N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue.1,2 We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow,3–6 underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair [OMIM 607721]7 shared the 4A>G missense change (Ser2Gly) in SHOC2 that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.
Nowadays, tobacco smoking is the cause of ~5-6 million deaths per year, counting 31% and 6% of all cancer deaths (affecting 18 different organs) in middle-aged men and women, respectively. Nicotine is the addictive component of tobacco acting on neuronal nicotinic receptors (nAChR). Functional nAChR, are also present on endothelial, haematological and epithelial cells. Although nicotine itself is regularly not referred to as a carcinogen, there is an ongoing debate whether nicotine functions as a 'tumour promoter'. Nicotine, with its specific binding to nAChR, deregulates essential biological processes like regulation of cell proliferation, apoptosis, migration, invasion, angiogenesis, inflammation and cell-mediated immunity in a wide variety of cells including foetal (regulation of development), embryonic and adult stem cells, adult tissues as well as cancer cells. Nicotine seems involved in fundamental aspects of the biology of malignant diseases, as well as of neurodegeneration. Investigating the biological effects of nicotine may provide new tools for therapeutic interventions and for the understanding of neurodegenerative diseases and tumour biology.
The conversion of the normal cellular prion protein (PrP C ) into the abnormal scrapie isoform (PrP Sc ) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP C and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP C we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP C and on PrP Sc formation. We found that ER-targeted anti-prion scFvs retain PrP C in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP C , with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP C acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP Sc accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.
We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic.Keywords: intracellular antibodies; scFv fragments; anti-p21Ras; phage display library.The antibody-based intracellular immunization strategy exploits the ectopic expression of recombinant antibodies to redirect antibodies to different intracellular compartments, to inhibit the function of selected antigens in different biological systems [1±3]. By using this approach the function of several intracellular gene products has been successfully inhibited in the cytoplasm, in the nucleus and in the secretory compartments [4±9]. Intracellular antibodies have been prospected as tools for gene therapy [10] and for functional genomics [11]. In a gene therapy setting, the therapeutic antibody is likely to be the outcome of a long and careful optimization process. On the other hand, in the context of functional genomics project, where intracellular antibodies could be used in a high throughput fashion to assess the function of genes coming from genome projects, the development of reliable methods linking the isolation of recombinant antibodies derived from phage libraries to their intracellular expression in mammalian cells is required. This strategy would require that a high proportion of the isolated antibodies are functional when expressed as intrabodies in the cell cytoplasm.The aim of our paper was to investigate what proportion of the antibody fragments isolated from phage libraries are effective as intrabodies and to assess whether in vivo inhibition of protein function is crucially dependent on a particular antibody with well defined properties or whether it can be obtained by a wide spectrum of single-chain variable fragments (scFv). To this aim we exploited the Ras system, a signal transduction protein which has been successfully inhibited with one intracellular antibody, derived from the well ch...
Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the central nervous system (CNS). The tumor suppressor protein p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. We investigated the role of p53 and related molecular mechanisms that support oxidative stress-induced apoptosis in glia. For this purpose, we exposed C6 glioma cells and primary cultures of rat cortical astrocytes to an H(2)O(2)-induced oxidative stress protocol followed by a recovery period. We evaluated the effects of pifithrin-alpha (PF-alpha), which has been reported to protect neurons from ischemic insult by specifically inhibiting p53 DNA-binding activity. Strikingly, PF-alpha was unable to prevent oxidative stress-induced astrocyte apoptosis. We demonstrate that p53 is able to mediate an apoptotic response by direct signaling at mitochondria, despite its transcriptional activity. The z-VAD-fmk-sensitive apoptotic response requires a caspase-dependent MDM-2 degradation, leading to p53 mitochondrial targeting accompanied by cytochrome c release and nucleosomal fragmentation.
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