The proto-oncogenic protein TAL1 controls TGF-β1 signaling through interaction with SMAD3

AbstractTGF-β1 is involved in many aspects of tissue development and homeostasis including hematopoiesis. The TAL1 transcription factor is also an important player of this latter process and is expressed very early in the myeloid and erythroid lineages. We previously established a link between TGF-β1 signaling and TAL1 by showing that the cytokine was able to induce its proteolytic degradation by the ubiquitin proteasome pathway. In this manuscript we show that TAL1 interacts with SMAD3 that acts in the pathway downstream of TGF-β1 association with its receptor. TAL1 expression strengthens the positive or negative effect of SMAD3 on various genes. Both transcription factors activate the inhibitory SMAD7 factor through the E box motif present in its transcriptional promoter. DNA precipitation assays showed that TAL1 present in Jurkat or K562 cells binds to this SMAD binding element in a SMAD3 dependent manner. SMAD3 and TAL1 also inhibit several genes including ID1, hTERT and TGF-β1 itself. In this latter case TAL1 and SMAD3 can impair the positive effect exerted by E47. Our results indicate that TAL1 expression can modulate TGF-β1 signaling by interacting with SMAD3 and by increasing its transcriptional properties. They also suggest the existence of a negative feedback loop between TAL1 expression and TGF-β1 signaling

Contribution à l'élaboration d'un modèle de la réparation de l'ADN par excision de nucléotide

Afin de préserver l intégrité de son ADN, support de l information génétique, la cellule a mis en place divers systèmes de réparation dont celui par excision-resynthèse de nucléotides (NER) qui s attache à éliminer toute lésion qui perturbe la structure première de la double hélice. Ces lésions sont générées par différents agents physiques et/ou chimiques tels que les UV, les médicaments antitumoraux ou les composés aromatiques polycycliques. Cette voie de réparation se déroule en 3 phases : la reconnaissance du dommage, la double incision et la resynthèse/ligation du nouveau brin d ADN. En vue d élucider le rôle des divers facteurs et de préciser leurs interactions fonctionnelles, nous avons développé un système de réparation in vitro mimant le mécanisme tel qu il s opère in vivo.Nous avons pu décrire comment s effectue la transition entre les étapes de double incision et de resynthèse, potentiellement risquée pour l intégrité de l ADN. Ainsi, nous avons identifié un nouveau rôle de XPG : celui-ci permet le recrutement de PCNA et la stabilisation du complexe de resynthèse en coopération avec RPA afin d assurer un recrutement correct de l ADN polymerase delta. L utilisation d extraits cellulaires de patients atteints de xeroderma pignemetosum et trichothiodystrophie confirme les rôles de ces facteurs et, à la lumière de ces résultats, permet d identifier certains défauts biochimiques de ces malades.Ce système nous a aussi permis d étudier la réparation d ADN endommagés par des adduits aromatiques polycycliques appelés BPDE, que l on trouve dans la fumée de cigarette et présentant un très fort potentiel cancérigène. Nous avons alors démontré que l efficacité de réparation des BPDE est directement corrélée à l efficacité de fixation d XPC/HR23B sur ces dommages. Ces travaux suggèrent finalement une explication au fort pouvoir cancérigène de certains composés de la fumée de cigarette comme le (+)trans-BPDE.In order to preserve the integrity of the DNA, the cell has set up various repair mechanisms. The one dealing with the damages induced by UV or bulky adducts such as aromatic polycyclic compounds, is called nucleotide excision repair ( NER). It can be divided in three steps : the damage recognition, the dual incision and the resynthesis/ligation of the new DNA strand. To elucidate the roles and the functional interactions of the various factors involved in this repair pathway, we set up an in vitro repair system mimicking the in vivo mechanism.With this tool, we were able to describe how the transition between the dual incision and the resynthesis occurs, highly hazardous step for the cell. Thus we highlighted a new role for the XPG endonuclease : il allows for the recruitment of PCNA and the stabilization of the resynthesis complex in association with RPA, enabling the correct coming of the DNA Pol . The use of XP and TTD patient cell line extracts corroborated these data.We also studied the repair of DNA damaged by aromatic polycyclic compounds known as BPDE and present in the cigarette smoke. We demonstrated that the repair efficiency of BPDE is directly correlated to the XPC/HR23B binding efficiency on the damaged DNA. These data also suggest an explanation for the high carcinogenic potential of the some of the cigarette smoke compounds such as (+)trans BPDE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The Complex Relationship between HTLV-1 and Nonsense-Mediated mRNA Decay (NMD)

International audienceBefore the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated

The fluorescent protein stability assay: an efficient method for monitoring intracellular protein stability

International audienceThe stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs

In silico pharmacological study of AQP2 inhibition by steroids contextualized to Ménière’s disease treatments

International audienceMénière’s disease (MD) is characterized by an abnormal dilatation of the endolymphatic compartment called endolymphatic hydrops and is associated with fluctuating hearing losses and vertigo. Corticosteroid treatment is typically administered for its anti-inflammatory effects to MD patients. However, we recently described for the first time a direct interaction of two corticosteroids (dexamethasone and cortisol) with human AQP2 which strongly inhibited water fluxes. From these initial studies, we proposed an AQPs Corticosteroids Binding Site (ACBS). In the present work, we tested the interaction of 10 molecules associated to the steroid family for this putative ACBS. We observed a wide diversity of affinity and inhibitory potential of these molecules toward AQP2 and discussed the implications for inner ear physiology. Among the tested compounds, cholecalciferol, calcitriol and oestradiol were the most efficient AQP2 water permeability inhibitors

Deciphering Molecular Mechanisms Involved in the Modulation of Human Aquaporins’ Water Permeability by Zinc Cations: A Molecular Dynamics Approach

Aquaporins (AQPs) constitute a wide family of water channels implicated in all kind of physiological processes. Zinc is the second most abundant trace element in the human body and a few studies have highlighted regulation of AQP0 and AQP4 by zinc. In the present work, we addressed the putative regulation of AQPs by zinc cations in silico through molecular dynamics simulations of human AQP0, AQP2, AQP4, and AQP5. Our results align with other scales of study and several in vitro techniques, hence strengthening the reliability of this regulation by zinc. We also described two distinct putative molecular mechanisms associated with the increase or decrease in AQPs’ water permeability after zinc binding. In association with other studies, our work will help deciphering the interaction networks existing between zinc and channel proteins

Plant Aquaporin Gating Is Reversed by Phosphorylation on Intracellular Loop D—Evidence from Molecular Dynamics Simulations

Aquaporins (AQPs) constitute a wide and ancient protein family of transmembrane channels dedicated to the regulation of water exchange across biological membranes. In plants, higher numbers of AQP homologues have been conserved compared to other kingdoms of life such as in animals or in bacteria. As an illustration of this plant-specific functional diversity, plasma membrane intrinsic proteins (PIPs, i.e., a subfamily of plant AQPs) possess a long intracellular loop D, which can gate the channel by changing conformation as a function of the cellular environment. However, even though the closure of the AQP by loop D conformational changes is well described, the opening of the channel, on the other hand, is still misunderstood. Several studies have pointed to phosphorylation events as the trigger for the transition from closed- to open-channel states. Nonetheless, no clear answer has been obtained yet. Hence, in order to gain a more complete grasp of plant AQP regulation through this intracellular loop D gating, we investigated the opening of the channel in silico through molecular dynamics simulations of the crystallographic structure of Spinacia oleracea PIP2;1 (SoPIP2;1). Through this technique, we addressed the mechanistic details of these conformational changes, which eventually allowed us to propose a molecular mechanism for PIP functional regulation by loop D phosphorylation. More precisely, our results highlight the phosphorylation of loop D serine 188 as a trigger of SoPIP2;1 water channel opening. Finally, we discuss the significance of this result for the study of plant AQP functional diversity

TFIIH enzymatic activities in transcription and nucleotide excision repair.

International audienceTranscription and nucleotide excision repair (NER) are two major mechanisms in which the transcription factor TFIIH plays a crucial role. In order to investigate its function, we first described a fast and efficient purification protocol of TFIIH from either HeLa cells or patient cell lines, as well as various in vitro enzymatic assays set up in our laboratory. All these enzymatic assays have been adapted to work on immobilized DNA, a powerful tool allowing for sequential protein incubations in various buffer conditions, without destabilizing protein complexes bound to the DNA. Runoff transcription assays performed with either whole cell extract or highly purified factors underline the role of TFIIH helicases (XPB and XPD) in the RNA synthesis. Moreover, the requirement of XPB and XPD in NER can also be investigated with various assays corresponding to the different steps of this process. The DNA opening assay (permanganate footprint) highlights DNA unwinding of the double-stranded DNA fragment within the repair complex, whereas the dual incision assay allows for detection of the double cut on both sides of the lesion. The gap-filling reaction following the cuts can be monitored as well with a DNA resynthesis assay. Futhermore, the use of immobilized DNA is of great interest to study the detailed mechanism in which TFIIH plays a central role. This chapter describes the ATP-independent recruitment of TFIIH on the damaged DNA previously recognized by XPC-hHR23B and the sequential arrival and departure of the repair proteins within the NER complex