Tuning the mechanical properties of self-assembled mixed-peptide tubes.

In this study, nano- and microscale fibrillar and tubular structures formed by mixing two aromatic peptides known to self-assemble separately, (diphenylalanine and di-D-2-napthylalanine) have been investigated. The morphology, mechanical strength and thermal stability of the tubular structures formed have been studied. The tubes are shown to consist of both peptides with some degree of nanoscale phase separation. The ability of the mixed peptides to form structures, which display variable mechanical properties dependent on the percentage composition of the peptides is presented. Such materials with tuneable properties will be required for a range of applications in nanotechnology and biotechnology.

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues.

The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, type II integral membrane protein, is retained in the late Golgi apparatus has been investigated. Prior work demonstrated that the 118-amino acid cytoplasmic domain is both necessary and sufficient for Golgi retention and that mutant or overexpressed DPAP A no longer retained in the Golgi was delivered directly to the vacuolar membrane (Roberts, C. J., S. F. Nothwehr, and T. H. Stevens. 1992. J. Cell Biol. 119:69-83). Replacement of the DPAP A transmembrane domain with a synthetic hydrophobic sequence did not affect either Golgi retention of DPAP A or vacuolar delivery of the retention-defective form of DPAP A. These results indicate that the DPAP A transmembrane domain is not involved in either Golgi retention or targeting of this membrane protein. A detailed mutational analysis of the cytoplasmic domain of DPAP A indicated that the most important elements for retention were within the eight residue stretch 85-92. A 10-amino acid region from DPAP A (81-90) was sufficient for Golgi retention of alkaline phosphatase, a type II vacuolar membrane protein. Detailed mutational analysis within this 10-amino acid sufficient region demonstrated that a Phe-X-Phe-X-Asp motif was absolutely required for efficient retention. The efficiency of Golgi retention via the DPAP A signal could be diminished by overexpression of wild type but not retention-defective versions of Kex2p, another late Golgi membrane protein, suggesting that multiple Golgi membrane proteins may be retained by a common machinery. These results imply a role for a cytoplasmic signal involving aromatic residues in retention of late Golgi membrane proteins in the yeast Saccharomyces cerevisiae.

Membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment.

The targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (DPAPs), DPAP B and DPAP A, which reside in the vacuole and the Golgi apparatus, respectively, were analyzed. No single domain of DPAP B is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. DPAP A was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast Golgi apparatus. The 118-amino acid cytoplasmic domain of DPAP A is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of DPAP B with that of DPAP A resulted in an immunolocalization pattern indistinguishable from that of wild type DPAP A. Overproduction of DPAP A resulted in its mislocalization to the vacuole, because cells expressing high levels of DPAP A exhibited vacuolar as well as Golgi staining. Deletion of 22 residues of the DPAP A cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. Thus, the cytoplasmic domain of DPAP A is both necessary and sufficient for Golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing DPAP A, resulted in transport to the vacuole. Like wild type DPAP B, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec1 mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. These data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway.

Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole.

We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2CO3 treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precursor form of DPAP B accumulates in sec mutants (Novick, P., C. Field, and R. Schekman. 1980. Cell. 21:205-215) that are blocked at the ER (sec18) or Golgi apparatus (sec7), but not at secretory vesicles (sec1). Immunolocalization of DPAP B in wild-type or sec1 mutant cells shows that the protein resides in the vacuolar membrane. However, it is present in non-vacuolar compartments in sec18 and sec7 cells, confirming that the delivery of DPAP B is blocked in these mutants. Interestingly, DPAP B appears to stain the nuclear envelope in a sec18 mutant, which is consistent with the accumulation of DPAP B in the ER membrane at the restrictive temperature. These results suggest that soluble and membrane-bound vacuolar proteins use the same stages of the secretory pathway for their transport.

Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation.

The function and regulation of DNA methylation in eukaryotes remain unclear. Genes affecting methylation were identified in the fungus Neurospora crassa. A mutation in one gene, dim-2, resulted in the loss of all detectable DNA methylation. Abnormal segregation of the methylation defects in crosses led to the discovery that the methylation mutants frequently generate strains with extra chromosomes or chromosomal parts. Starvation for S-adenosylmethionine, the presumed methyl group donor for DNA methylation, also produced aneuploidy. These results suggest that DNA methylation plays a role in the normal control of chromosome behavior.

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Genetic selection of peptide inhibitors of biological pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Insulin fibril nucleation: the role of prefibrillar aggregates.

Dynamic light scattering and Fourier transform infrared spectroscopy were used to study the formation of prefibrillar aggregates and fibrils of bovine pancreatic insulin at 60 degrees C and at pH 1. The kinetics of disintegration of the prefibrillar aggregates were also studied using these techniques after a quench to 25 degrees C. These experiments reveal that formation of prefibrillar aggregates is reversible under the solution conditions studied and show that it is possible to significantly reduce the nucleation (lag) times associated with the onset of fibril growth in bovine pancreatic insulin solutions by increasing the concentration of prefibrillar aggregates in solution. These results provide convincing evidence that less structured prefibrillar aggregates can act as fibril-forming intermediates.

The application of ToF-SIMS to the analysis of herbicide formulation penetration into and through leaf cuticles.

Understanding the movement of the active ingredient in relation to the other formulation components following application is crucial to an overall understanding of herbicide performance. We describe the novel use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) as a tool for following the movement of herbicide formulation components into and across plant cuticles. This technique provides new insights since it provides both high (sub-micron) spatial resolution combined with the chemical specificity associated with organic mass spectrometry. The components studied include the oligomeric ethoxylate surfactants Synperonic A7 and A20 and active ingredient Sulfosate (trimesium glyphosate). The movement of these molecules, both separately and when combined in a simple formulation, into the surface of Prunus laurocerasus leaves and across the isolated plant cuticle was investigated and clear differences in penetration/diffusion behaviour were identified. ToF-SIMS was uniquely able to (simultaneously) spatially resolve all the species involved, including the anion and cation components of the active ingredient. Also, using spectral reconstructions from the imaging raw data streams, the behaviour of individual oligomers within the surfactant distributions, could be assessed. The observations are discussed with reference to the action of surfactants identified in parallel micro-structural studies and the current understanding of herbicide uptake.

The impact of an emotionally expressive writing intervention on eating pathology in female students.

Introduction: Previous research demonstrating emotional influences on eating and weight suggest that emotionally expressive writing may have a significant impact on reducing risk of eating pathology. This study examined the effects of writing about Intensely Positive Experiences on weight and disordered eating during a naturalistic stressor. Method: Seventy-one female students completed an expressive or a control writing task before a period of exams. Both groups were compared on BMI (kg/m2) and the Eating Disorder Examination - Questionnaire (EDE-Q) before the writing task and at 8-week follow-up. A number of secondary analyses were also examined (to identify potential mediators) including measures of attachment, social rank, self-criticism and self-reassurance, stress and mood. Results: There was a significant effect of intervention on changes in the subscales of the EDE-Q (p = .03). Specifically, expressive writers significantly reduced their dietary restraint while those in the control group did not. There was no significant effect of the intervention on changes in BMI or the other subscales of the EDE-Q (Eating, Weight and Shape Concern). There was also no effect of writing on any of the potential mediators in the secondary analyses. Discussion: Emotionally expressive writing may reduce the risk of dietary restraint in women but these findings should be accepted with caution. It is a simple and light touch intervention that has the potential to be widely applied. However, it remains for future research to replicate these results and to identify the mechanisms of action.

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

The role of the 3-hydroxy 3-methylglutaryl coenzyme A reductase cytosolic domain in karmellae biogenesis.

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using beta-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae.

Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells. Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown. Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents. We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents. Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents. Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen.

Factors predictive of patient outcome following total wrist arthrodesis.

AIMS: Total wrist arthrodesis (TWA) produces a spectrum of outcomes. We investigated this by reviewing 77 consecutive TWA performed for inflammatory and post-traumatic arthropathies, wrist instability and as a salvage procedure. PATIENTS AND METHODS: All operations were performed by a single surgeon using a specifically designed pre-contoured dorsally applied non-locking wrist arthrodesis plate at a single centre. RESULTS: Median post-operative Buck-Gramcko Lohman (BGL), Disabilities of the Arm, Shoulder and Hand and Patient Rated Wrist Evaluation scores at six years (interquartile range (IQR) 3 to 11) were 9 (IQR = 6 to 10), 19 (IQR = 7 to 45) and 13 (IQR = 1 to 31) respectively. Polyarticular inflammatory arthritis and female gender were associated with poorer patient-reported outcomes, although the effect of gender was partly explained by higher rates of inflammatory disease among women. Return to work was negatively influenced by workers' compensation and non-inflammatory wrist pathology. There was no difference in complication rates for inflammatory and non-inflammatory indications. TAKE HOME MESSAGE: Polyarticular inflammatory arthritis is a risk factor for adverse patient-reported outcomes in TWA. Furthermore, when compared with patients without inflammatory arthritis, dorsally applied pre-contoured plates can be used for wrist arthrodesis in patients with inflammatory arthritis without an increased risk of complications. Cite this article: Bone Joint J 2016;98-B:647-53.

Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.