Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

Marikar, Faiz M. M. T.; Ma, Dong‐Lai; Ye, Jianqiang; Tang, Bo; Zheng, Wei-juan; Zhang, Jing; Lü, Min; Zhang, Hua

doi:10.1038/cmi.2008.59

Cited by 4 publications

(4 citation statements)

References 15 publications

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“…Human FADD‐his6 was expressed as an inclusion body in Escherichia coli BL21 (DE3) cells, while the FADD mutants and MBP‐fused caspase‐8 DED‐AB were majorly soluble. Wild‐type FADD purification was performed in denaturing conditions (22). Purification of FADD mutants and MBP‐fused procaspase‐8 DED‐AB was performed in nondenaturing conditions.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation

2015

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Assembly of a death-inducing signaling complex is a key event in the extrinsic apoptotic pathway, enabling activation of the caspase cascade and subsequent cell death. However, the molecular events governing DISC assembly have remained largely elusive because of the lack of information on mechanism and specificity regulating the death effector domain (DED)-DED interaction network. Using molecular modeling, mutagenesis, and biochemical and ex vivo experiments, we identified the precise binding interface and hot spots crucial for intermolecular DED chain assembly. Mutation of key interface residues (Leu42/Phe45) in procaspase-8 DED-A completely abrogated DED chain formation in HEK293 cells and prevented its association with FADD. A significant 2.6-3.6-fold reduction in procaspase-8 activation was observed in functional cell-death assays after substitution of the interfacial residues. Based on our results we propose a new model for DISC formation that refines the current understanding of the activation mechanism. Upon stimulation, FADD self-associates weakly via reciprocal interaction between helices α1/α4 and α2/α3 of the DED to form an oligomeric signaling platform that provides a stage for the initial recruitment of procaspase-8 through direct interaction with α1/α4 of DED-A, followed by sequential interaction mediated by helices α2/α5 of DED-B, to form the procaspase-8 DED chain that is crucial for its activation and subsequent cell death.

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Section: Methodsmentioning

confidence: 99%

Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation

2015

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“…The recombinant AAV-CD19BiTE contained the His-Tag sequence and to further validate the secretion of CD19BiTE after transfection, a His-Tag immuno uorescence analysis was performed as previous reported [22]. Placing sterile coverslips into six-well plates before transfection and adding 293T or HepG2 cells into plates.…”

Section: His-tag Immuno Uorescence Analysismentioning

confidence: 99%

In vivo expression of anti-CD19/CD3 BiTE by liver-targeted AAV for the treatment of B cell malignancies

Yang,

Song,

Liu

et al. 2024

Preprint

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Anti-CD19/CD3 bispecific T-cell engagers (CD19BiTE) has shown promising efficacy in patients with relapsed or refractory (r/r) B-cell malignancies. However, the short half-life of CD19BiTE necessitates long-term repeated administration with rest period, which not only increases the costs but also compromises the efficacy. Long-term and stable expression of CD19BiTE is crucial for achieving durable responses of B-cell malignancies. Adeno-associated virus (AAV)-mediated gene therapy has been demonstrated to achieve long-term efficacy for multiple diseases. Here, we generated liver-targeted AAV encoding CD19BiTE (AAV-CD19BiTE) and achieved sustained expression of CD19BiTE for more than six months. The results indicated that AAV-CD19BiTE could significantly reduce the tumor burdens in CD19+ B-cell malignancies xenograft model via a single injection of AAV-CD19BiTE. Meanwhile, more CD3+, CD4+, CD8+T, and activated CD8+T cells were observed in lymphoma microenvironment after therapy with AAV-CD19BiTE. In addition, AAV-CD19BiTE was also proved to have a strong antitumor activity in patient-derived xenograft (PDX) model of B-cell lymphoma. Altogether, in vivo expression of CD19BiTE circumvents the problem of short half-life and may hold promise as a new therapeutical strategy for CD19+ B-cell malignancies via a single injection of AAV.

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“…The appropriateness of recombination was evaluated by PCR and DNA sequencing (Boshang Company, China). The fusion proteins His-urease and His-tsaA were purified by affinity chromatography through a Ni 2+ -affinity column (Sigma, USA) after denaturation, as described previously (Marikar et al, 2008). Protein concentration was determined using the Bradford method and was adjusted to 0.6 g/ l. The protein sample was stored at -80°C until further analysis.…”

Section: Primersmentioning

confidence: 99%

Identification of S-nitrosylation of proteins of Helicobacter pylori in response to nitric oxide stress

Zhou

Sun

et al. 2011

J Microbiol.

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Innate and adaptive immune responses are activated in humans when Helicobacter pylori invades the gastric mucosa. Nitric oxide (NO) and reactive nitrogen species are important immune effectors, which can exert their functions through oxidation and S-nitrosylation of proteins. S-nitrosoglutathione and sodium nitroprus-side were used as NO donors and H. pylori cells were incubated with these compounds to analyze the inhibitory effect of NO. The suppressing effect of NO on H. pylori has been shown in vitro. Furthermore, the proteins modified by S-nitrosylation in H. pylori were identified through the biotin switch method in association with matrix-assisted laser desorption ionization/time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Five S-nitrosylated proteins identified were a chaperone and heat-shock protein (GroEL), alkyl hydroperoxide reductase (TsaA), urease alpha subunit (UreA), HP0721, and HP0129. Importantly, S-nitrosylation of TsaA and UreA were confirmed using purified recombinant proteins. Considering the importance of these enzymes in antioxidant defenses, adherence, and colonization, NO may exert its antibacterial actions by targeting enzymes through S-nitrosylation. Identification of protein S-nitrosylation may contribute to an understanding of the antibacterial actions of NO. Our findings provide an insight into potential targets for the development of novel therapeutic agents against H. pylori infection.

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Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

Cited by 4 publications

References 15 publications

Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation

Molecular basis of death effector domain chain assembly and its role in caspase‐8 activation

In vivo expression of anti-CD19/CD3 BiTE by liver-targeted AAV for the treatment of B cell malignancies

Identification of S-nitrosylation of proteins of Helicobacter pylori in response to nitric oxide stress

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