1.
Surveying proteolytic processes in human cancer microenvironments by microdialysis and activity-based mass spectrometry.
Source
Boston Biomedical Research Institute, Watertown, MA, USA. hardt@bbri.org.
Abstract
Purpose: We present a strategy to survey proteolytic processes that occur in human cancer microenvironments. Experimental design: In situ microdialysis during oral cancer surgery was combined with mass spectrometry-based proteomics to analyze interstitial fluid surrounding tumors and anatomically matched normal sites. Protease activity-based (18)O-profiling was utilized to reveal peptides that were processed by co-collected proteases ex vivo. Results: We demonstrated for the first time the use of microdialysis in humans to collect interstitial fluid from cancer microenvironments. Proteomic profiling identified proteases and inhibitors in the microdialysis samples. A subset ofpeptides displayed characteristic (18)O-isotope patterns that indicated processing by endogenous proteases. Conclusions and clinical relevance: The presented approach provides unprecedented views of in vivo targets of proteases without disrupting the cancer or surrounding tissue. The methodology can be broadly adapted to other physiological conditions in which proteolytic mediators are involved (e.g. arthritic joints, inflamed muscle, other types of cancer) and where a comparison of normal and pathological tissue is sought.
Copyright © 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22262628
- [PubMed - as supplied by publisher]
A universal strategy for preparing protected C-terminal peptides on the solid phase through an intramolecular click chemistry-based handle.
Source
Institute for Research in Biomedicine, Barcelona Science Park (PCB), Baldiri Reixac 10, 08028-Barcelona, Spain. albericio@irbbarcelona.org judit.tulla@irbbarcelona.org.
Abstract
A new universal strategy exploits DKP formation in a dipeptide moiety whose C-terminal residue is blocked by a leaving group. It enables both synthesis of C-terminal protected peptides that are useful for convergent synthesis of largepeptides and use of a C-terminal permanent protecting group that can be cleaved by catalytic hydrogenation to release the peptide.
The Most Common Mutation of KRT9, c.C487T (p.R163W), in Epidermolytic Palmoplantar Keratoderma in Two Large Chinese Pedigrees.
Source
Department of Biochemistry and Genetics, National Education Base for Basic Medical Sciences, Institute of Cell Biology, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China.
Abstract
Epidermolytic palmoplantar keratoderma (EPPK) is generally associated with dominant-negative mutations of the Keratin 9 gene (KRT9), and rarely with the Keratin 1 gene (KRT1). To date, a myriad of mutations has been reported with a high frequency of codon 163 mutations within the first exon of KRT9 in different populations. Notably, a distinct phenotypic heterogeneity, digital mutilation, was found recently in a 58-year-old female Japanese EPPK patient with p.R163W. Here, we report the most common mutation, c.C487T (p.R163W) of KRT9, in two large EPPK pedigrees from southeast China. The arginine residue in peptide position 163 remains almost constant in at least 47 intermediate filament proteins ranging from snail to human. A substitution in arginine alters both the charge and shape of the 1A rod domain and disrupts the function of the helix initiation motif of keratins, finally compromising the integrity of filaments and weakening their stability in the epidermis of palms and soles. We summarize the clinical symptoms of EPPK in Chinese and show that knuckle pads are associated with KRT9 mutations. We suggest that the frequency of p.R163W in Chinese EPPK patients (31.03%) is consistent with that in the general population (29.33%), and that codon 163 is truly a hotspot mutational site of KRT9. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.
Copyright © 2012 Wiley Periodicals, Inc.
- PMID:
- 22262370
- [PubMed - as supplied by publisher]
GLP-1, the Gut-Brain, and Brain-Periphery Axes.
Source
INSERM (Institut National de la Sante et de la Recherche Medicale), U1048, Institute of Metabolic and Cardiovascular Diseases Rangueil, University of Toulouse III (Paul-Sabatier), (C.C, R.B), and the Faculty of Pharmacy, Toulouse, France.
Abstract
Glucagon-like peptide 1 (GLP-1) is a gut hormone which directly binds to the GLP-1 receptor located at the surface of the pancreatic β-cells to enhance glucose-induced insulin secretion. In addition to its pancreatic effects, GLP-1 can induce metabolic actions by interacting with its receptors expressed on nerve cells in the gut and the brain. GLP-1 can also be considered as a neuropeptide synthesized by neuronal cells in the brain stem that release the peptide directly into the hypothalamus. In this environment, GLP-1 is assumed to control numerous metabolic and cardiovascular functions such as insulin secretion, glucose production and utilization, and arterial blood flow. However, the exact roles of these two locations in the regulation of glucose homeostasis are not well understood. In this review, we highlight the latest experimental data supporting the role of the gut-brain and brain-periphery axes in the control of glucose homeostasis. We also focus our attention on the relevance of β-cell and brain cell targeting by gut GLP-1 for the regulation of glucose homeostasis. In addition to its action on β-cells, we find that understanding the physiological role of GLP-1 will help to develop GLP-1-based therapies to control glycemia in type 2 diabetes by triggering the gut-brain axis or the brain directly. This pleiotropic action of GLP-1 is an important concept that may help to explain the observation that, during their treatment, type 2 diabetic patients can be identified as 'responders' and 'non-responders'.
Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography.
Source
Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle (Saale), Germany.
Abstract
A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith's surface are envisaged for the future development of monoliths with improved enrichment characteristics.
Targeted data extraction of the MS/MS spectra generated by data independent acquisition: a new concept for consistent and accurate proteome analysis.
Source
ETH Zurich - Institute of Molecular Systems Biology, Switzerland;
Abstract
Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify thepeptides generated by the proteolysis of a biological sample. However with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data independent acquisition method. For this study, the data was acquired on a fast, high-resolution Qq-TOF instrument by repeatedly cycling through 32 consecutive 25 Da precursor isolation windows (swaths). This acquisition set-up generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400-1200 m/z precursor range and the user defined retention time window. We show that suitable combinations of fragment ions extracted from these datasets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable to that of selected reaction monitoring, the gold-standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired datasets. This combination of unbiased, broad-range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics.
A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth.
Source
Department of Biochemistry, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA; Cancer Institute, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA.
Abstract
Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. We have developed a thermally responsive polypeptide, based on the sequence of Elastin-like polypeptide (ELP), that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. Following systemic administration, the ELP - delivered c-Myc inhibitory peptide was targeted to tumors using focused hyperthermia, and significantly reduced tumor growth in an orthotopic mouse model of breast cancer. This work provides a new modality for targeted delivery of a specific oncogene inhibitory peptide, and this strategy may be expanded for delivery of other therapeutic peptides or small molecule drugs.
Copyright © 2012. Published by Elsevier Ireland Ltd.
Evaluation of the long-term effects of gastric inhibitory polypeptide-ovalbumin conjugates on insulin resistance, metabolic dysfunction, energy balance and cognition in high-fat-fed mice.
Source
School of Biomedical Sciences Research Institute, SAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine BT52 1SA, UK.
Abstract
The effects of active immunisation with gastric inhibitory polypeptide (GIP) or (proline3)GIP-ovalbumin conjugates on insulin resistance, metabolic dysfunction, energy expenditure and cognition were examined in high-fat-fed mice. Normal mice were injected (subcutaneously) once every 14 d for 98 d with GIP-ovalbumin conjugates, with transfer to a high-fat diet on day 21. Active immunisation resulted in GIP antibody generation and significantly (P < 0·01 to P < 0·001) reduced circulating non-fasting plasma insulin concentrations compared to high-fat control mice from day 70 onwards. The glycaemic responses to intraperitoneal glucose or nutrient ingestion were significantly improved in all treated mice, with corresponding stimulated plasma insulin levels depressed compared to high-fat controls. These changes were associated with substantially (P < 0·001) improved glucose-lowering responses to exogenous insulin and decreases of muscle and fat TAG, pancreatic insulin, circulating total and LDL-cholesterol levels (P < 0·01 to P < 0·001). Treatment with GIP-ovalbumin conjugates was not associated with alterations in energy expenditure, indirect calorimetry or aspects of cognitive function. The observed changes were almost identical in GIP and (Pro3)GIP immunised mice and were independent of any effects on food intake or body weight. Further tests established that coupling of GIP peptides to ovalbumin abolished any intrinsic insulin-releasing or glucose-lowering activity. These results suggest that induction of GIP-neutralising antibodies with GIP-ovalbumin conjugates is an effective means of countering the metabolic abnormalities induced by high-fat feeding and does not adversely have an impact on a marker of cognition function or energy expenditure.
Convalescent Plasma Levels of TAFI Activation Peptide Predict Death and Recurrent Vascular Events in Ischemic Stroke Survivors.
Source
Institute of Neuroscience and Physiology, the Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
Wild-type amyloid beta 1-40 peptide induces Vascular Smooth Muscle Cell death independently from Matrix Metalloprotease activity.
Source
Paris 6 Pierre et Marie Curie University, UR4, Stress, vieillissement et Inflammation 7 quai Saint-Bernard, 75252 Paris, France Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, United States of America.
Abstract
Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-β (Aβ) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aβ peptidedeposits (composed mainly of wild-type (WT)Aβ(1-40) peptide in sporadic forms) induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated to their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and micro-hemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of MMP-2 activation in WT-Aβ(1-40) -treated VSMC and provide evidence that matrix metalloprotease (MMP) activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the wild-type Aβ(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity, nor hampering the expression of membrane-type1 MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aβ(1-40) -induced cell apoptosis. Our results, contrast those obtained using the Aβ(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
Copyright © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
A molecular carrier to transport and deliver cisplatin into endometrial cancer cells.
Source
NCI of Naples Pascale Foundation, Molecular Biology and Viral Oncogenesis C.N.R, Institute of Protein Biochemistry INBIOS srl, Naples, Italy University of Udine, Dept. of Statistics University of Naples Federico II, Dept. of Biological Sciences Second University of Naples, Dept. of Gynecology, Obstetrics and Reproduction.
Abstract
The leader peptide of a recombinant manganese superoxide dismutase (rMnSOD-Lp) acts as a molecular carrier. Clonogenic tests on normal (MRC-5) and endometrial adeno-carcinoma cells (HTB-112) were carried out in the presence of rMnSOD-Lp, cisplatin alone (CC) or cisplatin conjugated to the rMnSOD-Lp (rMnSOD-Lp-CC). The platinum delivered into the cells was measured by atomic spectrophotometric absorbance. The treatments on tumor and normal cells were finally evaluated by LM and TM microscopy. Tumor cell death in the case of 0.5 μM cisplatin on its own was minimal, while in the presence of 0.5 μM rMnSOD-Lp-CC, no tumor cells survived. Atomic absorbance analysis showed that rMnSOD-Lp-CC delivered approximately 4 times more cisplatin into HTB-112 cells than the amount delivered using cisplatin alone. By LM observation, the cells treated with rMnSOD-Lp-CC showed signs of nuclear and cytoplasmic fragmentation, i.e. apoptosis induced by the treatment. The therapeutic effect of rMnSOD-Lp-CC on endometrial cancer cells was significant, while on the normal cells it showed only a minimal toxicity. We believe that rMnSOD-Lp deserves to be considered as a molecular carrier to deliver cisplatin directly into tumor cells, thus transforming its antireplicative activity into a specific and selective antitumor agent. © 2012 John Wiley & Sons A/S.
© 2012 John Wiley & Sons A/S.
Emerging role of ER quality control in plant cell signal perception.
Source
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
Abstract
The endoplasmic reticulum quality control (ER-QC) is a conserved mechanism in surveillance of secreted signaling factors during cell-to-cell communication in eukaryotes. Recent data show that the ER-QC plays important roles in diverse cell-to-cell signaling processes during immune response, vegetative and reproductive development in plants. Pollen tube guidance is a precisely guided cell-cell communication process between the male and female gametophytes during plant reproduction. Recently, the female signal has been identified as small secreted peptides, but how the pollen tube responds to this signal is still unclear. In this review, we intend to summarize the role of ER-QC in plants and discuss the recent advances regarding our understanding of the mechanism of pollen tube response to the female signals.
Contributions of Individual Amino Acid Residues to the Endogenous CLV3 Function in Shoot Apical Meristem Maintenance in Arabidopsis.
Source
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, China.
Abstract
As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.
Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel.
Source
Albrecht-Kossel-Institute for Neuroregeneration, University of Rostock.
Abstract
The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.(1-5) Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.(6-8) Here we used PuraMatrix(9,10) (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.(7,11-14) PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free. Recently we have studied the influence of the PM-concentration on the formation of the scaffold.(13) In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by βIII-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells(13), however the concentration might be adapted to other cell types.(12) The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.
Structural changes in influenza virus at low pH characterized by cryo-electron tomography.
Source
Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.
Abstract
Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the post-fusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed; larger particles representing fused virions appear; the HA spikes become conspicuously disorganized; a layer of M1 matrix protein is no longer resolved on most virions; and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear respectively to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH.
Endothelin in hypertension: an update.
Source
aLady Davis Institute for Medical Research bDepartment of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montréal, Québec, Canada.
Abstract
PURPOSE OF REVIEW:
The purpose of this review of the vascular biology of endothelin-1 (ET-1) is the presentation of recent data including the use of endothelin-receptor antagonists for the treatment of hypertension.
RECENT FINDINGS:
Recent discoveries regarding the pharmacology of ET-1 in the vascular wall and its effect on signalling transduction and gene expression in vascular smooth muscle cells are reviewed, as well as mechanisms controlling blood pressure in normal conditions and in hypertension, discovered using genetically modified models. Finally, studies of endothelin antagonists for treatment of hypertension will be summarized.
SUMMARY:
Pharmacological studies demonstrate that calcitonin gene-related peptide is a physiological antagonist of ET-1 that terminates the long-lasting contraction induced by ET-1. ET-1-induced rise in [Ca]i involves the newly described stromal-interaction molecule-1/orai1 pathway to increase store-operated calcium entry. Sensitization of contractile proteins to calcium during ET-1-induced contraction of vascular smooth muscle cells includes activation of p63Rho guanine nucleotide exchange factor and increase in O-GlcNAcylation, a form of posttranslational modification. Genetically modified mice have demonstrated that endothelial ET-1 is involved in the regulation of normal blood pressure and development of vascular disease. Gene expression induced by endothelial overexpression of ET-1 in mice demonstrated upregulation of lipid metabolism, inflammatory and signal transduction genes. Crossing these mice with apoE mice was associated with acceleration of atherosclerosis on a high-fat diet and blood pressure elevation. Finally, the DORADO clinical trial has demonstrated that the ETA-receptor antagonist darusentan is able to decrease the blood pressure of patients with refractory hypertension.
Novel Nonphosphorylated Peptides with Conserved Sequences Selectively Bind to Grb7 SH2 Domain with Affinity Comparable to Its Phosphorylated Ligand.
Source
National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing, China.
Abstract
The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. Thesepeptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.
Mechanism and Function of Drosophila capa GPCR: A Desiccation Stress-Responsive Receptor with Functional Homology to Human NeuromedinU Receptor.
Source
Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Abstract
The capa peptide receptor, capaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-capa-1 and -2 (capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptidefamily are of interest. Capa peptide induces calcium signaling via capaR with EC(50) values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. capaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal -FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca(2+)]i) levels via capaR. In vivo, NMU-25 increases [Ca(2+)]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca(2+)]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca(2+)]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, capaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly.
Antiapoptotic Actions of Exendin-4 against Hypoxia and Cytokines Are Augmented by CREB.
Source
Section of Endocrinology (K.V., S.P.), Veterans Affairs Medical Center, Denver, Colorado 80220; Department of Medicine (K.V., S.P.), University of Colorado Denver, Aurora, Colorado 80045; Schulze Diabetes Institute (A.N.B., G.L., B.J.H.), Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455; and Department of Pediatrics (A.A.), National Jewish Health, Denver, Colorado 80206.
Abstract
Islets isolated from cadaveric donor pancreas are functionally viable and can be transplanted in diabetic patients to reduce insulin requirements. This therapeutic approach is less efficient because a significant portion of functional islets is lost due to oxidative stress, inflammation, and hypoxia. Exendin-4, a glucagon-like peptide-1 receptor agonist, is known to improve islet survival through activation of the transcription factor, cAMP response element binding protein (CREB). However, isolated human islets are exposed to several stresses known to down-regulate CREB. The objective of the present study was to determine whether the cytoprotective actions of exendin-4 in human islets can be augmented by increasing the levels of CREB. Simulation of ischemia/reperfusion injury and exposure to hypoxic conditions in cultured human islets resulted in decreased CREB activation and induction of apoptosis. Islets were transduced with adenoviral CREB followed by exposure to exendin-4 as a strategy for improving their survival. This combination increased the levels of several proteins needed for β-cell survival and function, including insulin receptor substrate-2, Bcl-2, and baculoviral IAP repeat-containing 3, and suppressed the expression of proapoptotic and inflammatory genes. A combination of CREB and exendin-4 exerted enhanced antiapoptotic action in cultured islets against hypoxia and cytokines. More significantly, transplantation of human islets transduced with adenoviral CREB and treated with exendin-4 showed improved glycemic control over a 30-d period in diabetic athymic nude mice. These observations have significant implications in the therapeutic potential of exendin-4 and CREB in the islet transplantation setting as well as in preserving β-cell mass of diabetic patients.
Disruption of the Murine Glp2r Impairs Paneth Cell Function and Increases Susceptibility to Small Bowel Enteritis.
Source
Department of Medicine, Mt. Sinai Hospital, Samuel Lunenfeld Research Institute (S.-J.L., J.L., K.K.L., D.H., B.Y., D.J.D.), and the Department of Cell and Systems Biology (H.M., D.S.G.), University of Toronto, Toronto Ontario, Canada M5G 1X5.
Abstract
Exogenous glucagon-like peptide-2 receptor (GLP-2R) activation elicits proliferative and cytoprotective responses in the gastrointestinal mucosa and ameliorates experimental small and large bowel gut injury. Nevertheless, the essential physiological role(s) of the endogenous GLP-2R remain poorly understood. We studied the importance of the GLP-2R for gut growth, epithelial cell lineage allocation, the response to mucosal injury, and host-bacterial interactions in Glp2r(-/-) and littermate control Glp2r(+/+) mice. Glp2r(-/-) mice exhibit normal somatic growth and preserved small and large bowel responses to IGF-I and keratinocyte growth factor. However, Glp2r(-/-) mice failed to up-regulate intestinal epithelial c-fos expression in response to acute GLP-2 administration and do not exhibit changes in small bowel conductance or small or large bowel growth after administration of GLP-2R agonists. The crypt and villus compartment and the numbers and localization of Paneth, enteroendocrine, and goblet cells were comparable in Glp2r(+/+) vs. Glp2r(-/-) mice. Although the severity and extent of colonic mucosal injury in response to 3% oral dextran sulfate was similar across Glp2r genotypes, Glp2r(-/-) mice exhibited significantly increased morbidity and mortality and increased bacterial translocation after induction of enteritis with indomethacin and enhanced mucosal injury in response to irinotecan. Moreover, bacterial colonization of the small bowel was significantly increased, expression of Paneth cell antimicrobial gene products was reduced, and mucosal bactericidal activity was impaired in Glp2r(-/-) mice. Although the Glp2r is dispensable for gut development and the response to colonic injury, Glp2r(-/-) mice exhibit enhanced sensitivity to small bowel injury, and abnormal host-bacterial interactions in the small bowel.
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