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  • Open Access

Characterization of the TNFR1-d2 protein: Implication in TNF receptor associated periodic syndrome (TRAPS)?

  • 1, 2,
  • 2, 3,
  • 4,
  • 5, 6,
  • 2, 3, 6,
  • 1, 2, 6 and
  • 1, 2
Pediatric Rheumatology201513 (Suppl 1) :P39

https://doi.org/10.1186/1546-0096-13-S1-P39

  • Published:

Keywords

  • Internal Ribosome Entry Site
  • Confocal Fluorescence Microscopy
  • T50M Mutation
  • Recurrent Fever
  • Fluorescence Microscopy Analysis

Introduction

Binding of TNF to TNF receptor 1 (TNFR1) induces both the survival pathway by activation of the NF-kB transcription factor, and the death pathway by apoptosis. Mutations in the TNFR1 gene (TNFSFR1A) are responsible for the auto-inflammatory disease TRAPS, a dominantly inherited hereditary recurrent fever. Various defects such as defective TNFR1 receptor shedding, protein misfolding, NF-kB activation, or apoptosis have been associated with the pathogenesis of TRAPS.

Previously, we have identified TNFR1-d2, an exon2-spliced transcript of TNFRSF1A. TNFR1-d2 is expressed in a tissue-specific manner in contrast to ubiquitous expression of the full-length TNFR1 transcript.

Objectives

This study aimed to analyze the TNFR1-d2 protein expression and its function in NF-kB signalling pathways and to investigate the possible role of TNFR1-d2 in TRAPS physiopathology.

Materials and methods

Translation analyses of TNFR1-d2 were performed in HEK293T by over-expression of different TNFR1-d2 cDNA constructs fused to the Flag tag. HEK293T transfected cells were used to measure internal ribosome entry site (IRES) activity and NF-κB-activation by luciferase assays. Subcellular localization of the TNFR1-d2 fused to the GFP protein was studied in MCF7 cells, followed by staining of different cellular compartments and confocal fluorescence microscopy analysis.

Results

We showed that TNFR1-d2 is translated from an alternative start codon due to an IRES activity created by the exons 1 and 3 junction. The methionine 109 located in exon 4 in-frame with TNFR1 was used, resulting in a putative new protein isoform lacking its N-terminal region. Subcellular localization showed that the full-length and TNFR1-d2 proteins shared the same intracellular localization to the Golgi complex. Since the c.224C>T (p.Pro75Leu, P46L) and c.236C>T (p.Thr79Met, T50M) mutations in exon 3 lie in close vicinity to a strong Kozak consensus sequence, we hypothesized that these 2 sequence variants could affect TNFR1-d2 translation. Interestingly, we found that only TNFR1-d2 carrying the severe T50M mutation was translated through the mutated codon which induced a decrease of the IRES activity. Moreover, whereas overexpression of wild type TNFR1-d2 was not associated with increased NF-κB transcriptional activity, TNFR1-d2-T50M seemed to increase NF-κB activity as compared to the empty vector.

Conclusion

Our results support that the TNFR1-d2-T50M translation defect could lead to a gain-of-function of TNFR1-d2, suggesting that TNFR1-d2 may account for the physiopathology of TRAPS in patients carrying the T50M mutation, which is associated with a severe TRAPS phenotype.

Authors’ Affiliations

(1)
Laboratoire des maladies rares et auto-inflammatoires, Hopital A. de Villeneuve, Montpellier, France
(2)
Inserm / Chu, U1183 Montpellier, France
(3)
Département de génétique médicale, Hopital A. de Villeneuve, Montpellier, France
(4)
CHU Caremeau, Pole psychiatrie, Nimes, France
(5)
Institut de génétique moléculaire, Montpellier, France
(6)
Université, Montpellier, France

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