The scaffold protein KSR1, a novel therapeutic target for the treatment of Merlin-deficient tumors

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1's function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.

Microscale mesoarrays created by dip-pen nanolithography for screening of protein-protein interactions

Using microarrays to probe protein-protein interactions is becoming increasingly attractive due to their compatibility with highly sensitive detection techniques, selectivity of interaction, robustness and capacity for examining multiple proteins simultaneously. The major drawback to using this approach is the relatively large volumes and high concentrations necessary. Reducing the protein array spot size should allow for smaller volumes and lower concentrations to be used as well as opening the way for combination with more sensitive detection technologies. Dip-Pen Nanolithography (DPN) is a recently developed technique for structure creation on the nano to microscale with the capacity to create biological architectures. Here we describe the creation of miniaturised microarrays, 'mesoarrays', using DPN with protein spots 400× smaller by area compared to conventional microarrays. The mesoarrays were then used to probe the ERK2-KSR binding event of the Ras/Raf/MEK/ERK signalling pathway at a physical scale below that previously reported. Whilst the overall assay efficiency was determined to be low, the mesoarrays could detect KSR binding to ERK2 repeatedly and with low non-specific binding. This study serves as a first step towards an approach that can be used for analysis of proteins at a concentration level comparable to that found in the cellular environment.

Using secondary knowledge to support decision tree classification of retrospective clinical data

Retrospective clinical data presents many challenges for data mining and machine learning. The transcription of patient records from paper charts and subsequent manipulation of data often results in high volumes of noise as well as a loss of other important information. In addition, such datasets often fail to represent expert medical knowledge and reasoning in any explicit manner. In this research we describe applying data mining methods to retrospective clinical data to build a prediction model for asthma exacerbation severity for pediatric patients in the emergency department. Difficulties in building such a model forced us to investigate alternative strategies for analyzing and processing retrospective data. This paper describes this process together with an approach to mining retrospective clinical data by incorporating formalized external expert knowledge (secondary knowledge sources) into the classification task. This knowledge is used to partition the data into a number of coherent sets, where each set is explicitly described in terms of the secondary knowledge source. Instances from each set are then classified in a manner appropriate for the characteristics of the particular set. We present our methodology and outline a set of experiential results that demonstrate some advantages and some limitations of our approach. © 2008 Springer-Verlag Berlin Heidelberg.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway

The extracellular signal-regulated kinase (ERK) pathway participates in the control of numerous cellular processes, including cell proliferation. Since its activation kinetics are critical for to its biological effects, they are tightly regulated. We report that the protein translation factor, eukaryotic translation initiation factor 3, subunit a (eIF3a), binds to SHC and Raf-1, two components of the ERK pathway. The interaction of eIF3a with Raf-1 is increased by ß-arrestin2 expression and transiently decreased by epidermal growth factor (EGF) stimulation in a concentration-dependent manner. The EGF-induced decrease in Raf-1-eIF3a association kinetically correlates with the time course of ERK activation. eIF3a interferes with Raf-1 activation and eIF3a downregulation by small interfering RNA enhances ERK activation, early gene expression, DNA synthesis, expression of neuronal differentiation markers in PC12 cells, and Ras-induced focus formation in NIH 3T3 cells. Thus, eIF3a is a negative modulator of ERK pathway activation and its biological effects.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

Distributive tactile sensing using fibre Bragg grating sensors for biomedical applications

Artificial tactile sensing systems using the distributive tactile sensing technique and fibre Bragg grating sensors are presented. A one-dimensional arrangement, with possible applications in an endoscope, is compared with a similar arrangement using conventional electronic sensors. A two-dimensional sensing surface is described, with potential applications in human balance and gait analysis, capable of detecting simultaneously the position and shape of an object placed upon it. It is believed that this work represents the first use of fibre Bragg grating sensors in a distributive sensing regime.

Diabetic retinopathy and blockade of the renin–angiotensin system:new data from the DIRECT study programme

The pathogenesis and medical management of diabetic retinopathy is reviewed. The importance of good control of blood glucose and blood pressure remain key elements in the prevention and treatment of diabetic retinopathy, and a number of specific metabolic pathways have been identified that may be useful additional targets for therapeutic intervention. Trial data, however, aimed specifically to answer the questions of optimum medical management are limited, so the DIRECT study of renin-angiotensin blockade using oral candesartan 32 mg daily is a welcome addition to our knowledge. This arose from the promising improvement of retinopathy outcomes in the EUCLID study of lisinopril in type I diabetes. In DIRECT, 5 years of candesartan treatment in type I diabetes reduced the incidence of retinopathy by two or more steps (EDTRS) in severity by 18% (P = 0.0508) and, in a post hoc analysis, reduced the incidence of retinopathy by three-step progression by 35% (P = 0.034). In type I diabetes patients there was no effect on progression of established retinopathy. In contrast, in type II diabetes, 5 years of candesartan treatment resulted in 34% regression of retinopathy (P ≤0.009). Importantly, an overall significant change towards less-severe retinopathy was noted in both type I and II diabetes (P0.03). Although there is still no absolute proof that these effects were specific to RAS blockade, or just an effect of lower blood pressure, it is reasonable to conclude that candesartan has earned a place in the medical management of diabetic retinopathy, to prevent the problem in type I diabetes and to treat the early stages in type II diabetes. © 2010 Macmillan Publishers Limited All rights reserved.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Compartmentalization of the MAPK scaffold protein KSR1 modulates synaptic plasticity in hippocampal neurons

ERK1/2 is required for certain forms of synaptic plasticity, including the long-term potentiation of synaptic strength. However, the molecular mechanisms regulating synaptically localized ERK1/2 signaling are poorly understood. Here, we show that the MAPK scaffold protein kinase suppressor of Ras 1 (KSR1) is directly phosphorylated by the downstream kinase ERK1/2. Quantitative Western blot analysis further demonstrates that expression of mutated, feedback-deficient KSR1 promotes sustained ERK1/2 activation in HEK293 cells in response to EGF stimulation, compared to a more transient activation in control cells expressing wild-type KSR1. Immunocytochemistry and confocal imaging of primary hippocampal neurons from newborn C57BL6 mice further show that feedback phosphorylation of KSR1 significantly reduces its localization to dendritic spines. This effect can be reversed by tetrodotoxin (1 μM) or PD184352 (2 μM) treatment, further suggesting that neuronal activity and phosphorylation by ERK1/2 lead to KSR1 removal from the postsynaptic compartment. Consequently, electrophysiological recordings in hippocampal neurons expressing wild-type or feedback-deficient KSR1 demonstrate that KSR1 feedback phosphorylation restricts the potentiation of excitatory postsynaptic currents. Our findings, therefore, suggest that feedback phosphorylation of the scaffold protein KSR1 prevents excessive ERK1/2 signaling in the postsynaptic compartment and thus contributes to maintaining physiological levels of synaptic excitability. © FASEB.