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"Thorpe, Peter"
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Endocrinology of social relationships
This edited work considers the increasing evidence that hormones are as important to social as to reproductive behaviours.
Book
Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications
Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the
Saccharomyces cerevisiae
(budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.
Journal Article
The origin, deployment, and evolution of a plant-parasitic nematode effectorome
Plant-parasitic nematodes constrain global food security. During parasitism, they secrete effectors into the host plant from two types of pharyngeal gland cells. These effectors elicit profound changes in host biology to suppress immunity and establish a unique feeding organ from which the nematode draws nutrition. Despite the importance of effectors in nematode parasitism, there has been no comprehensive identification and characterisation of the effector repertoire of any plant-parasitic nematode. To address this, we advance techniques for gland cell isolation and transcriptional analysis to define a stringent annotation of putative effectors for the cyst nematode
Heterodera schachtii
at three key life-stages. We define 717 effector gene loci: 269 “known” high-confidence homologs of plant-parasitic nematode effectors, and 448 “novel” effectors with high gland cell expression. In doing so we define the most comprehensive “effectorome” of a plant-parasitic nematode to date. Using this effector definition, we provide the first systems-level understanding of the origin, deployment and evolution of a plant-parasitic nematode effectorome. The robust identification of the effector repertoire of a plant-parasitic nematode will underpin our understanding of nematode pathology, and hence, inform strategies for crop protection.
Journal Article
Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA
The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism.
Journal Article
Cell cycle dependent methylation of Dam1 contributes to kinetochore integrity and faithful chromosome segregation
The kinetochore, a megadalton structure composed of centromeric ( CEN ) DNA and protein complexes, is required for faithful chromosome segregation in eukaryotes. The evolutionarily conserved Dam1/DASH complex (Ska1 in metazoans) is one of the essential protein sub-complexes of the budding yeast kinetochore. Previous studies showed that methylation of lysine residue 233 in Dam1 by Set1 is important for haploid growth as mutation of lysine 233 to alanine results in lethality. In this study, we report that Set1-mediated cell cycle dependent Dam1 lysine methylation contributes to kinetochore assembly and chromosomal stability. Our results show that Dam1 methylation is cell cycle regulated with the highest levels of methylation in metaphase. Consistent with these results, co-immunoprecipitation experiments revealed an interaction between Dam1 with Set1 in metaphase cells. Set1 has been shown to colocalize with Jhd2, a histone lysine demethylase which demethylates Set1-methylated histones. Affinity purification-based mass spectroscopy of Jhd2 associated proteins identified seven of the ten subunits of the Dam1 complex; an association of Jhd2 with non-histone proteins, such as Dam1 has not been previously reported. We confirmed the interaction of Jhd2 with Dam1 and showed that cells overexpressing JHD2 exhibit reduced levels of methylated lysine in Dam1 in wild type and UBP8 deletion strains, growth defects in kinetochore mutants, reduced levels of kinetochore proteins at CEN chromatin, defects in kinetochore biorientation and chromosome missegregation. In summary, we have shown that cell cycle dependent methylation of Dam1 plays a crucial role in the maintenance of kinetochore assembly for faithful chromosome segregation.
Journal Article
Comparative transcriptomics and proteomics of three different aphid species identifies core and diverse effector sets
Background
Aphids are phloem-feeding insects that cause significant economic losses to agriculture worldwide. While feeding and probing these insects deliver molecules, called effectors, inside their host to enable infestation. The identification and characterization of these effectors from different species that vary in their host range is an important step in understanding the infestation success of aphids and aphid host range variation. This study employs a multi-disciplinary approach based on transcriptome sequencing and proteomics to identify and compare effector candidates from the broad host range aphid
Myzus persicae
(green peach aphid) (genotypes O, J and F), and narrow host range aphids
Myzus cerasi
(black cherry aphid) and
Rhopalosiphum padi
(bird-cherry oat aphid).
Results
Using a combination of aphid transcriptome sequencing on libraries derived from head versus body tissues as well as saliva proteomics we were able to predict candidate effectors repertoires from the different aphid species and genotypes. Among the identified conserved or core effector sets, we identified a significant number of previously identified aphid candidate effectors indicating these proteins may be involved in general infestation strategies. Moreover, we identified aphid candidate effector sequences that were specific to one species, which are interesting candidates for further validation and characterization with regards to species-specific functions during infestation. We assessed our candidate effector repertoires for evidence of positive selection, and identified 49 candidates with DN/DS ratios >1. We noted higher rates of DN/DS ratios in predicted aphid effectors than non-effectors. Whether this reflects positive selection due to co-evolution with host plants, or increased neofunctionalization upon gene duplication remains to be investigated.
Conclusion
Our work provides a comprehensive overview of the candidate effector repertoires from three different aphid species with varying host ranges. Comparative analyses revealed candidate effectors that are most likely are involved in general aspects of infestation, whereas others, that are highly divergent, may be involved in specific processes important for certain aphid species. Insights into the overlap and differences in aphid effector repertoires are important in understanding how different species successfully infest different ranges of plant species.
Journal Article
Unifying the mechanism of mitotic exit control in a spatiotemporal logical model
The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell–cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.
Journal Article
Synergistic Control of Kinetochore Protein Levels by Psh1 and Ubr2
The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.
Journal Article
ScreenGarden: a shinyR application for fast and easy analysis of plate-based high-throughput screens
Background
Colony growth on solid media is a simple and effective measure for high-throughput genomic experiments such as yeast two-hybrid, synthetic dosage lethality and Synthetic Physical Interaction screens. The development of robotic pinning tools has facilitated the experimental design of these assays, and different imaging software can be used to automatically measure colony sizes on plates. However, comparison to control plates and statistical data analysis is often laborious and pinning issues or plate specific growth effects can lead to the detection of false-positive growth defects.
Results
We have developed ScreenGarden, a shinyR application, to enable easy, quick and robust data analysis of plate-based high throughput assays. The code allows comparisons of different formats of data and different sized arrays of colonies. A comparison of ScreenGarden with previous analysis tools shows that it performs, at least, equivalently. The software can be run either via a website or offline via the RStudio program; the code is available and can be modified by expert uses to customise the analysis.
Conclusions
ScreenGarden provides a simple, fast and effective tool to analyse colony growth data from genomic experiments.
Journal Article