Three structural motifs in the invariant chain (li) control the intracellular transport of class II major histocompatibility complex molecules. An endoplasmic reticulum retention signal in the full-length li suggests a role for li in the alpha-beta heterodimer assembly. Another signal motif directs a truncated li, alone or associated with individual class II chains, to a degradation compartment by a pathway circumventing the Golgi. When this truncated li binds alpha-beta dimers, a third signal dominates, directing the complex by way of the Golgi to vesicles in the cell periphery, which may represent a subcompartment of recycling endosomes.
We investigated the molecular events involved in the long-lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet-induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716-treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high-fat regimen and which are resistant to obesity, supporting a CB1 receptor-mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the beta-oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity-related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.
Professional antigen-presenting cells (APCs) have a distinct compartment in which class II molecules are proposed to acquire antigenic peptides. Genetic evidence suggests that human leukocyte antigen (HLA)-DM, an unusual class II molecule, participates in this process. Peptide acquisition was reconstituted in nonprofessional APCs by transfection of class II, invariant chain (li), and H-2M, the murine equivalent of DM. The H-2M heterodimer appeared in an endosomal compartment, not at the cell surface, and the localization was independent of li. The data presented show that H-2M, class II, and li are the minimally required components for efficient formation of stable class II-peptide complexes, and thus for a functional class II compartment.
The nonpolymorphic human class II molecule HLA-DM (DM) has been found to play a key role in antigen presentation by MHC class II molecules. HLA-DM and its murine equivalent H2-M are located intracellularly and are absent from the cell surface. In transfected HeLa cells, H2-M was transported to an endosomal compartment in the absence of invariant chain. A tyrosine-based targeting motif in the cytoplasmic tail of H2-M beta was responsible for the endosomal location and, if this tyrosine was mutated, H2-M accumulated at the cell surface. In the presence of invariant chain the mutated H2-M was redistributed to endosomes. The targeting motif of H2-M appeared not to be crucial for efficient peptide loading of class II, but if the invariant chain targeting motif also was removed, peptide loading decreased drastically. Thus, the targeting motif of H2-M appears to be supplementary, rather than essential for class II-peptide association.
The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, aSaccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.
The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N-and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). The viral Tax protein has been proposed to contribute to the proliferation and the transformation of T cells by HTLV-1 (reviewed in references 23 and 54). Tax is involved in transcriptional regulation of several cellular genes and is also a potent transactivator of transcription from the viral long terminal repeat (LTR) promoter (15,21,25,73). Three 21-bp repeats located in the U3 region of the LTR are sufficient to confer transactivation of transcription by Tax (12,26,63,69). These conserved repeats, called Tax-responsive elements (TxREs), can be subdivided into three motifs known as A, B, and C. The central domains B correspond to imperfect cyclic AMP response elements (CRE) which are able to interact in vitro with multiple members of the activating transcription factor/CREbinding protein (ATF/CREB) family (4,7,24,70,77,89). The domains A and C of TxREs can also be recognized by cellular factors, such as AP-2, HEB-1, 66).To activate transcription of the HTLV-1 genome, Tax does not bind specifically to DNA (8, 28, 38) but interacts with cellular ATF/CREB factors bound to the LTR. These factors are characterized by basic-leucine zipper (bZIP) C-terminal structures required for DNA binding and protein dimerization. Tax enhances the binding affinity of these bZIP transcriptional factors for the 21-bp motifs (3,24,81,85,90), probably by stabilizing the LTR-bound complexes through direct contacts with nucleotides flanking domain B (43, 48, 52). It has also been suggested that Tax could increase the DNA binding activity of ATF/CREB factors by promoting dimerization of their bZIP domain (3,39,81). Then, the LTR-associated Tax molec...
Oxaliplatin (Eloxatin) is a third-generation platinum derivative with an in vitro and in vivo spectrum of activity distinct from that of cisplatin, especially in colon cancer cells. Here, we studied the molecular basis of this difference on the HCT-116 human colon carcinoma cell line (mismatch repair-deficient, wild-type functional p53). Oxaliplatin inhibited HCT-116 cell proliferation with greater efficacy than cisplatin. At comparable concentrations, cisplatin slowed down the replication phase and activated the G 2 -M checkpoint, whereas oxaliplatin activated the G 1 -S checkpoint and completely blocked the G 2 -M transition. With the aim of finding oxaliplatinspecific target genes and mechanisms differing from those of cisplatin, we established the transcriptional signatures of both products on HCT-116 cells using microarray technology. Based on hierarchical clustering, we found that (a) many more genes were modulated by oxaliplatin compared with cisplatin and (b) among the 117 modulated genes, 79 were regulated similarly by both drugs and, in sharp contrast, 38 genes were dose dependently down-regulated by oxaliplatin and, conversely, up-regulated or unaffected by cisplatin. Interestingly, several cell cycle -related genes encoding proteins involved in DNA replication and G 2 -M progression belong to this latter group. RNA modulations, confirmed at the protein level, were in accordance with oxaliplatin-and cisplatin-induced cell cycle variations. Beyond the identification of genes affected by both drugs, the identified oxaliplatin-specific target genes could be useful as predictive markers for evaluating and comparing the efficacy and molecular pharmacology of platinum drugs.[Mol Cancer Ther
The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N-and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.
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