Alena Pance

Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor

Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150–268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFΔ150–268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF–HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.

Secretory granule biogenesis in sympathoadrenal cells : Identification of a granulogenic determinant in the secretory prohormone chromogranin A

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules ∼180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.

Heat shock enhances transcriptional activation of the murine inducible nitric oxide synthase gene

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivofootprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ‐898/9, at a putative partial heat shock element (HSE) and at ‐893/4, a site bordering an E‐box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E‐box and a partial HSE within the iNOS enhancer. Binding to the E‐box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heat shock factor‐1 (HSF‐1). Mutations at the HSE revealed the importance of HSF‐1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever‐associated gram‐negative infection.

A repressor in the proximal human inducible nitric oxide synthase promoter modulates transcriptional activation

The human inducible nitric oxide synthase (iNOS or NOSII) gene is regulated through an extended and complex promoter. In this study, the transcriptional regulation of human NOSII is investigated in the human colon cell line HCT‐8R. Stimulation with a cytokine mix (interferon‐γ, interleukin 1‐β, and tumor necrosis factor α) induces NOSII mRNA accumulation, as well as promoter activity in these cells. Several random deletions were performed within the proximal 7 kb of the promoter, which led to the identification of a region, whose deletion provokes a marked increase in transcriptional activity upon cytokine stimulation. Furthermore, this region is shown to repress a viral‐driven luciferase construct, mainly at basal levels. An AP‐1‐like sequence present in this region that is specifically recognized by nuclear proteins is shown to be involved in the repressive effect. This element is capable of repressing a viral promoter, and its deletion augments cytokine‐stimulated transcription. These findings are confirmed in various cell lines and suggest a general mechanism for the control of basal levels of NOSII expression, to avoid unnecessary toxicity under normal conditions.

A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory‐deficient PC12 cells

The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down‐regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1‐silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant‐negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the β3 subunit of the voltage‐gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re‐expressed. Granule and synaptic vesicle proteins were not re‐expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post‐transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory‐deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

Hemopoietic-specific Sf3b1 -K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3′ splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

A PC12 variant lacking regulated secretory organelles: aberrant protein targeting and evidence for a factor inhibiting neuroendocrine gene expression.

Abstract: A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein‐converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine β‐hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine‐specific gene expression.

Nitric oxide and hormones in breast cancer: allies or enemies?

Unlike other types of cancer, tumors of the breast are greatly influenced by steroid hormones. The effect of estrogen and progesterone depends on the presence of their specific receptors and these constitute important parameters in determining the aggressiveness of the tumor, the feasibility of certain therapies and the prediction of relapse. The molecular mechanisms of steroid hormone action have not been fully elucidated but recent findings implicate the nitric oxide (NO) pathway in some of these effects. Both hormones can regulate the nitric oxide synthases (NOS) and, in turn, the NO produced has profound consequences on tumor cell homeostasis. On one hand, estrogen increases the activity of endothelial NOS (eNOS or NOSIII), while progesterone activates inducible NOS (iNOS or NOSII) expression. The data presented suggest that the low levels of NO produced by NOSIII mediate the proliferative effect of estrogen. On the other hand, the increase in apoptosis in response to progesterone could implicate the high levels of NO produced by induction of NOSII expression. Understanding of the mechanisms and interactions of steroid hormones with the NO pathway could lead to the development of new approaches and strategies for the effective treatment of breast cancer.

Antitumoral Effects of Lipid A: Preclinical and Clinical Studies

Cancer is the second cause of death in developed countries. Surgery and radiotherapy are widely used in the treatment of localized and attainable tumors. Chemotherapy and immunotherapy are used as adjuvant treatments or as therapies for advanced-stage diseases. However, chemotherapy often leads to a high relapse rate because of drug resistance, which might be overcome by immunotherapy. When the immune system is not destroyed by chemotherapy, it is able to recognize tumor-specific antigens and eventually can eliminate the cancer cells. One of the means of achieving this goal is the use of lipopolysaccharides (LPSs). LPSs are components of the outer membrane of Gram-negative bacteria and are composed of a polysaccharide, an oligosaccharide core, and lipid A. These compounds have the property of inducing the secretion of various cytokines, such as tumor necrosis factor-α (TNF-α),1interferon-γ (IFN-γ),2interleukin 1-β (IL1-β),3and interleukin-6 (IL-6),4as well as activating immune cells, including neutrophils,5macrophages,6and CD4 and CD8 T lymphocytes that infiltrate the tumors.7,8Furthermore, these compounds have been shown to decrease suppressive cytokines such as transforming growth factor-β (TGF-β).4,9 The first assays with bacterial extracts containing LPS to be used as a treatment for cancer were performed in clinical trials in 1898.10A half-century later, the antitumoral effect was attributed to LPSs in murine subcutaneous tumors,11and eventually it was demonstrated that this effect is due to the lipid A component of LPSs.12Because lipid A and its derivatives are less toxic than LPSs, most anticancer treatments aimed at activating an immune response against tumors have been developed with the use of natural lipid A or synthetic analogs, either alone or as adjuvants, to enhance the efficacy of therapeutic anticancer vaccines. Animal models permit the investigation of the mechanisms of the …

Distinct domains of the sodium channel β3-subunit modulate channel-gating kinetics and subcellular location

Electrical excitability in neurons depends on the expression and activity of voltage-gated sodium channels in the neuronal plasma membrane. The ion-conducting alpha-subunit of the channel is associated with auxiliary beta-subunits of which there are four known types. In the present study, we describe the first detailed structure/function analysis of the beta3-subunit. We correlate the effect of point mutations and deletions in beta3 with the functional properties of the sodium channel and its membrane-targeting behaviour. We show that the extracellular domain influences sodium channel gating properties, but is not required for the delivery of beta3 to the plasma membrane when expressed with the alpha-subunit. In contrast, the intracellular domain is essential for correct subunit targeting. Our results reveal the crucial importance of the Cys21-Cys96 disulphide bond in maintaining the functionally correct beta3 structure and establish a role for a second putative disulphide bond (Cys2-Cys24) in modulating channel inactivation kinetics. Surprisingly, our results imply that the wild-type beta3 molecule can traverse the secretory pathway independently of the alpha-subunit.