Publications on this page begin in 2012. Some staff have a record of earlier publications on their personal pages.
BACKGROUND:Molecular markers have transformed our understanding of the heterogeneity of breast cancer and have allowed the identification of genomic profiles of estrogen receptor (ER)-α signaling. However, our understanding of the transcriptional profiles of ER signaling remains inadequate. Therefore, we sought to identify the genomic indicators of ER pathway activity that could supplement traditional immunohistochemical (IHC) assessments of ER status to better understand ER signaling in the breast tumors of individual patients.
MATERIALS AND METHODS:We reduced ESR1 (gene encoding the ER-α protein) mRNA levels using small interfering RNA in ER(+) MCF7 breast cancer cells and assayed for transcriptional changes using Affymetrix HG U133 Plus 2.0 arrays. We also compared 1034 ER(+) and ER(-) breast tumors from publicly available microarray data. The principal components of ER activity generated from these analyses and from other published estrogen signatures were compared with ESR1 expression, ER-α IHC, and patient survival.
RESULTS:Genes differentially expressed in both analyses were associated with ER-α IHC and ESR1 mRNA expression. They were also significantly enriched for estrogen-driven molecular pathways associated with ESR1, cyclin D1 (CCND1), MYC (v-myc avian myelocytomatosis viral oncogene homolog), and NFKB (nuclear factor kappa B). Despite their differing constituent genes, the principal components generated from these new analyses and from previously published ER-associated gene lists were all associated with each other and with the survival of patients with breast cancer treated with endocrine therapies.
CONCLUSION:A biomarker of ER-α pathway activity, generated using ESR1-responsive mRNAs in MCF7 cells, when used alongside ER-α IHC and ESR1 mRNA expression, could provide a method for further stratification of patients and add insight into ER pathway activity in these patients.
INTRODUCTION:Rheumatic fever (RF) incidence among New Zealand (NZ) individuals of Polynesian (Māori and Pacific) ancestry remains among the highest in the world. Polymorphisms in the IL-6, IL1RN, and CTLA4 genes have been associated with RF, and their products are modulated by new medications. Confirmation of these previous associations could help guide clinical approaches. We aimed to test IL-6, IL-1RA (IL1RN), and CTLA4 functional SNPs in 204 rheumatic heart disease (RHD) patients and 116 controls of Māori and Pacific ancestry.
MATERIAL AND METHOD:Self-reported ancestry of the eight great-grandparents defined ancestry of participants. Severity of carditis was classified according to the 2012 World Heart Federation guideline for the echocardiographic diagnosis of RHD. The IL-6 promoter rs1800797, IL1RN rs447713 and CTLA4 rs3087243 SNPs were genotyped by Taqman. Correlations were assessed by logistic regression analysis adjusting for gender and ancestry.
RESULTS:The IL-6 rs1800797 variant was significantly associated with RHD with carriers of the GG genotype 6.09 (CI 1.23; 30.23) times more likely to develop RHD than the carriers of the AA genotype (P=0.027). No significant associations with RHD were found for the IL1RN rs447713 and CTLA4 rs3087243 SNPs. Patients carrying the G allele (GG plus AG genotype) for the IL1RN rs447713 SNP had 2.36 times (CI 1.00; 5.56) more severe carditis than those without this allele (the AA genotype) (P=0.049).
CONCLUSION:The IL-6 promoter rs1800797 (-597G/A) SNP may influence susceptibility to RHD of people of Māori and Pacific ancestry living in NZ. The IL1RN rs447713 SNP may influence the severity of carditis in this population.
This study investigated some physicochemical properties of keratin extracted from Merino wool using five chemical extraction methods: alkali hydrolysis, sulfitolysis, reduction, oxidation, and extraction using ionic liquid. The ionic liquid method produced the highest protein yield (95%), followed by sulfitolysis method (89%), while the highest extraction yield was obtained with the reduction method (54%). The lowest yield was obtained with the oxidation method (6%). The oxidation extract contained higher molecular weight (>40 kDa) protein components, whereas the alkali hydrolysis extract contained protein material of <10 kDa. The sulfitolysis, reduction, and ionic liquid extracts contained various protein components between 3.5 and 60 kDa. Keratin obtained from various extraction methods had different yield, morphology, and physicochemical properties. None of the samples were toxic to L929 fibroblast cells up to a concentration of 2.5 mg/mL. Apart from the alkali hydrolysis extract, all other keratin extracts (reduction, sulfitolysis, ionic liquid, and oxidation) showed Fourier transform infrared adsorption peaks attributed to the sulfitolysis–oxidation stretching vibrations of cysteine-S-sulfonated residues, with the oxidation extract showing the highest content of cysteine-S-sulfonated residues. This study indicates that the properties of the keratin extract obtained vary depending on the extraction method used, which has implications for use in structural biomaterial applications.
Covalent modification of substrate proteins with ubiquitin is the end result of an intricate network of protein-protein interactions. The inherent ability of the E1, E2, and E3 proteins of the ubiquitylation cascade (the ubiquitin writers) to interact with ubiquitin facilitates this process. Importantly, contact between ubiquitin and the E2/E3 writers is required for catalysis and the assembly of chains of a given linkage. However, ubiquitin is also an activator of ubiquitin-writing enzymes, with many recent studies highlighting the ability of ubiquitin to regulate activity and substrate modification. Here, we review the interactions between ubiquitin-writing enzymes and regulatory ubiquitin molecules that promote activity, and highlight the potential of these interactions to promote processive ubiquitin transfer.
Objectives: Lipoprotein(a) (Lp(a)) is a low density lipoprotein (LDL)-like lipoprotein associated with an apolipoprotein(a) (apo(a)). The catabolic fate of Lp(a) in the liver remains controversial. Our study aims to determine the intracellular trafficking and catabolism of Lp(a) within HepG2 cells.
Methods: Lp(a) was purified from normolipidemic healthy individuals using density gradient ultracentrifugation and fast protein liquid chromatography. HepG2 cells were treated with 5 μg.ml-1 of Lp(a) and the uptake and intracellular localisation was studied using western blot analysis and confocal microscopy.
Results: Lp(a) was maximally internalised by HepG2 cells after 2 hours and mostly absent at 24 hours. Upon internalisation Lp(a), as detected by anti-apo(a) antibody, localises to early endosomes and the trans-Golgi network. Unlike LDL, Lp(a) did not colocalise with lysosomes. Golgi localisation of Lp(a) lead us to investigate the possibility of Lp(a) recycling. Interestingly, Lp(a) was resecreted by HepG2 cells into the media both as Lp(a) and free apo(a) as present in cell lysates. Furthermore, Lp(a) colocalised with the recycling marker transferrin. Investigation of factors regulating Lp(a) uptake showed that uptake was abolished by chelation of extracellular calcium and significantly reduced by inhibition of L-type calcium channels with Nifidipine and Verapmil.
Conclusions: Endocytosis of Lp(a) follows the early endosome-trans-Golgi network recycling trafficking route in liver cells. Lp(a) does not follow the lysosomal degradation pathway. Lp(a) uptake was inhibited by blocking L-type calcium channels suggesting Lp(a) internalisation is controlled by calcium dependent receptors. These findings may have implications for the manipulation of Lp(a) levels.
Monoubiquitylation of histone H2B is a post-translational mark that plays key roles in regulation of transcription and genome stability. In humans, attachment of ubiquitin to lysine 120 of histone H2B depends on the activity of the E2 ubiquitin-conjugating enzyme, Ube2B, and the really interesting new gene (RING) E3 ligases, RING finger protein (RNF) 20 and RNF40. To better understand the molecular basis of this modification, we have solved the crystal structure of the RNF20 RING domain and show that it is a homodimer that specifically interacts with the Ube2B~Ub conjugate. By mutating residues at the E3-E2 and E3-ubiquitin interfaces, we identify key contacts required for interaction of the RNF20 RING domain with the Ube2B~Ub conjugate. These mutants were used to generate a structure-based model of the RNF20-Ube2B~Ub complex that reveals differences from other RING-E2~Ub complexes, and suggests how the RNF20-Ube2B~Ub complex might interact with its nucleosomal substrate. Additionally, we show that the RING domains of RNF20 and RNF40 can form a stable heterodimer that is active. Together, our studies provide new insights into the mechanisms that regulate RNF20-mediated ubiquitin transfer from Ube2B.
Life has existed on the Earth for approximately four billion years. The sheer depth of evolutionary time, and the diversity of extant species, makes it tempting to assume that all the key biochemical innovations underpinning life have already happened. But we are only a little over halfway through the trajectory of life on our planet. In this Opinion piece, we argue: (i) that sufficient time remains for the evolution of new processes at the heart of metabolic biochemistry and (ii) that synthetic biology is providing predictive insights into the nature of these innovations. By way of example, we focus on engineered solutions to existing inefficiencies in energy generation, and on the complex, synthetic regulatory circuits that are currently being implemented.
In one of the final stages of cyanobacterial Photosystem II (PS II) assembly, binding of up to four extrinsic proteins to PS II stabilizes the oxygen-evolving complex (OEC). Growth of cyanobacterial mutants deficient in certain combinations of these thylakoid- lumen-associated polypeptides is sensitive to changes in environmental pH, despite the physical separation of the membrane-embedded PS II complex from the external environment. In this perspective we discuss the effect of environmental pH on OEC function and photoautotrophic growth in cyanobacteria with reference to pH-sensitive PS II mutants lacking extrinsic proteins. We consider the possibilities that, compared to pH 10.0, pH 7.5 increases susceptibility to PS II-generated reactive oxygen species (ROS) causing photoinhibition and reducing PS II assembly in some mutants, and that perturbations to channels in the lumenal regions of PS II might alter the accessibility of water to the active site as well as egress of oxygen and protons to the thylakoid lumen. Reduced levels of PS II in these mutants, and reduced OEC activity arising from the disruption of substrate/product channels, could reduce the trans-thylakoid pH gradient (ΔpH), leading to the impairment of photosynthesis. Growth of some PS II mutants at pH 7.5 can be rescued by elevating CO2 levels, suggesting that the pH-sensitive phenotype might primarily be an indirect result of back-pressure in the electron transport chain that results in heightened production of ROS by the impaired photosystem.
Secreted amyloid precursor protein-α (sAPPα) is a neurotrophic and neuroprotective molecule which can enhance learning and synaptic plasticity. Aging is associated with memory decline and impaired long-term potentiation (LTP). SAPPα therefore has potential as a nootropic agent which could be used to offset age-related cognitive decline. In this study we investigated the effects of sAPPα on spatial memory tasks and LTP in aged and young Long-Evans rats. Two hippocampus-dependent tasks were employed to measure spatial memory that is susceptible to impairments during aging. Aged rats showed a mild deficit in the novel object location task, but memory was significantly enhanced by bilateral intrahippocampal injections of sAPPα. There was no effect on the performance of young animals. In the watermaze task, however, sAPPα did not alleviate age-related decline in spatial memory. In subsequent electrophysiological experiments, LTP was impaired in slices from aged animals, but plasticity was rescued in a concentration-dependent manner by exogenous sAPPα administration. In contrast, LTP was impaired in young animals by sAPPα. Overall, these data support the hypothesis that sAPPα has therapeutic potential as a treatment for age-related cognitive decline.
The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen.