Identification of Chlamydial Iron-Responsive Proteins during Intracellular Growth.

Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent cause of bacterial sexually transmitted disease. Genital chlamydial infections, marked by chronic, intense inflammation, can lead to genital tissue scarring and infertility and is a contributing factor to development of pelvic inflammatory disease and ectopic pregnancy. Iron is required as a cofactor for numerous highly conserved pathways, and nearly all studied organisms rely on iron for growth. In response to iron restriction, the chlamydial developmental cycle arrests at the intracellular reticulate body stage, resulting in a phenomenon termed persistence. Persistence likely plays a role in chlamydial pathogenesis through the expression of virulence factors and antigens in addition to sustaining chronic infection; however, little is known concerning how chlamydiae respond to iron limitation at the molecular level, and no systems for iron acquisition have been identified in Chlamydia. This dissertation presents an investigation into the chlamydial response to iron restriction. Chlamydial heat shock protein 60 (cHsp60) has been implicated in development of the more severe disease sequelae and has been found to increase in expression following iron restriction; however, three cHsp60 homologues were identified following the sequencing of the chlamydial genome. Here, iron restriction is shown to increase expression of cHsp60-2 but not the two other homologs, cHsp60-1 or -3. Next, in order to investigate an alternate model for restricting iron availability to chlamydiae, a cell line with inducible expression of recombinant ferroportin, a eukaryotic iron efflux protein, was examined. Lastly, 10 chlamydial proteins differentially expressed during growth in iron-restricted host cells were identified by proteomic analysis of radiolabeled proteins followed by mass spectrometry analysis; transcripts encoding 5 iron responsive proteins were examined across a timecourse of infection and revealed increased transcript levels at 18 and/or 24 hours post infection. Together, these studies have examined the molecular response of chlamydiae to reduced iron availability and have underlined the importance for pathways involved in protection against oxidative damage and adaptation to stress

NUAK2 is a critical YAP target in liver cancer

Hippo-YAP pathway plays an important role in cancers; however the in vivo relevance of YAP/TAZ target genes is unclear. Here, the authors show that NUAK2 is a target of YAP and participates in a feedback loop to maximize YAP activity. Inhibition of NUAK2 suppresses YAP-driven hepatomegaly and liver cancer growth, offering a new target for cancer therapy

Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associ- ated with Parkinson’s disease, chronic inflammation and mycobac- terial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobac- terial control independently of autophagy. In vivo, LRRK2 defi- ciency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2- dependent cellular pathway that controls M. tuberculosis replica- tion by regulating phagosome maturation

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

International audienceMutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser 65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embry-onic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser 111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser 111 of each of the Rabs, we demonstrate that Rab Ser 111 phosphoryla-tion occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolari-sation. We provide evidence that Rab8A GTPase Ser 111 phosphory-lation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser 111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser 65. We further show mechanistically that phosphorylation at Ser 111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/ 8B/13 at Ser 111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease

IFNs Modify the Proteome of <i>Legionella</i>-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria

The Influence of professional doctorates on practice and the workplace

This paper investigates the influence that undertaking a professional doctorate has on the practice of the graduate and their workplace or organisation. There is a growing literature on how undertaking such advanced development influences the individual graduate at the personal and professional level but there is little evidence of a wider impact on practice in general or at the organisational level. This study seeks to address this issue through a qualitative study of practitioners from a range of professional arenas and sectors who have graduated from a professional doctorate within the past 10 years. Through thematic analysis of semi-structured interviews and the candidates’ project reports we explore their experience of applying their learning within their workplaces providing insight into the level and degree of influence such development can have on organisational contexts. The paper does not focus on the academic or personal impact of their experience as the intention of these particular doctoral researchers is the creation of new knowledge embedded in practice

Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection

Maize Genomes to Fields: 2014 and 2015 field season genotype, phenotype, environment, and inbred ear image datasets

Objectives: Crop improvement relies on analysis of phenotypic, genotypic, and environmental data. Given large, well-integrated, multi-year datasets, diverse queries can be made: Which lines perform best in hot, dry environments? Which alleles of specific genes are required for optimal performance in each environment? Such datasets also can be leveraged to predict cultivar performance, even in uncharacterized environments. The maize Genomes to Fields (G2F) Initiative is a multi-institutional organization of scientists working to generate and analyze such datasets from existing, publicly available inbred lines and hybrids. G2F’s genotype by environment project has released 2014 and 2015 datasets to the public, with 2016 and 2017 collected and soon to be made available. Data description: Datasets include DNA sequences; traditional phenotype descriptions, as well as detailed ear, cob, and kernel phenotypes quantified by image analysis; weather station measurements; and soil characterizations by site. Data are released as comma separated value spreadsheets accompanied by extensive README text descriptions. For genotypic and phenotypic data, both raw data and a version with outliers removed are reported. For weather data, two versions are reported: a full dataset calibrated against nearby National Weather Service sites and a second calibrated set with outliers and apparent artifacts removed