Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains

<p>Abstract</p> <p>Background</p> <p>We previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form.</p> <p>Results</p> <p>Several bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway.</p> <p>Conclusion</p> <p>Our observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.</p

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching

AbstractThe HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to arginine reduces markedly the steady state amount of the protein, but does not impair its ability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity

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

Ancient DNA Identification of Early 20th Century Simian T-Cell Leukemia Virus Type 1

The molecular identification of proviruses from ancient tissues (and particularly from bones) remains a contentious issue. It can be expected that the copy number of proviruses will be low, which magnifies the risk of contamination with retroviruses from exogenous sources. To assess the feasibility of paleoretrovirological studies, we attempted to identify proviruses from early 20th century bones of museum specimens while following a strict ancient DNA methodology. Simian T-cell leukemia virus type 1 sequences were successfully obtained and authenticated from a Chlorocebus pygerythrus specimen. This represents the first clear evidence that it will be possible to use museum specimens to better characterize simian and human T-tropic retrovirus genetic diversity and analyze their origin and evolution, in greater detail

Decreased expression of the translation factor eIF3e induces senescence in breast cancer cells via suppression of PARP1 and activation of mTORC1

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To translate or to degrade? The role of INT6 in histone mRNA translation and Nonsense Mediated mRNA Decay

Différentes observations montrent que la protéine INT6 humaine possède une activité suppresseur de tumeurs. Il a été démontré que chez l homme le gène int6 était sous-exprimé dans environ 30% des cancers du poumon non à petits cellules et que cette sous-expression était un facteur de mauvais pronostic. Des expériences de criblage double hybride avec INT6 comme appât ont identifié une protéine nommée SLIP1 (SLBP Interacting Protein 1). Un effet de SLIP1 sur la traduction des ARN messager des histones a été montré. Les travaux que j ai menés indiquent qu INT6 en interagissant avec SLIP intervient dans le contrôle de la stabilité et de la traduction des ARNs codant pour les histones. Un knockdown d INT6 provoque une baise des niveaux des histones endogènes sans avoir un effet au niveau d ARN. Mes études, en révélant un nouveau mécanisme de dans lequel INT6 joue un rôle direct, permettent ainsi de faire le lien entre d une part les fonctions connues de cette protéine dans la traduction et son contrôle et d autre part les effets oncogéniques connus de son altération. Par ailleurs, l étude de la fonction d INT6 dans les cellules humaines réalisée par ARN interférence montre une inhibition de la dégradation des ARNm possédant un codon stop prématuré par la voie du Nonsense Mediated mRNA Decay (NMD). Nous avons étudié son action par rapport aux ARNs HTLV-1. Nous avons observé une stabilisation significative des cibles de NMD. Ceci démontre que la protéine Tax interfère avec cette voie de dégradation des ARN d une part en empêchant l interaction entre UPF1 et INT6 et d autre part en interagissant lui-même avec la protéine UPF1 phosphorylée. En agissant sur le NMD, Tax intervient à un niveau post transcriptionel qui pourrait avantager la réplication virale et aussi permettre la tolérance cellulaire aux mutations liées à l'effet mutagénique établi de Tax.Several observations show that the human protein INT6 has a tumour suppressor activity.It has been demonstrated that in humans the expression of the int6 gene is reduced in about 30% of non-small cell lung cancers and that this under-expression is linked with a bad prognosis. Yeast two hybrid screening experiments using INT6 as bait have led to the identication ofa protein named SLIP1 (SLBP Interacting Protein 1). An effect of SLIP1 on the translation of histone messenger RNAs has been shown. My work indicates that INT6 through its interaction with SLIP1 plays a role in the controlof the stability and translation of histone mRNAs. INT6 knockdown induces a reduction of the level of endogenous histones without affecting that of their mRNAs. My studies, by revealing a new histone translation mechanism in which INT6 plays a direct role, establish a connection between on one side the known functions of this protein in translation and its control and on the other the known oncogenic effects of its alteration. The study of the function of INT6 in human cells by RNA interference resulted in inhibition of the degradation of mRNAs containing a premature termination codon by the Nonsense Mediated mRNA Decay pathway (NMD). Since we identified the HTLV-1 protein Tax as an interactor of INT6, and since some features of HTLV-1 mRNAs suggest that they may be potential NMD targets, we have studied the effect of Tax on the NMD pathway.We have observed that Tax significantly stabilizes NMD targets.We have demonstrated that the interference of Tax with the NMD pathway is mediated through first the perturbation of the interaction between INT6 and the NMD factor UPF1 and second through direct interaction between Tax and phosphorylated UPF1.Through its effect on NMD, Tax may favor viral replication at a post-transcriptional level as well as enable cellular tolerance for some mutations resulting from the established mutagenic activity of Tax.LYON-ENS Sciences (693872304) / SudocSudocFranceF

ANALYSE DE LA FONCTION DES PROTEINES PDZ INTERAGISSANT AVEC L'ONCOPROTEINE TAX DU VIRUS HTLV-I

LES RETROVIRUS SONT RESPONSABLES DE NOMBREUSES PATHOLOGIES HUMAINES OU ANIMALES. HTLV-I (HUMAN T-CELL LEUKEMIA VIRUS TYPE I) EST LE PREMIER RETROVIRUS DECOUVERT CHEZ L'HOMME. L'INFECTION PAR HTLV-I PROVOQUE DIVERS SYNDROMES PROLIFERATIFS ET DEGENERATIFS COMME L'ATL (ADULT T-CELL LEUKEMIA) ET LA TSP/HAM (TROPICAL SPASTIC PARAPARESIS / HTLV-I ASSOCIATED MYELOPATHY). HTLV-I CODE NOTAMMENT POUR LA PROTEINE TAX. IL EST ADMIS QUE C'EST CETTE PROTEINE QUI CONFERE AU VIRUS SON POUVOIR TRANSFORMANT. TAX INTERAGIT AVEC DES PROTEINES CONTENANT LE MOTIF D'INTERACTION PROTEINE-PROTEINE PDZ. CES PROTEINES INTERVIENNENT DANS LA FORMATION DES JONCTIONS CELLULAIRES, DES SYNAPSES ET DANS L'ASSEMBLAGE DE CERTAINS RECEPTEURS. CERTAINES SONT AUSSI CAPABLES DE RELAYER LES SIGNAUX EXTRACELLULAIRES, ET DE PARTICIPER AU CONTROLE DE LA PROLIFERATION. AU COURS DE MA THESE JE ME SUIS ATTACHEE A ANALYSER LA FONCTION DE QUELQUES PROTEINES CELLULAIRES A DOMAINES PDZ, LES TIPS (TAX INTERACTING PROTEIN), DANS LE BUT DE MIEUX COMPRENDRE SI L'INTERACTION ENTRE TAX ET CES PROTEINES POUVAIT ETRE IMPORTANTE POUR LE POTENTIEL ONCOGENIQUE DE CETTE PROTEINE VIRALE. J'AI TOUT D'ABORD ANALYSE LA FONCTION DE LA PROTEINE PDZ TIP-1. ELLE INTERAGIT AVEC LA RHOTEKINE, EFFECTUER DE LA PETITE GTPASE RHOA. PAR DES EXPERIENCES DE CO-IMMUNOPRECIPITATION, J'AI MONTRE QUE LA RHOTEKINE INTERAGIT A LA FOIS AVEC RHOA LIEE AU GTP, ET AVEC LA PROTEINE PDZ TIP-1. D'AUTRE PART, LE COMPLEXE FORME PAR CES TROIS PROTEINES CONDUIT A UNE ACTIVATION MARQUEE DE LA SEQUENCE ENHANCER SRE. LES ETUDES REALISEES SUR TIP-15, QUI CORRESPOND A UNE FORME TRONQUEE DE PSD-95, INDIQUE QU'ELLE INTERAGIT AVEC L'ONCOGENE NET1, GEF DES GTPASES RHO. NET1 POTENTIALISE LA CAPACITE DE RHOA A FORMER DES FIBRES DE STRESS ET A ACTIVER LE SRE. TAX INTERAGIT DONC AVEC DEUX PROTEINES PDZ DIFFERENTES QUI SONT CEPENDANT IMPLIQUEES TOUTES DEUX DANS L'ACTIVATION DU SRE PAR RHOA. J'AI ENSUITE ETUDIE LA PROTEINE PDZ TIP-2. ELLE INTERAGIT AVEC TAX ET AVEC LA PROTEINE VIRALE E6 DE HPV-18. L'ANALYSE DES DONNEES OBTENUES ET CONNUES INDIQUENT QUE LA CAPACITE DE TAX A INTERAGIR AVEC LES PROTEINES A DOMAINES PDZ, POURRAIT JOUER UN ROLE MAJEUR POUR CONFERER SON POUVOIR TRANSFORMANT A LA PROTEINE VIRALE. DU FAIT DES FONCTIONS DE CES PROTEINES PDZ, IL EST POSSIBLE QUE LEUR LIAISON A TAX SOIT A L'ORIGINE DE CERTAINS DESORDRES PROLIFERATIFS OU DEGENERATIFS ASSOCIEES A HTLV-I.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Etude fonctionnelle de la protéine Int-6 et caractérisation de ses interactions avec eIF3, le protéasome 26S et le COP9 Signalosome

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Altération de la fonction de la protéine à domaine PDZ TIP2/GIPC par les oncoprotéines virales Tax de HTLV-1 et E6 de HPV18

LYON-ENS Sciences (693872304) / SudocSudocFranceF