349 research outputs found
Functions of hepatitis C virus non-structural protein 2
No full textAn estimated 170 million people are infected with hepatitis C virus (HCV). 15-30% of HCV-induced chronic hepatitis progresses to cirrhosis within years to decades after infection, and 3-4% of them will develop hepatocellular carcinoma. There is no vaccine available, and current HCV therapy of pegylated interferon-! in combination with ribavirin leads to a sustained response only in about 50% of infected patients. The HCV non-structural protein NS2 (MW 23 kDa) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. The N terminal region of NS2 interacts with membranes whereas the Cterminal region, together with the N-terminal third of NS3, forms the NS2-3 protease. NS2 is not required for RNA synthesis, although cleavage at the NS2/3 junction is necessary for replication. Further, NS2 has been shown to interact with a number of viral and host proteins; it has been reported to activate transcription factors, inhibit apoptosis, and is a substrate for host kinase phosphorylation and proteosomic degradation. NS2 determinants and their respective function in the HCV life cycle were investigated. Based on the crystal structure of the post-cleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication by preventing NS2/3 cleavage. Supplementary assays indicated a dimerization defect for these mutant constructs, which prevented cleavage and RNA replication. In contrast, alterations in the dimer interface and at the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity, but decreased infectious virus production.
Analysis of the NS2/3 cleavage site revealed an additional function for several residues besides cleavage, notably in infectious virus production. A more comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal residue in infectious particle production. Structural data suggests that the C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the NS2 folding and disrupt potential protein-protein interactions important for infectious particle production. Further, we established an NS2- 3 interaction model based on the solved NS2pro and NS3 crystal structures and assessed the importance of proximal residues for viral propagation. We were able to show genetic interactions between the viral proteins NS2 and E1 as well as NS2 and NS3. Additionally a pull down assay of strep-tagged NS2 followed by a mass spectrometry analysis divulged the physical interactions between NS2-E2, and NS2- NS3. Numerous host cell proteins could be identified to interact with NS2, involved in various pathways such as membrane trafficking, actin/myosin interactions or actin polymerization. In this study we dissected the residues of NS2 involved in its multiple essential roles and interactions in the HCV life cycle and established NS2 as a new viable target for HCV-specific inhibitors for future anti-viral therapeutics
Molecular endocrinology of vitamin D on the epigenome level
No full textThe molecular endocrinology of vitamin D is based on the facts that i) its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the nuclear receptor vitamin D receptor (VDR) and ii) the transcription factor VDR is the unique target of 1,25(OH)2D3 in the nucleus. Short-term alterations of the epigenome are primarily changes in the post-translational modification status of nucleosome-forming histone proteins, the consequences of which are i) a local increase or decrease in chromatin accessibility and ii) the activation or repression of gene transcription. Vitamin D has via VDR a direct effect on the expression of several hundred primary target genes implying numerous effects on the epigenome. Next-generation sequencing methods, such as ChIP-seq and FAIRE-seq, were applied to cellular model systems of vitamin D signaling, such as THP-1 human monocytes, and provided data for a chromatin model of vitamin D signaling. Key points of this model are that i) in the absence of ligand VDR binds to a limited number of loci within accessible chromatin, ii) a stimulation with ligand increases the number of DNA-bound VDR molecules, iii) VDR's access to genomic DNA is supported by pioneer factors, such as PU.1 in monocytes, iv) VDR binding leads to local opening of chromatin and v) the binding strength of topologically associating domain anchor forming CCCTC-binding factor sites upstream and downstream of prominent VDR binding sites is changing in response to ligand stimulation. This model provides the present basis of the molecular endocrinology of vitamin D and will be in future refined by the integration of vitamin D-sensitive chromatin markers and other genome-wide data, such as the 1,25(OH)2D3-sensitive binding of co-factors, chromatin modifying enzymes and chromatin remodeling proteins
A Pleiotropic Nuclear Hormone Labelled Hundred Years Ago Vitamin D
No full textThis year we are celebrating 100 years of the naming of vitamin D, but the molecule is, in fact, more than one billion years old [...
Vitamin D in the Context of Evolution
No full textFor at least 1.2 billion years, eukaryotes have been able to synthesize sterols and, therefore, can produce vitamin D when exposed to UV-B. Vitamin D endocrinology was established some 550 million years ago in animals, when the high-affinity nuclear receptor VDR (vitamin D receptor), transport proteins and enzymes for vitamin D metabolism evolved. This enabled vitamin D to regulate, via its target genes, physiological process, the first of which were detoxification and energy metabolism. In this way, vitamin D was enabled to modulate the energy-consuming processes of the innate immune system in its fight against microbes. In the evolving adaptive immune system, vitamin D started to act as a negative regulator of growth, which prevents overboarding reactions of T cells in the context of autoimmune diseases. When, some 400 million years ago, species left the ocean and were exposed to gravitation, vitamin D endocrinology took over the additional role as a major regulator of calcium homeostasis, being important for a stable skeleton. Homo sapiens evolved approximately 300,000 years ago in East Africa and had adapted vitamin D endocrinology to the intensive exposure of the equatorial sun. However, when some 75,000 years ago, when anatomically modern humans started to populate all continents, they also reached regions with seasonally low or no UV-B, i.e., and under these conditions vitamin D became a vitamin
The Moral Rubicon. A Study of the Principles of Sanctity of Life and Quality of Life in Bioethics.
No full textThe principles of sanctity of life and quality of life are often appealed to in medical decisions at the "edges of life." Notwithstanding their importance in bioethics, these principles are still badly understood. This study is an attempt to clarify their normative significance and content. Because these principles pertain to the more general question of the value of life, the study begins by exploring the possibility of formulating such a value in today's secular pluralistic health-care. This context is often marked by intractable moral disagreement on issues such as abortion and euthanasia. Despite this disagreement, it is argued that human life might, after all, be considered as an "ultimate" value. The author interprets the sanctity-of-life principle as affirming that human life is not self-possessed and is inviolable from the moment of conception. The quality-of-life principle, on the other hand, is interpreted as insisting that life is valuable only if it can serve the person whose life it is to acquire other values. The last chapter explores the relationship between the principles. It is argued that, despite their differences, they can be considered as compatible and that sanctity of life should receive priority in the formulation of any theory of respect for human life
Genome-wide (over)view on the actions of vitamin D
No full textFor a global understanding of the physiological impact of the nuclear hormone α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the analysis of the genome-wide locations of its high affinity receptor, the transcription factor vitamin D receptor (VDR), is essential. Chromatin immunoprecipitation sequencing (ChIP-seq) in GM10855 and GM10861 lymphoblastoid cells, undifferentiated and lipopolysaccharide-differentiated THP-1 monocytes, LS180 colorectal cancer cells and LX2 hepatic stellate cells revealed between 1,000 and 13,000 VDR-specific genomic binding sites. The harmonized analysis of these ChIP-seq datasets indicates that the mechanistic basis for the action of the VDR is independent of the cell type. Formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) data highlight accessible chromatin regions, which are under control of 1,25(OH)2D3. In addition, public data, such as from the ENCODE project, allow to relate the genome-wide actions of VDR and 1,25(OH)2D3 to those of other proteins within the nucleus. For example, locations of the insulator protein CTCF suggest a segregation of the human genome into chromatin domains, of which more than 1,000 contain at least one VDR binding site. The integration of all these genome-wide data facilitates the identification of the most important VDR binding sites and associated primary 1,25(OH)2D3 target genes. Expression changes of these key genes can serve as biomarkers for the actions of vitamin D3 and its metabolites in different tissues and cell types of human individuals. Analysis of primary tissues obtained from vitamin D3 intervention studies using such markers indicated a large inter-individual variation for the efficiency of vitamin D3 supplementation. In conclusion, a genome-wide (over)view on the genomic locations of VDR provides a broader basis for addressing vitamin D’s role in health and disease
- …
