bioorg Chem ; 76: 294-302, 2018 02.
Article in English | MEDLINE | ID: mdl-29223806

ABSTRACT

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.


Subject(s)
Cytochromes b5/metabolism , Escherichia coli/metabolism , Interferon alpha-2/metabolism , Recombinant Fusion Proteins/metabolism , Acetylation , Antiviral Agents/pharmacology , Cell Line, Tumor , Cytochromes b5/pharmacology , Escherichia coli/genetics , Humans , Interferon alpha-2/genetics , Interferon alpha-2/pharmacology , Methionine/metabolism , Mutation , Phenylalanine/metabolism , Protein Domains , Protein Engineering , Protein Processing, Post-Translational , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology
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