No description available.

Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.

No description available.

Par-4 is a leucine zipper domain protein that induces apoptosis on its own in certain cancer cells and in Ras-transformed cells, but not in normal or immortalized cells. Par-4 induces apoptosis by activation of the Fas death receptor pathway and co-parallel inhibition of NF-B transcription activity. Cells that are resistant to apoptosis by Par-4 alone, however, are greatly sensitized by Par-4 to the action of other pro-apoptotic insults such as growth factor withdrawal, TNF, ionizing radiation, intracellular calcium elevation, or those involved in neuronal degeneration such as Alzheimer's, Parkinson's, Huntington's and Stroke. Previous studies have suggested that the apoptosis-sensitization potential of Par-4 is dependent upon inhibition of PKC or WT1 cell survival function by direct interaction between the leucine zipper domain at the carboxy-terminus of Par-4 and the zinc finger domains of PKC or WT1. In this study, I performed structure-function analysis using GFP-fusion proteins and deletion mutants to identify the functional localization and domains of Par-4 that are essential for apoptosis induction. My findings suggest that apoptosis by Par-4 is dependent on its translocation to the nucleus for induction of apoptosis. A bipartite nuclear localization signal sequence corresponding to amino acids 137-155 was necessary for nuclear translocation of Par-4. Importantly, the core residues 137-204 in the center part of Par-4 were necessary and sufficient to induce Fas pathway activation, inhibition of nuclear NF-B transcription activity and apoptosis. These findings imply that binding of Par-4 via its leucine zipper domain to other proteins is dispensable for apoptosis by Par-4.

Ime4 is the catalytic subunit of a conserved methyltransferase (MTase) complex found in yeast, S. cerevisiae. This complex is responsible for creating the RNA modification N6- methyladenosine (m6A), the most common post-transcriptional modification in higher eukaryotes. There is evidence to suggest that m6A is an important mediator of gene expression control within the cell and has been associated with a diverse array of phenotypic effects, notably as a conserved determinant of cell fate. The MTase complex is known to be a nuclear protein, the compartment where it is believed to carry out most of its methylation activity. Recently, the nuclear localization signals (NLS) of the subunits of the human MTase complex were experimentally identified, whereas the NLSs of the yeast MTase complex remain unknown. Here, we have experimentally identified the amino acid sequence 517RKYQEFMKSKTGTSHTGTKKIDKK540, located within the C-terminal region, as a putative bipartite NLS for Ime4.

La mitogaligine est une protéine codée par le gène galig, un gène inducteur de la mort cellulaire. Préalablement, il a été montré au laboratoire que cette protéine mitochondriale pouvait induire la mort cellulaire selon une voie alternative de l’apoptose. Nos études indiquent que la mitogaligine peut également être adressée au noyau où elle exerce aussi une activité cytotoxique. Le signal d’adressage nucléaire est non conventionnel et lié à une répétition d’acides aminés. Il n’est fonctionnel que si le signal de localisation mitochondriale est aboli. La fonction cytotoxique de la mitogaligine pourrait donc être régulée par son adressage subcellulaire. De nombreuses protéines apoptotiques sont régulées par des modifications post-traductionnelles. La mitogaligine possède différents sites de phosphorylation potentiels. Plusieurs d’entre eux ont été mutés afin de mettre en évidence une éventuelle implication de cette fonction dans l’activité de la mitogaligine. La mitogaligine non mutée subit une ou plusieurs coupures protéolytiques. La mutation des résidus thréonines 29 et 73 en résidus phospho-mimétiques diminue cette protéolyse mais aussi la cytotoxicité de la protéine. Le même type de mutations de la thréonine 38 et la sérine 39 provoque une délocalisation de la protéine. La dernière partie de la thèse s’intéresse plus particulièrement à l’interaction fonctionnelle entre la mitogaligine et Mcl-1, une protéine anti-apoptotique de la famille-Bcl-2. La coexpression de galig et Mcl-1 réduit la mort cellulaire induite par l’expression de galig et la surexpression de galig réduit la production endogène de Mcl-1. Ces résultats montrent une implication de galig dans une voie de régulation de l’apoptose. Galig pourrait donc représenter une cible thérapeutique pour restaurer la mort cellulaire dans des cellules surexprimant des protéines antiapoptotiques de la famille Bcl-2. / Mitogaligin is a protein encoded by galig, a cell death gene. Previously, it has been shown in our laboratory, that this mitochondrial protein induces cell death through an alternative pathway of apoptosis. In this report, we show that mitogaligin can also be addressed to the nucleus where it exhibits also a cytotoxic activity. The nuclear localization signal is not conventional, based on a repetition of amino acids, and function only if the mitochondrial localization signal is inactivated. Thus, the cytotoxicity of mitogaligin could be regulated by its subcellular localization. A large number of apoptotic proteins are regulated by post-translational modifications. Mitogaligin has several potential sites of phosphorylation. Some of them have been mutated to highlight the possible role of phosphorylation in the mitogaligin activity. The wild type protein undergoes one or several proteolytic cuts. Replacement of the threonines 29 and 73 by phospho-mimetic residue decrease mitogaligin proteolysis and also mitogaligin cytotoxicity. The same type of mutation on threonine 38 and serine 39 induces a re-localization of mitogaligin outside mitochondria. The last part of the manuscript focuses on the functional interaction between mitogaligin and Mcl-1, an anti-apoptotic protein of the Bcl-2 family. Coexpression of galig and Mcl-1 significantly reduces galig cell death and overexpression of galig reduces production of endogenous Mcl-1. Therefore, galig could represent a therapeutic target in order to restore cell death in cells overexpressing anti-apoptotic proteins of the Bcl-2 family.

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

SHP-1 is a protein tyrosine phosphatase that often targets the phosphatidylinositol 3'-kinase (PI3K)/Akt signalling pathway. PI3K/Akt signalling regulates cell growth and survival, proliferation and differentiation. Growth factor-stimulated PI3K phospholipid production at the plasma membrane helps to recruit 3'-phosphoinositide-dependent protein kinase-1 (PDK1) and Akt, where PDK1 phosphorylates and activates the pro-survival kinase Akt.<p>Tyrosine phosphorylation of PDK1 may regulate its function and, perhaps more importantly, its nuclear localization. Yet, it is unclear how PDK1 is imported into the nucleus as it does not contain a nuclear localization signal (NLS), although it does contain a nuclear export signal (NES). Interestingly, several tyrosines in PDK1 are targets for Src kinase and are putative target motifs for SHP-1, which does have an NLS.<p>Hypothesis: SHP-1 and PDK1 form a tyrosine-dependent, nucleo-cytoplasmic shuttling complex. <p>Removal of serum from C6 glioma cell cultures induces a platelet-derived growth factor receptor (PDGFR)-sensitive redistribution of PI3K lipid kinase activity to the nucleus. PDK1 tyrosine phosphorylation and its association with SHP-1 are also increased, as is the accumulation of both SHP-1 and PDK1 in the nucleus. Site-directed mutagenesis of tyrosine residues in PDK1 reveals that tyrosine 9 (Tyr9) and Tyr376 are important for the interaction of PDK1 with SHP1, whereas Tyr333 and Tyr 373 are not. Using pharmacological and genetic manipulations, it was demonstrated that SHP-1 and PDK1 shuttle between the nucleus and cytoplasm, and that the C-terminal-expressed NLS of SHP-1 facilitates shuttling, while dephosphorylation of PDK1 Tyr9 and Tyr376 regulates the rate of PDK1 (and by virtue of association, SHP-1) export from the nucleus. The SHP-1/PDK1 complex, which is constitutive in most cell lines, is functionally relevant as indicated by its requirement for NGF-induced differentiation of preneuronal cells to a neuronal phenotype.

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.