Distinct overlapping sequences at the carboxy-terminus of merlin regulate its tumour suppressor and morphogenic activity

The Neurofibromatosis 2 (NF2) gene product merlin is a tumour suppressor, which in addition to inhibiting cell proliferation regulates cell morphology. The morphogenic properties of merlin may play a role in tumour suppression, as patient-derived tumour cells demonstrate cytoskeletal abnormalities. However, it is still unclear how these functions are linked. The N-terminal FERM-domain of merlin is highly homologous to the oncogenic protein ezrin, while the C-termini are less conserved, suggesting that the opposite effect of the proteins on proliferation could be mediated by their distinct C-terminal regions. In this study we characterize the role of the most C-terminal residues of merlin in the regulation of proliferation, cytoskeletal organization, phosphorylation and intramolecular associations. In addition to the two full-length merlin isoforms and truncating mutations found in patients, we focused on the evolutionally conserved C-terminal residues 545-547, also harbouring disease-causing mutations. We demonstrate that merlin induces cell extensions, which result from impaired retraction of protrusions rather than from increased formation of filopodia. The residues 538-568 were found particularly important for this morphogenic activity. The results further show that both merlin isoforms are able to equally inhibit proliferation, whereas C-terminal mutants affecting residues 545-547 are less effective in growth suppression. This study demonstrates that the C-terminus contains distinct but overlapping functional domains important for regulation of the morphogenic activity, intramolecular associations and cell proliferation.     

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.     

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms

The tumor‐suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N‐terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain‐containing proteins. RING domains bind and activate E2 ubiquitin‐conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer‐associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.     

Validation of an ELISA for urinary dopamine: applications in monitoring treatment of dopamine‐related disorders

Dopamine is a catecholamine that serves as a neurotransmitter in the central and peripheral nervous system. Non‐invasive, reliable, and high‐throughput techniques for its quantification are needed to assess dysfunctions of the dopaminergic system and monitor therapies. We developed and validated a competitive ELISA for direct determination of dopamine in urine samples. The method provides high specificity, good accuracy, and precision (average inter‐assay variation < 12%). The analysis is not affected by general urinary components and structurally related drugs and metabolites. The correlation between ELISA and LC‐MS/MS analyses was very good (r = 0.986, n = 28). The reference range was 64–261 μg/g Cr (n = 64). Week‐to‐week biological variations of second morning urinary dopamine under free‐living conditions were 23.9% for within‐ and 35.5% for between‐subject variation (n = 10). The assay is applied in monitoring Parkinson's disease patients under different treatments. Urinary dopamine levels significantly increase in a dose‐dependent manner for Parkinson's disease patients under l ‐DOPA treatment. The present ELISA provides a cost‐effective alternative to chromatographic methods to monitor patients receiving dopamine restoring treatment to ensure appropriate dosing and clinical efficacy. The method can be used in pathological research for the assessment of possible peripheral biological markers for disorders related to the dopaminergic system.     

Small mammals cause non-trophic effects on habitat and associated snails in a native system

Legacy effects occur when particular species or their interactions with others have long-lasting impacts, and they are increasingly recognized as important determinants of ecological processes. However, when such legacy effects have been explicitly explored, they most often involve the long-term direct effects of species on systems, as opposed to the indirect effects. Here, we explore how a legacy of small mammal exclusion on the abundance of a shrub, bush lupine (Lupinus arboreus), influences the abundance of a native land snail (Helminthoglypta arrosa) in coastal prairie and dune habitats in central California. The factors that limit populations of land snails are very poorly known despite the threats to the persistence of this group of species. In grasslands, prior vole (Microtus californicus) exclusion created long-lasting gains in bush lupine abundance, mediated through the seedbank, and was associated with increased snail numbers (10×) compared to control plots where mammals were never excluded. Similar plots in dune habitat showed no difference in snail numbers due to previous mammal exclusion. We tested whether increased competition for food, increased predation, and/or lower desiccation explained the decline in snail numbers in plots with reduced lupine cover. Tethering experiments supported the hypothesis that voles can have long-lasting impacts as ecosystem engineers, reducing woody lupine habitat required for successful aestivation by snails. These results add to a growing list of studies that have found that non-trophic interactions can be limiting to invertebrate consumers.     

Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula

The physiological mechanisms controlling plant responses to dynamic changes in ambient solar ultraviolet (UV) radiation are not fully understood: this information is important to further comprehend plant adaptation to their natural habitats. We used the fluorimeter Dualex to estimate in vivo the epidermal flavonoid contents by measuring epidermal UV absorbance (A(375) ) in Betula pendula Roth (silver birch) leaves of different ages under altered UV. Seedlings were grown in a greenhouse for 15 days without UV and transferred outdoors under three UV treatments (UV-0, UV-A and UV-A+B) created by three types of plastic film. After 7 and 13 days, Dualex measurements were taken at adaxial and abaxial epidermis of the first three leaves (L1, L2 and L3) of the seedlings. After 14 days, some of the seedlings were reciprocally swapped amongst the treatments to study the accumulation of epidermal flavonoids in the youngest unfolded leaves (L3) during leaf expansion under changing solar UV environments. A(375) of the leaves responded differently to the UV treatment depending on their position. UV-B increased the A(375) in the leaves independently of leaf position. L3 quickly adjusted A(375) in their epidermis according to the UV they received and these adjustments were affected by previous UV exposure. The initial absence of UV-A+B or UV-A, followed by exposure to UV-A+B, particularly enhanced leaf A(375) . Silver birch leaves modulate their protective pigments in response to changes in the UV environment during their expansion, and their previous UV exposure history affects the epidermal-absorbance achieved during later UV exposure.     

Metabolic regulation in progression to autoimmune diabetes

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes.