The Dark Side of EGFP: Defective Polyubiquitination

Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-κB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered

A Novel TRAF6 Binding Site in MALT1 Defines Distinct Mechanisms of NF-{kappa}B Activation by API2{middle dot}MALT1 Fusions

The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.status: publishe

Impact of increasing lymph node yield on staging, morbidity and survival after esophagectomy for esophageal adenocarcinoma

Background: Extended lymphadenectomy during esophagectomy for esophageal cancer may increase survival, but also increase morbidity. This study analyses the influence of lymph node yield after transthoracic esophagectomy for esophageal adenocarcinoma on the number of positive lymph nodes, pathological N-stage, complications and survival. Materials and methods: Consecutive patients undergoing transthoracic esophagectomy for esophageal adenocarcinoma between 2010 and 2020 were prospectively recorded (follow-up until January 2022). Lymph node yield was analyzed as continuous and dichotomous variable (≤30 vs. ≥31 nodes). The effect of lymph node yield on number of positive lymph nodes, complications, disease-free (DFS) and overall survival (OS) was assessed in multivariable regression analyses. Results: 585 patients were included. Median lymph node yield increased from 25 (IQR 20–34) in 2010 to 39 (IQR 32–50) in 2020. Higher lymph node yield was associated with more positive lymph nodes (≥31 vs. ≤30 IRR 1.39, 95%CI 1.11–1.75). In 258 (y)pN + patients, the percentage of (y)pN3-stage increased with 14% between patients with ≤30 and ≥ 31 lymph nodes examined (p 0.014). Higher lymph node yield was not associated with more complications. Superior survival was seen in patients with ≥31 vs. ≤30 lymph nodes examined [DFS: HR 0.73, 95%CI 0.58–0.93, OS: HR 0.71, 95%CI 0.55–0.93)]. Conclusions: A lymph node yield of 31 or higher was associated with upstaging and superior survival after esophagectomy for esophageal adenocarcinoma, without increasing morbidity. Extended lymphadenectomy may therefore be regarded as an important part of the multimodal treatment of esophageal cancer

Clinical implications of chyle leakage following esophagectomy

The clinical consequences of chyle leakage following esophagectomy are underexposed. The aim of this study was to investigate the clinical implications of chyle leakage following esophagectomy. This retrospective study of prospectively collected data included patients who underwent transthoracic esophagectomy in 2017-2020. Routinely, the thoracic duct was resected en bloc as part of the mediastinal lymphadenectomy. Chyle leakage was defined as milky drain fluid for which specific treatment was initiated and/or a triglyceride level in drain fluid of ?1.13?mmol/L, according to the Esophagectomy Complications Consensus Group (ECCG) classification. Primary endpoints were the clinical characteristics of chyle leakage (type, severity and treatment). Secondary endpoints were the impact of chyle leakage on duration of thoracic drainage and hospital stay. Chyle leakage was present in 43/314 patients (14%), of whom 24 (56%) were classified as severity A and 19 (44%) as severity B. All patients were successfully treated with either medium chain triglyceride diet (98%) or total parental nutrition (2%). There were no re-interventions for chyle leakage during initial admission, although one patient needed additional pleural drainage during readmission. Patients with chyle leakage had 3?days longer duration of thoracic drainage (bias corrected accelerated (BCa) 95%CI:0.46-0.76) and 3?days longer hospital stay (BCa 95%CI:0.07-0.36), independently of the presence of other complications. Chyle leakage is a relatively frequent complication following esophagectomy. Postoperative chyle leakage was associated with a significant longer duration of thoracic drainage and hospital admission. Nonsurgical treatment was successful in all patients with chyle leakage

EGFP blocks Lys63- and Lys48-linked polyubiquitination.

<div><p>(<b>A</b>) 293T cells transfected with the indicated constructs and treated for 4 hours with 20 ng/ml TNF-α (lane 5 and 6) were immunoblotted with anti-Flag (API2-MALT1), anti-HA (HA-Ub-K63) and anti-EGFP antibodies (left panel) or anti-IKKγ immunoprecipitates were immunoblotted with anti-Flag (IKKγ) or anti-HA (ubiquitin) (right panel).</p> <p>(<b>B</b>) EGFP affects K48-linked polyubiquitination.</p> <p>293T cells were transfected with a Ubiquitin construct with only Lys48 available for polymerization (HA-Ub-K48), treated for 4 hours with 20 ng/ml TNF-α or left untreated and cell lysates were immunoblotted with anti-HA (Ub-K48) and anti-EGFP antibodies.</p> <p>Fluorescence intensities (Excitation 485/Emission 520 nm) for EGFP and pmaxGFP were comparable (∼100 fold higher then background values), expression of pDs-Red was confirmed by Fluorescence microscopy.</p> <p>(<b>C</b>) EGFP stabilizes exogenous API2-Myc in 293T cells via reduction of its Lys48-linked auto-ubiquitination and proteasomal degradation.</p> <p>(<b>D</b>) Stable expression of EGFP in the merkel cell carcinoma cell line MCC14.2 reduces polyubiquitination and enhances endogenous p53 expression levels.</p> <p>The average ratio and standard deviation of p53 to actin signals are given (three independent experiments), (Ub)ⁿ : polyubiquitinated proteins.</p></div

EGFP affects polyubiquitination-dependent processes <b><i>in vivo</i></b>.

<div><p>(<b>A</b>) Western blots of spleen and liver extracts from FVB wild-type (wt) and EGFP transgenic mice detected with antibodies against Ubiquitin, EGFP and actin (loading control).</p> <p>(<b>B</b>) EGFP reduces phosphorylation of IκB-α in anti-IgM/anti-CD40 stimulated B-lymphocytes purified from EGFP mice.</p> <p>Experiments performed in triplicate, a representative image of one experiment is shown.</p> <p>The average and standard deviations of ratios of IκB-α-P to actin relative to un-stimulated cells are given.</p> <p>(<b>C</b>) The deficit of B220⁺/CD40⁺ B-cells in the bone marrow of API2-MALT1 mice is restored in EGFP/API2-MALT1 double transgenic mice.</p> <p>Experiments performed in triplicate, the average and standard deviations are depicted. eMalt1: endogenous Malt1, *a-specific band (<b>D</b>) EGFP mice show increased p53 levels in the liver and have enhanced p53/p21 responses upon γ-irradiation induced DNA damage.</p> <p>The average and standard deviations of the ratios of p53/p21 to actin signals relative to that of sample 1 are given.</p> <p>(<b>E</b>) Recombinant EGFP (rEGFP) does not prevent polyubiquitination of a Biotin-Lysozyme substrate <i>in vitro</i>. (Ub)ⁿ: polyubiquitinated proteins.</p></div

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population