Characterisation of GLUT4 trafficking in HeLa cells: Comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)- containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA–GLUT4–GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA–GLUT4–GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells

The South Asian genome

Genetics of disease Microarrays Variant genotypes Population genetics Sequence alignment AllelesThe genetic sequence variation of people from the Indian subcontinent who comprise one-quarter of the world's population, is not well described. We carried out whole genome sequencing of 168 South Asians, along with whole-exome sequencing of 147 South Asians to provide deeper characterisation of coding regions. We identify 12,962,155 autosomal sequence variants, including 2,946,861 new SNPs and 312,738 novel indels. This catalogue of SNPs and indels amongst South Asians provides the first comprehensive map of genetic variation in this major human population, and reveals evidence for selective pressures on genes involved in skin biology, metabolism, infection and immunity. Our results will accelerate the search for the genetic variants underlying susceptibility to disorders such as type-2 diabetes and cardiovascular disease which are highly prevalent amongst South Asians.Whole genome sequencing to discover genetic variants underlying type-2 diabetes, coronary heart disease and related phenotypes amongst Indian Asians. Imperial College Healthcare NHS Trust cBRC 2011-13 (JS Kooner [PI], JC Chambers)

Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrPSc, suggesting that these domains of cellular prion protein (PrPC) serve as docking sites for PrPSc during prion propagation. To examine the role of polybasic domains in the context of full-length PrPC, we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrPSc molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrPSc prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrPSc molecules. Remarkably, ΔC-PrPSc and other polybasic domain PrPSc molecules displayed diminished or absent biological infectivity relative to wild-type PrPSc, despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrPSc prions interact with PrPC molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrPSc propagation. Furthermore, polybasic domain deficient PrPSc molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrPSc molecules may not depend on a single stereotypic mechanism, but that normal PrPC/PrPSc interaction through polybasic domains may be required to generate prion infectivity

Molecular Interactions between Prions as Seeds and Recombinant Prion Proteins as Substrates Resemble the Biological Interspecies Barrier In Vitro

Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrPC) into the pathogenic isoform (PrPSc). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble α-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrPSc as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrPSc from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

<p>Abstract</p> <p>Background</p> <p>The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as <it>E. coli </it>due to the presence of multiple pathways for their reduction.</p> <p>Results</p> <p>Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of <it>E. coli </it>even without the disruption of genes involved in disulfide bond reduction, for example <it>trxB </it>and/or <it>gor</it>. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an <it>E. coli </it>strain with the reducing pathways intact, than in the commercial Δ<it>gor </it>Δ<it>trxB </it>strain rosetta-gami upon co-expression of Erv1p.</p> <p>Conclusions</p> <p>Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of <it>E. coli </it>and open up new possibilities for the use of <it>E. coli </it>as a microbial cell factory.</p

In Situ Photodegradation of Incorporated Polyanion Does Not Alter Prion Infectivity

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrPSc aggregates in vivo [1], [2]. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrPSc, we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrPC substrate, PC-oligo was incorporated into physical complexes with PrPSc molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrPSc molecules alone

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172

In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Amyloid-Associated Nucleic Acid Hybridisation

Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution Kd, as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA) fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution