Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 × 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor-γ coactivator-1α (ET ∼8.5-fold, ST ∼10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET ∼26-fold, ST ∼39-fold), vascular endothelial growth factor (VEGF; ET ∼4.5-fold, ST ∼4-fold), and muscle atrophy F-box protein (MAFbx) (ET ∼2-fold, ST ∼0.4-fold) mRNA increased in both groups, whereas MyoD (∼3-fold), myogenin (∼0.9-fold), and myostatin (∼2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (∼7-fold, P < 0.01) and MyoD (∼0.7-fold) increased, whereas MAFbx (∼0.7-fold) and myostatin (∼0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Neuropsychological development in children with early and continuously treated phenylketonuria : association with biochemical markers

PKU is a genetically inherited inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase. The failure of this enzyme causes incomplete metabolism of protein ingested in the diet, specifically the conversion of one amino acid, phenylalanine, to tyrosine, which is a precursor to the neurotransmitter dopamine. Rising levels of phenylalanine is toxic to the developing brain, disrupting the formation of white matter tracts. The impact of tyrosine deficiency is not as well understood, but is hypothesized to lead to a low dopamine environment for the developing brain. Detection in the newborn period and continuous treatment (a low protein phe-restricted diet supplemented with phenylalanine-free protein formulas) has resulted in children with early and continuously treated PKU now developing normal I.Q. However, deficits in executive function (EF) are common, leading to a rate of Attention Deficit Hyperactivity Disorder (ADHD) up to five times the norm. EF worsens with exposure to higher phenylalanine levels, however recent research has demonstrated that a high phenylalanine to tyrosine ratio (phenylalanine:tyrosine ratio), which is hypothesised to lead to poorer dopamine function, has a more negative impact on EF than phenylalanine levels alone. Research and treatment of PKU is currently phenylalanine-focused, with little investigation of the impact of tyrosine on neuropsychological development. There is no current consensus as to the veracity of tyrosine monitoring or treatment in this population. Further, the research agenda in this population has demonstrated a primary focus on EF impairment alone, even though there may be additional neuropsychological skills compromised (e.g., mood, visuospatial deficits). The aim of this PhD research was to identify residual neuropsychological deficits in a cohort of children with early and continuously treated phenylketonuria, at two time points in development (early childhood and early adolescence), separated by eight years. In addition, this research sought to determine which biochemical markers were associated with neuropsychological impairments. A clinical practice survey was also undertaken to ascertain the current level of monitoring/treatment of tyrosine in this population. Thirteen children with early and continuously treated PKU were tested at mean age 5.9 years and again at mean age 13.95 years on several neuropsychological measures. Four children with hyperphenylalaninemia (a milder version of PKU) were also tested at both time points and provide a comparison group in analyses. Associations between neuropsychological function and biochemical markers were analysed. A between groups analysis in adolescence was also conducted (children with PKU compared to their siblings) on parent report measures of EF and mood. Minor EF impairments were evident in the PKU group by age 6 years and these persisted into adolescence. Life-long exposure to high phenylalanine:tyrosine ratio and/or low tyrosine independent of phenylalanine were significantly associated with EF impairments at both time points. Over half the children with PKU showed severe impairment on a visuospatial task, and this was associated only with concurrent levels of tyrosine in adolescence. Children with PKU also showed a statistically significant decline in a language comprehension task from 6 years to adolescence (going from normal to subnormal), this deficit was associated with lifetime levels of phenylalanine. In comparison, the four children with hyperphenylalaninemia demonstrated normal function at both time points, across all measures. No statistically significant differences were detected between children with PKU and their siblings on the parent report of EF and mood. However, depressive symptoms were significantly correlated with: EF; long term high phe:tyr exposure; and low tyrosine levels independent of phenylalanine. The practice survey of metabolic clinics from 12 countries indicated a high level of variability in terms of monitoring/treatment of tyrosine in this population. Whilst over 80% of clinics surveyed routinely monitored tyrosine levels in their child patients, 25% reported treatment strategies to increase tyrosine (and thereby lower the phenylalanine:tyrosine ratio) under a variety of patient presentation conditions. Overall, these studies have shown that EF impairments associated with PKU provide support for the dopamine-deficiency model. A language comprehension task showed a different trajectory, serving a timely reminder that non-EF functions also remain vulnerable in this population; and that normal function in childhood does not guarantee normal function by adolescence. Mood impairments were associated with EF impairments as well as long term measures of phenylalanine:tyrosine and/or tyrosine. The implications of this research for enhanced clinical guidelines are discussed given varied current practice.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7was shown to interact directly in vivo with themolecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.