Phenotype-specific store-operated calcium entry and the differentiation response in neuroblastoma cells /

PhD ThesisUnderstanding the fundamental molecular mechanisms that control the proliferationdifferentiation cellular switch and maintenance of the differentiated state is needed to fully harness the therapeutic potential for highly detrimental diseases such as cancer and neurodegenerative disorders. Intracellular free Ca2+ plays an essential role in the differentiation process and, more specifically, the ubiquitous Ca2+ signalling pathway; storeoperated Ca2+ entry (SOCE) is altered with differentiation. The SH-SY5Y neuroblastoma cancer cell line was utilised in this study to investigate the role of SOCE in phenotype-specific differentiation responses using morphological, biochemical and functional single cell, Ca2+ imaging techniques. Neuroblastoma is a paediatric malignancy of the sympathetic nervous system that is comprised of immature neural crest cells. Retinoic acid is used to treat neuroblastoma patients however many respond poorly, leading to aggressive disease progression. The SHSY5Y neuroblastoma-derived cell line consists of three morphologically distinct phenotypes; immature neuroblastic N-type cells, non-neuronal S-type cells and putative intermediate Itype cells, which exhibit variable tumourgenicity and can be induced to differentiate using 9- cis-retinoic acid (9cRA). The 9cRA-induced differentiation response of N-type and S-type populations involved morphological changes accompanied by an uncoupling of SOCE from Ca2+ store release that could be observed from the first day of 9cRA treatment. SOCE down-regulation was attributed to changes in expression and localisation of the CRAC channel protein Orai1 and the Ca2+ sensing protein STIM1. The extent of SOCE uncoupling was influenced in N-type and I-type cells but not S-type cells by the predominant background cell environment. Conditioned media from proliferating and differentiating N-type and S-type populations was also able to influence cell phenotype and the associated SOCE responses. This study describes how the 9cRA-induced differentiation response occurred in a multi-step manner in N-type populations and in gradual manner in S-type populations and raises the possibility that SOCE proteins could potentially be utilised as drug targets in neuroblastoma treatment or neurodegenerative disease therapy

SPR-measured dissociation kinetics of PROTAC ternary complexes influence target degradation rate

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation <i>in vitro</i> using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4^BD2 or Brd2^BD2 and VHL. Equivalent complexes with Brd3^BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation

The structure of protostellar envelopes derived from submillimeter continuum images

High dynamic range imaging of submillimeter dust emission from the envelopes of eight young protostars in the Taurus and Perseus star-forming regions has been carried out using the SCUBA submillimeter camera on the James Clerk Maxwell Telescope. Good correspondence between the spectral classifications of the protostars and the spatial distributions of their dust emission is observed, in the sense that those with cooler spectral energy distributions also have a larger fraction of the submillimeter flux originating in an extended envelope compared with a disk. This results from the cool sources having more massive envelopes rather than warm sources having larger disks. Azimuthally-averaged radial profiles of the dust emission are used to derive the power-law index of the envelope density distributions, p (defined by rho proportional to r^-p), and most of the sources are found to have values of p consistent with those predicted by models of cloud collapse. However, the youngest protostars in our sample, L1527 and HH211-mm, deviate significantly from the theoretical predictions, exhibiting values of p somewhat lower than can be accounted for by existing models. For L1527 heating of the envelope by shocks where the outflow impinges on the surrounding medium may explain our result. For HH211-mm another explanation is needed, and one possibility is that a shallow density profile is being maintained in the outer envelope by magnetic fields and/or turbulence. If this is the case star formation must be determined by the rate at which the support is lost from the cloud, rather than the hydrodynamical properties of the envelope, such as the sound speed.Comment: Accepted for publication in the Astrophysical Journa

Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple

<p>Abstract</p> <p>Background</p> <p>Integrating plant genomics and classical breeding is a challenge for both plant breeders and molecular biologists. Marker-assisted selection (MAS) is a tool that can be used to accelerate the development of novel apple varieties such as cultivars that have fruit with anthocyanin through to the core. In addition, determining the inheritance of novel alleles, such as the one responsible for red flesh, adds to our understanding of allelic variation. Our goal was to map candidate anthocyanin biosynthetic and regulatory genes in a population segregating for the red flesh phenotypes.</p> <p>Results</p> <p>We have identified the <it>Rni </it>locus, a major genetic determinant of the red foliage and red colour in the core of apple fruit. In a population segregating for the red flesh and foliage phenotype we have determined the inheritance of the <it>Rni </it>locus and DNA polymorphisms of candidate anthocyanin biosynthetic and regulatory genes. Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs) in the candidate genes were also located on an apple genetic map. We have shown that the MdMYB10 gene co-segregates with the <it>Rni </it>locus and is on Linkage Group (LG) 09 of the apple genome.</p> <p>Conclusion</p> <p>We have performed candidate gene mapping in a fruit tree crop and have provided genetic evidence that red colouration in the fruit core as well as red foliage are both controlled by a single locus named <it>Rni</it>. We have shown that the transcription factor MdMYB10 may be the gene underlying <it>Rni </it>as there were no recombinants between the marker for this gene and the red phenotype in a population of 516 individuals. Associating markers derived from candidate genes with a desirable phenotypic trait has demonstrated the application of genomic tools in a breeding programme of a horticultural crop species.</p

Liberal warriors and the violent colonial logics of “partnering and advising"

Building on the feminist literature that traces the (re)production of militarized masculinities in and through military interventions, this article details some of the ways British soldiering subjects are being shaped in today's counterinsurgency context. Required now to be both nation builders and war fighters, contemporary soldiers are a “softer,” less masculinized subjectivity, and what Alison Howell has termed “liberal warriors.” British troops with their long history of colonialism and frequent overseas military campaigns are understood to be particularly suited to this role. Taking the British military's involvement in the “partnering and advising” of the Afghan National Army (ANA), this article pays attention to the interlocking gendered, raced, and sexualized discourses through which the British/Afghan encounter is experienced. Exploring first British troops' preoccupation with the perceived femininity and homosexuality of their Afghan counterparts, and second, Afghan hypermasculinity as demonstrated by the characterizations of their violent and chaotic fighting tactics, colonial logics are revealed. While British liberal warriors come to know “who they are” through these logics, (mis)represented Afghan soldiers are rendered increasingly vulnerable to the very “real,” very material violences of war

Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation.

Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). Here we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1

Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation.

Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). Here we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1

Adiposity and cardiovascular outcomes in three-year-old children of participants in UPBEAT, an RCT of a complex intervention in pregnant women with obesity

Background: Maternal obesity is associated with offspring cardiometabolic risk. UPBEAT was a randomised controlled trial of an antenatal diet and physical activity intervention in 1555 women with obesity. The intervention was associated with lower gestational weight gain, healthier diet and metabolic profile in pregnancy, and reduced infant adiposity at six months. Objective: We have investigated whether the UPBEAT intervention influenced childhood cardiometabolic outcomes or was associated with sustained improvements in maternal lifestyle 3-years after delivery. Methods: In UPBEAT mother-child dyads at the 3-year follow-up, we assessed childhood blood pressure, resting pulse rate, and adiposity (body mass index, skinfold thicknesses, body fat, waist and arm circumferences) and maternal diet, physical activity, and anthropometry. Results: 514 three-year-old children attended the appointment (49% intervention, 51% standard care). There was no difference in the main outcome of interest, subscapular skinfold thickness, between the trial arms (−0.30 mm, 95% confidence interval: −0.92, 0.31). However, the intervention was associated with a lower resting pulse rate (−5 bpm [−8.41, −1.07]). There was also a non-significant lower odds of overweight/obesity (OR 0.73; 0.50, 1.08). Maternal dietary improvements observed in the UPBEAT trial, including glycaemic load and saturated fat were maintained 3-years postpartum. Conclusion: This study has demonstrated that an antenatal dietary and physical activity intervention in women with obesity is associated with lower offspring pulse rate and sustained improvement in maternal diet. Whilst larger than previous cohorts, there remains potential for bias from attrition and these findings require validation in future cohorts

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules