Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell-cell and cell-extracellular matrix interactions remain relatively unexplored. Methods and Results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein-protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness. Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.
Acute Lymphoblastic Leukemia (ALL) non-random fusions influence clinical outcome and alter the accumulation of MTX-PGs in vivo. Analysis of primary ALL samples uncovered subtype-specific patterns of folate gene expression. Using an FPGS-luciferase reporter gene assay, we determined that E2A-PBX1 and TEL-AML1 expression decreased FPGS transcription. ChIP assays uncovered HDAC1, AML1, mSin3A, E2F, and Rb interactions with the FPGS promoter region. We demonstrate that FPGS expression is epigenetically regulated through binding of selected ALL fusions to a multiprotein complex, which also controls the cell cycle dependence of FPGS expression. This study provides insights into the pharmacogenomics of MTX in ALL subtypes.
The liver is the major site of xenobiotic metabolism and detoxification. Primary cultures of hepatocytes are a vital tool in the development of new therapeutic agents but their utility is hindered by the rapid loss of phenotype. Hepatocytes cultured in a sandwich of extracellular matrix protein maintain better hepatic function compared with cells cultured as a monolayer but a wide-ranging proteomics study of the differences in cultures has never been performed. We characterize the changing phenotype of rat hepatocytes in primary culture using iTRAQ proteomics and systems biology network analysis of the identified, significantly regulated, proteins. A total of 754 unique proteins were identified from 4 independent experiments. Of these, 413 proteins were common to at least 3 experiments and 328 proteins were identified in all experiments. Both culture systems displayed altered expression of many common proteins. Network analysis showed that the primary functions of these proteins were in metabolic pathways, immune responses and cytoskeleton remodelling. Monolayer cultures uniquely regulate proteins mapping to pathways of oxidative stress and cell migration, whereas sandwich culture affected translation regulation and apoptosis pathways. These experiments provide a detailed proteomics data set to direct further work into maintaining hepatic phenotype using cultured primary hepatocytes and stem cell derived hepatocyte-like cells.
The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2(-/-)) are highly susceptible to xenobiotic-mediated toxicity, but the precise molecular basis of enhanced toxicity is unknown. Oligonucleotide array studies suggest that a wide range of gene products is altered constitutively, however no equivalent proteomics analyses have been conducted. To define the range of Nrf2-regulated proteins at the constitutive level, protein expression profiling of livers from Nrf2(-/-) and wild type mice was conducted using both stable isotope labelling (iTRAQ) and gel electrophoresis methods. To establish a robust reproducible list of Nrf2-dependent proteins, three independent groups of mice were analysed. Correlative network analysis (MetaCore) identified two predominant groups of Nrf2-regulated proteins. As expected, one group comprised proteins involved in phase II drug metabolism, which were down-regulated in the absence of Nrf2. Surprisingly, the most profound changes were observed amongst proteins involved in the synthesis and metabolism of fatty acids and other lipids. Importantly, we show here for the first time, that the enzyme ATP-citrate lyase, responsible for acetyl-CoA production, is negatively regulated by Nrf2. This latter finding suggests that Nrf2 is a major regulator of cellular lipid disposition in the liver.
The HSPs (hereditary spastic paraplegias) are genetic conditions in which there is distal degeneration of the longest axons of the corticospinal tract, resulting in spastic paralysis of the legs. The gene encoding spartin is mutated in Troyer syndrome, an HSP in which paralysis is accompanied by additional clinical features. There has been controversy over the subcellular distribution of spartin. We show here that, at steady state, endogenous spartin exists in a cytosolic pool that can be recruited to endosomes and to lipid droplets. Cytosolic endogenous spartin is mono-ubiquitinated and we demonstrate that it interacts via a PPXY motif with the ubiquitin E3 ligases AIP4 [atrophin-interacting protein 4; ITCH (itchy E3 ubiquitin protein ligase homologue] [corrected] and AIP5 (WWP1). Surprisingly, the PPXY motif, AIP4 and AIP5 are not required for spartins ubiquitination, and so we propose that spartin acts as an adaptor for these proteins. Our results suggest that spartin is involved in diverse cellular functions, which may be of relevance to the complex phenotype seen in Troyer syndrome.
One of the greatest challenges of the post-genomic era is the construction of a more comprehensive human protein interaction map. While this process may take many years to complete, the development of stringent high throughput techniques and the emergence of complementary assays mean that the aim of building a detailed binary map of the human interactome is now a very realistic goal. In particular, methods which facilitate the analysis of large numbers of membrane-protein interactions mean that it will be possible to construct more extensive networks, which in turn provide new insights into the functional connectivity between intra- and extra-cellular processes. This is important as many therapeutic strategies are designed to elicit effects via tractable cell-surface proteins. Therefore, the construction of maps depicting the complexity of trans-cellular communication networks will not only improve our understanding of physiological processes, it will also aid the design of rational therapeutic strategies, with fewer potential side effects. This review aims to provide a basic insight into the approaches currently being used to construct binary human protein interaction networks, with particular reference to newer techniques, which have the potential to extend network coverage and aid the conditional annotation of interactome-scale protein interaction maps.
The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.
VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the ?-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to ?-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of ?-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type ?-adaptin but not by ?-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.
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