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

BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport

A great deal of sub-cellular organelle positioning, and essentially all minus-ended organelle transport, depends on cytoplasmic dynein, but how dynein's function is regulated is not well understood. BicD is established to play a critical role in mediating dynein function-loss of BicD results in improperly localized nuclei, mRNA particles, and a dispersed Golgi apparatus-however exactly what BicD's role is remains unknown. Nonetheless, it is widely believed that BicD may act to tether dynein to cargos. Here we use a combination of biophysical and biochemical studies to investigate BicD's role in lipid droplet transport during Drosophila embryogenesis.Functional loss of BicD impairs the embryo's ability to control the net direction of droplet transport; the developmentally controlled reversal in transport is eliminated. We find that minimal BicD expression (near-BicD(null)) decreases the average run length of both plus and minus end directed microtubule (MT) based transport. A point mutation affecting the BicD N-terminus has very similar effects on transport during cellularization (phase II), but in phase III (gastrulation) motion actually appears better than in the wild-type.In contrast to a simple static tethering model of BicD function, or a role only in initial dynein recruitment to the cargo, our data uncovers a new dynamic role for BicD in actively regulating transport. Lipid droplets move bi-directionally, and our investigations demonstrate that BicD plays a critical-and temporally changing-role in balancing the relative contributions of plus-end and minus-end motors to control the net direction of transport. Our results suggest that while BicD might contribute to recruitment of dynein to the cargo it is not absolutely required for such dynein localization, and it clearly contributes to regulation, helping activation/inactivation of the motors

Differential requirement of MALT1 for BAFF-induced outcomes in B cell subsets

B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor κB (NF-κB) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-κB signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-κB2 (p100), p100 degradation, and RelB nuclear translocation in B220+ B cells. This corresponds with impaired survival of MALT1−/− marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1−/− MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor–mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell–intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction

Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

International audienceBACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader

Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2

Loss of c-IAP2 ubiquitin ligase activity, which occurs in the lymphoma-causing c-IAP2/MALT1 fusion protein, activates non-canonical NF-κB signaling and results in B cell abnormalities characteristic of MALT lymphoma

Alcohol, tobacco and breast cancer – collaborative reanalysis of individual data from 53 epidemiological studies, including 58 515 women with breast cancer and 95 067 women without the disease

Alcohol and tobacco consumption are closely correlated and published results on their association with breast cancer have not always allowed adequately for confounding between these exposures. Over 80% of the relevant information worldwide on alcohol and tobacco consumption and breast cancer were collated, checked and analysed centrally. Analyses included 58 515 women with invasive breast cancer and 95 067 controls from 53 studies. Relative risks of breast cancer were estimated, after stratifying by study, age, parity and, where appropriate, women's age when their first child was born and consumption of alcohol and tobacco. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, (8.4 g per day and 5.0 g per day, respectively). Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32 (1.19–1.45, P<0.00001) for an intake of 35–44 g per day alcohol, and 1.46 (1.33–1.61, P<0.00001) for ⩾45 g per day alcohol. The relative risk of breast cancer increased by 7.1% (95% CI 5.5–8.7%; P<0.00001) for each additional 10 g per day intake of alcohol, i.e. for each extra unit or drink of alcohol consumed on a daily basis. This increase was the same in ever-smokers and never-smokers (7.1% per 10 g per day, P<0.00001, in each group). By contrast, the relationship between smoking and breast cancer was substantially confounded by the effect of alcohol. When analyses were restricted to 22 255 women with breast cancer and 40 832 controls who reported drinking no alcohol, smoking was not associated with breast cancer (compared to never-smokers, relative risk for ever-smokers=1.03, 95% CI 0.98–1.07, and for current smokers=0.99, 0.92–1.05). The results for alcohol and for tobacco did not vary substantially across studies, study designs, or according to 15 personal characteristics of the women; nor were the findings materially confounded by any of these factors. If the observed relationship for alcohol is causal, these results suggest that about 4% of the breast cancers in developed countries are attributable to alcohol. In developing countries, where alcohol consumption among controls averaged only 0.4 g per day, alcohol would have a negligible effect on the incidence of breast cancer. In conclusion, smoking has little or no independent effect on the risk of developing breast cancer; the effect of alcohol on breast cancer needs to be interpreted in the context of its beneficial effects, in moderation, on cardiovascular disease and its harmful effects on cirrhosis and cancers of the mouth, larynx, oesophagus and liver

MALT1, BCL10 and FOXP1 in salivary gland mucosa-associated lymphoid tissue lymphomas

In view of the certain anatomic site-dependent frequency of chromosomal translocations involved in extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) pathogenesis, 17 salivary gland MALT lymphoma cases were analyzed for MALT1 and FOXP1 translocations. B cell CLL/lymphoma 10 (BCL10) and forkhead box PA (FOXP1) protein expression were studied by immunohistochemistry and translocations identified using fluorescence in situ hybridization (FISH)-specific probes FOXP1, t(11;18)(q21;q21)/API2-MALT1 and t(14;18)(q32;q21)/IgH-MALT1. None of the 11 analyzed cases showed FOXP1 rearrangement or amplification. The t(11;18) was present in five of 13 cases and the t(14;18) in three of 13 cases. MALT1 translocations were mostly mutually exclusive except in a single case. FOXP1 protein expression showed differences in the proportion of tumor cells with nuclear expression but not in their intensity, with the exception of one case where very intense nuclear staining was noted. BCL10 nuclear expression was present in four of 17 cases, two of which lacked t(11;18). Our results suggest that MALT1-specific translocations and FOXP1 rearrangements are not commonly involved in pathogenesis. A case with strong FOXP1 protein expression indicates the possibility that the upregulation of FOXP1 expression is significant in a small subset of salivary gland MALT lymphomas. Also a single case in which both MALT1 translocations were present indicates that these are not always mutually exclusive

Megakaryoblastic leukemia with an N-ras mutation and late acquisition of a Philadelphia chromosome.

A patient is described with de novo acute non-lymphocytic leukemia of megakaryoblastic lineage with tri-lineage myelodysplasia. This patient was studied cytogenetically and using molecular genetic techniques throughout her clinical course. She had an N-ras mutation at diagnosis which persisted despite a bone marrow transplant, and acquired a Philadelphia chromosome associated with a P190 BCR-ABL transcript at clinical relapse 3 months post-transplantatio