DOCSLIB.ORG

Misincorporation Proteomics Technologies: a Review

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Misincorporation Proteomics Technologies: a Review proteomes Review Misincorporation Proteomics Technologies: A Review Joel R. Steele 1,2 , Carly J. Italiano 2 , Connor R. Phillips 2, Jake P. Violi 1,2, Lisa Pu 2 , Kenneth J. Rodgers 2 and Matthew P. Padula 1,* 1 Proteomics Core Facility and School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] (J.R.S.); [email protected] (J.P.V.) 2 Neurotoxin Research Group, School of Life Sciences, The University of Technology Sydney, Ultimo, NSW 2007, Australia; [email protected] (C.J.I.); [email protected] (C.R.P.); [email protected] (L.P.); [email protected] (K.J.R.) * Correspondence: [email protected] Abstract: Proteinopathies are diseases caused by factors that affect proteoform conformation. As such, a prevalent hypothesis is that the misincorporation of noncanonical amino acids into a pro- teoform results in detrimental structures. However, this hypothesis is missing proteomic evidence, specifically the detection of a noncanonical amino acid in a peptide sequence. This review aims to outline the current state of technology that can be used to investigate mistranslations and misincor- porations whilst framing the pursuit as Misincorporation Proteomics (MiP). The current availability of technologies explored herein is mass spectrometry, sample enrichment/preparation, data analysis techniques, and the hyphenation of approaches. While many of these technologies show potential, our review reveals a need for further development and refinement of approaches is still required. Keywords: misincorporation; non protein amino acids; post translational modifications Citation: Steele, J.R.; Italiano, C.J.; 1. Introduction Phillips, C.R.; Violi, J.P.; Pu, L.; The “central dogma” of molecular biology suggests that the translation of one gene Rodgers, K.J.; Padula, M.P. results in the expression of a single protein [1]. However, translated proteins are known to Misincorporation Proteomics exist as multiple biological variants or proteoforms [2]. These proteoforms are the result of Technologies: A Review. Proteomes modifications to the polypeptide chain, including the addition, subtraction, or alteration 2021 9 , , 2. https://doi.org/10.3390/ of chemical groups. Such modifications can endow proteoforms with biological activity proteomes9010002 or an altered function varying from the original proteoform [3]. Any modification that occurs to a proteoform once already translated is termed a post-translational modification Received: 16 December 2020 (PTM), generating a new and distinct proteoform, adding the advantage of complexity to Accepted: 18 January 2021 Published: 21 January 2021 the proteome [4–6]. Additionally, and importantly for this review, variant proteoforms may be generated Publisher’s Note: MDPI stays neutral by mechanisms other than post-translational modification. During protein translation, with regard to jurisdictional claims in an incorrect amino acid may be inserted into the growing peptide chain, resulting in a published maps and institutional affil- modification in the final proteoform. Such errors generate new, non-native proteoforms iations. that have the potential to cause harm to the cell [7,8]. The mistranslational error rate reported in Escherichia coli (E. coli) is between 0.5–5% at any amino acid position [8]. This occurrence of errors in translation during synthesis can generate a new proteoform in the same way that cleavage or PTM can e.g., N-terminal methionine excision [8–10]. However, instead of providing an advantage to the cell, proteoforms produced through mistranslation Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. often present a burden, as they are unpredictably generated with the resulting non-native This article is an open access article proteoforms prone to misfolding [11,12]. To overcome this, the cell has sophisticated distributed under the terms and machinery to identify and degrade these proteoforms [13]. conditions of the Creative Commons The “misincorporation” of incorrect amino acids into a proteoform need not be limited Attribution (CC BY) license (https:// to the 20 canonical amino acids used in proteoform synthesis. Thousands of synthetic and creativecommons.org/licenses/by/ naturally occurring nonprotein amino acids (NPAAs) exist, also referred to as “nonpro- 4.0/). teogenic”, “noncanonical”, “noncoded”, or “non-natural” [14]. The infiltration of NPAAs Proteomes 2021, 9, 2. https://doi.org/10.3390/proteomes9010002 https://www.mdpi.com/journal/proteomes Proteomes 2021, 9, x FOR PEER REVIEW 2 of 20 Proteomes 2021, 9, 2 2 of 20 “nonproteogenic”, “noncanonical”, “noncoded”, or “non-natural” [14]. The infiltration of NPAAs into the protein translation process adds further opportunity for mistakes to be madeinto the during protein translation, translation resulting process in addsdistinct further and unintentionally opportunity for produced mistakes proteoforms. to be made Suchduring misincorporation translation, resulting can cause in distinct aberrant and modifications unintentionally to proteoform produced proteoforms. structure, with Such bi- ologicalmisincorporation ramifications can causefor the aberrant cell or organism modifications that can to proteoform perturb normal structure, cellular with function. biological It ramifications for the cell or organism that can perturb normal cellular function. It has has been shown that misincorporation of an NPAA can alter the 3D structure of a pro- been shown that misincorporation of an NPAA can alter the 3D structure of a proteoform, teoform, resulting in aggregation [15]. Such misfolding and aggregation of proteoforms is resulting in aggregation [15]. Such misfolding and aggregation of proteoforms is known known to be a hallmark of numerous degenerative neurological diseases [16] and for some to be a hallmark of numerous degenerative neurological diseases [16] and for some dis- diseases, an association already exists between exposure to NPAAs and disease develop- eases, an association already exists between exposure to NPAAs and disease development. ment. This includes motor neuron disease (MND) [17,18], multiple sclerosis [19,20] and This includes motor neuron disease (MND) [17,18], multiple sclerosis [19,20] and neuro- neurolathyrism [21,22]. As such, the exploration of NPAA misincorporation into pro- lathyrism [21,22]. As such, the exploration of NPAA misincorporation into proteoforms to teoforms to date has largely focused on their underlying potential to trigger proteinopa- date has largely focused on their underlying potential to trigger proteinopathies and cause thies and cause neurodegeneration. To describe the study of NPAA misincorporation into neurodegeneration. To describe the study of NPAA misincorporation into the proteome, we the proteome, we introduce the term Misincorporation Proteomics (MiP), whereby during introduce the term Misincorporation Proteomics (MiP), whereby during protein translation, proteina genetically translation, encoded a genetically canonical aminoencoded acid canonical is replaced amino by a acid NPAA. is replaced by a NPAA. 2. Amino Acid Misincorporation Protein translation translation involves involves the the cognate cognate amino amino acid acid (AA) (AA) being being charged charged by its by appro- its ap- propriatepriate aminoacyl aminoacyl tRNA tRNA synthetase synthetase (aaRS; (aaRS; Figure Figure 1). 1The). The charging charging is enabled is enabled by bythe thehy- drolysishydrolysis of ofadenosine adenosine triphosphate triphosphate (ATP) (ATP) and and allows allows the formationformation ofof an an AA/aaRS/AMP AA/aaRS/AMP complex. The cognate transfertransfer RNARNA (tRNA)(tRNA) thenthen bindsbinds to to this this aaRS/AA/AMP aaRS/AA/AMP complex and an aminoacyl ester bond forms, transferringtransferring the aminoacyl group to the tRNA and releasing AMP [[23,24].23,24]. Figure 1. TheThe transfer transfer of ofamino amino acids acids (AA) (AA) onto onto tRNA tRNA through through tRNA tRNA synthetase synthetase (aaRS) (aaRS) in the in pres- the presenceence of ATP. of ATP. ATP ATP is converted is converted to AMP to AMP activating activating thethe aaRS aaRS with with the the AA, AA, an antRNA tRNA is then is then bound bound to theto the AA AA of ofthe the AA/aaRS/AMP AA/aaRS/AMP complex complex subseque subsequentlyntly the thenow now charged charged tRNA tRNA dissociates. dissociates. Correct binding of amino acid to its aaRS is dependent on the fit between the amino acid and enzyme binding pocket [25]. If an incorrect amino acid binds the aaRS, this can be removed by a series of proofreading functions known as pre- and post-transfer Proteomes 2021, 9, x FOR PEER REVIEW 3 of 20 Proteomes 2021, 9, 2 3 of 20 Correct binding of amino acid to its aaRS is dependent on the fit between the amino acid and enzyme binding pocket [25]. If an incorrect amino acid binds the aaRS, this can be removed by a series of proofreading functions known as pre- and post-transfer editing (Figureediting (Figure2). Pre-transfer2). Pre-transfer editing editing occurs occurs before before tRNA tRNA binds binds to the to charged the charged amino amino acid. acid. Re- movalRemoval of the of the charged charged amino amino acid acid has has been been suggested suggested to to occur occur via via various various mechanisms, mechanisms, includingincluding selective selective release release by by the the aaRS aaRS enzyme, enzyme, translocation translocation to a to separate a separate hydrolytic hydrolytic ed-
Load more

Recommended publications
  • Monitoring Flavor Quality, Composition and Ripening Changes of Cheddar Cheese Using Fourier-Transform Infrared Spectroscopy
    MONITORING FLAVOR QUALITY, COMPOSITION AND RIPENING CHANGES OF CHEDDAR CHEESE USING FOURIER-TRANSFORM INFRARED SPECTROSCOPY DISSERTATION Presented in Partial Fulfillment of the Requirements for Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anand S. Subramanian, M.S. ***** The Ohio State University 2009 Dissertation Committee: Approved by Dr. Luis E. Rodriguez-Saona, Advisor Dr. W. James Harper _______________________ Dr. V.M Balasubramaniam Advisor Dr. David B. Min Food Science and Nutrition Graduate Program Copyright by Anand Swaminathan Subramanian 2009 ABSTRACT Cheese flavor develops during the ripening process, when complex changes take place, leading to the formation of flavor compounds, including amino acids and organic acids. However, cheese ripening is not completely understood, making it difficult to produce cheese of uniform flavor quality. Currently, cheese characteristics are determined using sensory panels and chromatography, which are expensive, time- consuming, and laborious. The objective was to develop a rapid and cost-effective technique based on Fourier-transform infrared spectroscopy (FTIR) for simultaneous analysis of cheese composition and flavor quality and identify some of the biochemical changes occurring during ripening. Cheddar cheese samples were obtained from a cheese manufacturer, along with their composition, age and flavor quality data. The samples were powdered using liquid nitrogen and water-soluble compounds were extracted using water, chloroform and ethanol. The extracts were analyzed by reverse phase HPLC for 3 organic acids, GC-FID for 20 amino acids, and FTIR to collect the mid-infrared spectra (4000-700 cm-1). The collected spectra were correlated with flavor quality and composition data to develop classification models based on soft independent modeling of class analogy (SIMCA) and regression models based on partial least squares (PLS).
    [Show full text]
  • Chemical Methods for the Characterization of Proteolysis in Cheese During Ripening Plh Mcsweeney, Pf Fox
    Chemical methods for the characterization of proteolysis in cheese during ripening Plh Mcsweeney, Pf Fox To cite this version: Plh Mcsweeney, Pf Fox. Chemical methods for the characterization of proteolysis in cheese during ripening. Le Lait, INRA Editions, 1997, 77 (1), pp.41-76. hal-00929515 HAL Id: hal-00929515 https://hal.archives-ouvertes.fr/hal-00929515 Submitted on 1 Jan 1997 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1997) 77, 41-76 41 © ElseviernNRA Review Chemical methods for the characterization of proteolysis in cheese during ripening PLH McSweeney, PF Fox Department of Food Chemistry, University College, Cork, Ireland Summary - Proteolysis is the principal and most complex biochemical event which occurs during the maturation of most cheese varieties. Proteolysis has been the subject of much study and a range of analytieal techniques has been developed to assess its extent and nature. Methods for assessing pro- teolysis can he c1assified under two broad headings: non-specifie and specifie techniques, both of which are reviewed. Non-specifie techniques include the quantitation of nitrogen soluble in various extrac- tants or precipitants and the Iiberation of reactive groups.
    [Show full text]
  • Searching for Inhibitors of the Protein Arginine Methyl Transferases: Synthesis and Characterisation of Peptidomimetic Ligands
    SEARCHING FOR INHIBITORS OF THE PROTEIN ARGININE METHYL TRANSFERASES: SYNTHESIS AND CHARACTERISATION OF PEPTIDOMIMETIC LIGANDS by ASTRID KNUHTSEN B. Sc., Aarhus University, 2009 M. Sc., Aarhus University, 2012 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2016 © Astrid Knuhtsen, 2016 UNIVERSITY OF COPENH AGEN FACULTY OF HEALTH A ND MEDICAL SCIENCES PhD Thesis Astrid Knuhtsen Searching for Inhibitors of the Protein Arginine Methyl Transferases: Synthesis and Characterisation of Peptidomimetic Ligands December 2015 This thesis has been submitted to the Graduate School of The Faculty of Health and Medical Sciences, University of Copenhagen ii Thesis submission: 18th of December 2015 PhD defense: 11th of March 2016 Astrid Knuhtsen Department of Drug Design and Pharmacology Faculty of Health and Medical Sciences University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Denmark and Faculty of Pharmaceutical Sciences University of British Columbia 2405 Wesbrook Mall BC V6T 1Z3, Vancouver Canada Supervisors: Principal Supervisor: Associate Professor Jesper Langgaard Kristensen Department of Drug Design and Pharmacology, University of Copenhagen, Denmark Co-Supervisor: Associate Professor Daniel Sejer Pedersen Department of Drug Design and Pharmacology, University of Copenhagen, Denmark Co-Supervisor: Assistant Professor Adam Frankel Faculty of Pharmaceutical
    [Show full text]
  • Designing Peptidomimetics
    CORE Metadata, citation and similar papers at core.ac.uk Provided by UPCommons. Portal del coneixement obert de la UPC DESIGNING PEPTIDOMIMETICS Juan J. Perez Dept. of Chemical Engineering ETS d’Enginyeria Industrial Av. Diagonal, 647 08028 Barcelona, Spain 1 Abstract The concept of a peptidomimetic was coined about forty years ago. Since then, an enormous effort and interest has been devoted to mimic the properties of peptides with small molecules or pseudopeptides. The present report aims to review different approaches described in the past to succeed in this goal. Basically, there are two different approaches to design peptidomimetics: a medicinal chemistry approach, where parts of the peptide are successively replaced by non-peptide moieties until getting a non-peptide molecule and a biophysical approach, where a hypothesis of the bioactive form of the peptide is sketched and peptidomimetics are designed based on hanging the appropriate chemical moieties on diverse scaffolds. Although both approaches have been used in the past, the former has been more widely used to design peptidomimetics of secretory peptides, whereas the latter is nowadays getting momentum with the recent interest in designing protein-protein interaction inhibitors. The present report summarizes the relevance of the information gathered from structure-activity studies, together with a short review on the strategies used to design new peptide analogs and surrogates. In a following section there is a short discussion on the characterization of the bioactive conformation of a peptide, to continue describing the process of designing conformationally constrained analogs producing first and second generation peptidomimetics. Finally, there is a section devoted to review the use of organic scaffolds to design peptidomimetics based on the information available on the bioactive conformation of the peptide.
    [Show full text]
  • THE SPORULATION of PERONOSPORA TABAOINA ADAM. on TOBACCO LEAF DISKS by C
    EFFECTS OF METABOLITES AND ANTlMETABOLITES ON THE SPORULATION OF PERONOSPORA TABAOINA ADAM. ON TOBACCO LEAF DISKS By C. J. SHEI'HERD* and M. MANDRYK* [Manuscript received ~arch 13, 1964] Summary . The effects of 148 metabolites and a~tinietabolites on the sporulation or" Peronospora tabacina Adam. on leaf disks of Nieotiand tubacum cv. Virginia Gold. have been determined. (1) Normal metabolites, with the exception of flavin adenine dinucleotide, had slight althoU:gh statistically significant effects on sporulation intensIty, which suggests that inhibition-nutrition phenomena play no part in the sporulation process of P. tabacina. (2) Seven uracil analogues had an inhibitory effect on sporulation, and the reversal of inhibition by uracil suggests the active involvement of this compound in the sporulation process. (3) Canavanine at a final concentrat.ion of 120 ftgjml showed complete inhibition of sporulation. The reversal of the canavanine inhibition of sporulation by arginine, citrulline, and ornithine suggests the involvement of arginine and the functioning of an ornithine cycle in the sporulating system. (4) White, instead of the normal blue, conidia were produced in the presence of a number of sUlphur-containing compounds. It is suggested that this phenomenon depends on the chelating properties of these compounds towards copper ions, with the subsequent inactivation of tyrosinase activity in the conidia. (5) Sporulation intensities of 68 X 10'-}53 X 104 conidia per sqnare centimetre of leaf area were observed during the present study. I. INTRODUCTION Clayton and Gaines (1933), Armstrong and Sumner (1935), and Dixon, McLean, and Wolf (1936) have shown that sporulation by Peronospora tabacina Adam. occurred only under conditions of high humidity.
    [Show full text]
  • Ratio of Phosphate to Amino Acids
    National Institute for Health and Care Excellence Final Neonatal parenteral nutrition [D10] Ratio of phosphate to amino acids NICE guideline NG154 Evidence reviews February 2020 Final These evidence reviews were developed by the National Guideline Alliance which is part of the Royal College of Obstetricians and Gynaecologists FINAL Error! No text of specified style in document. Disclaimer The recommendations in this guideline represent the view of NICE, arrived at after careful consideration of the evidence available. When exercising their judgement, professionals are expected to take this guideline fully into account, alongside the individual needs, preferences and values of their patients or service users. The recommendations in this guideline are not mandatory and the guideline does not override the responsibility of healthcare professionals to make decisions appropriate to the circumstances of the individual patient, in consultation with the patient and/or their carer or guardian. Local commissioners and/or providers have a responsibility to enable the guideline to be applied when individual health professionals and their patients or service users wish to use it. They should do so in the context of local and national priorities for funding and developing services, and in light of their duties to have due regard to the need to eliminate unlawful discrimination, to advance equality of opportunity and to reduce health inequalities. Nothing in this guideline should be interpreted in a way that would be inconsistent with compliance with those duties. NICE guidelines cover health and care in England. Decisions on how they apply in other UK countries are made by ministers in the Welsh Government, Scottish Government, and Northern Ireland Executive.
    [Show full text]
  • L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of Alzheimer's Disease
    bioRxiv preprint doi: https://doi.org/10.1101/354290; this version posted June 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. L-norvaline reverses cognitive decline and synaptic loss in a murine model of Alzheimer's disease. Baruh Polis1,2*, Kolluru D Srikanth2, Evan Elliott3, Hava Gil-Henn2 , Abraham O. Samson1 1 Drug Discovery Laboratory, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, 1311502, Israel. 2 Laboratory of Cell Migration and Invasion, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, 1311502, Israel. 3 Laboratory of Molecular and Behavioral Neuroscience, The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, 1311502, Israel. * Correspondence should be addressed to: Baruh Polis, The Azrieli Faculty of Medicine, Bar-Ilan University, 8th Henrietta Szold Street, P.O. Box 1589, Safed 1311502, Israel. Phone: +972525654451, e-mail: [email protected] Keywords: Alzheimer’s disease, L-norvaline, L-arginine, arginase, memory, amyloid beta, ribosomal protein S6 kinase beta-1, mTOR. 1 bioRxiv preprint doi: https://doi.org/10.1101/354290; this version posted June 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract The urea cycle plays a role in the pathogenesis of Alzheimer’s disease (AD).
    [Show full text]
  • Effect of L-Norvaline on the Small Intestinal
    Effect of L-norvaline on the small intestinal wall blood perfusion in a model of acute segmental mesenteric thrombosis Effecto de L-norvalina en la perfusión sanguínea de la pared del intestino delgado en el modelo de trombosis mesentérica segmentaria aguda Elena N. Bezhina; Sergey A. Alekhin; Elena B. Artyushkova; Angelika Y. Orlova; Lev N. Sernov; Tatyana A. Denisuk; Anna A. Peresypkina 1Belgorod State University, Pobedy St., 85, Belgorod, 308015, Russia *Corresponding author: Sergey A. Alekhin, Belgorod State University, Pobedy St., 85, Belgorod, 308015, Russia; e-mail: [email protected] Received/Recibido: 06/28/2020 Accepted/Aceptado: 07/15/2020 Published/Publicado: 09/09/2020 DOI: 10.5281/zenodo.4266263 Abstract Introduction. Ischemic and reperfusion injury play one of the before occlusion of segmental mesenteric arteries. The most important roles in the pathogenesis of many disorders. speed of microcirculation was measured using laser Doppler Especially severe changes in the wall of the small intestine flowmetry by Biopac systems MP100 with TSD144 probe and are observed in acute mesenteric thrombosis, and restora- Acknowledge 3.9.0 program. tion of blood flow to the ischemic tissue initiates a cascade of events that may lead to additional cell injury known as reper- Results. Arginase inhibitor, L-norvaline, in doses of 5, 10, 15, fusion injury. This reperfusion damage frequently exceeds the 20 mg/kg decreased the level of post-occlusive hyperemia original ischemic insult. L-norvaline, as an arginase inhibitor from 1846.25± 54.97 BPU to 1738.49± 42.67, 1622.91± 17.15, was shown to be potentially strategy to combat hepatic isch- and 1412,88 ± 38,08 BPU, respectively.
    [Show full text]
  • Enzymatic Aminoacylation of Trna with Unnatural Amino Acids
    Enzymatic aminoacylation of tRNA with unnatural amino acids Matthew C. T. Hartman, Kristopher Josephson, and Jack W. Szostak* Department of Molecular Biology and Center for Computational and Integrative Biology, Simches Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114 Edited by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved January 24, 2006 (received for review October 21, 2005) The biochemical flexibility of the cellular translation apparatus applicable to screening large numbers of unnatural amino acids. offers, in principle, a simple route to the synthesis of drug-like The commonly used ATP-PPi exchange assay, although very modified peptides and novel biopolymers. However, only Ϸ75 sensitive, does not actually measure the formation of the AA- unnatural building blocks are known to be fully compatible with tRNA product (13). A powerful assay developed by Wolfson and enzymatic tRNA acylation and subsequent ribosomal synthesis of Uhlenbeck (14) allows the observation of AA-tRNA synthesis modified peptides. Although the translation system can reject even with unnatural amino acids through the use of tRNA which substrate analogs at several steps along the pathway to peptide is 32P-labeled at the terminal C-p-A phosphodiester linkage (4, synthesis, much of the specificity resides at the level of the 15). Because the assay is based on the separation of AMP and aminoacyl-tRNA synthetase (AARS) enzymes that are responsible esterified AA-AMP by TLC, each amino acid analog must be for charging tRNAs with amino acids. We have developed an AARS tested in a separate assay mixture; moreover, this assay cannot assay based on mass spectrometry that can be used to rapidly generally distinguish between tRNA charged with the desired identify unnatural monomers that can be enzymatically charged unnatural amino acid or with contaminating natural amino acid.
    [Show full text]
  • Design and Synthesis of Novel Prodrugs to Modulate Gaba Receptors in Cancer Hui Zhang
    DESIGN AND SYNTHESIS OF NOVEL PRODRUGS TO MODULATE GABA RECEPTORS IN CANCER HUI ZHANG Thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University for the degree of Doctor of Philosophy March 2017 Declaration It is hereby declared that this thesis and the research work upon which it is based were conducted by the author, Hui Zhang. Hui Zhang Abstract of Thesis GABA (gamma-amino butanoic acid) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA has been found to play an inhibitory role in some cancers, including colon carcinoma, cholangiocarcinoma, and lung adenocarcinoma. Growing evidence shows that GABAB receptors are involved in tumour development. The expression level of GABAB receptors was found to be upregulated in some human tumours, including the pancreas, and cancer cell lines, suggesting that GABAB receptors may be potential targets for cancer therapy and diagnosis. In this research programme, several diverse series of potential anticancer prodrugs of GABA and GABA receptor-targeting agents have been rationally designed and synthesised for selective activation in the tumour microenvironment. In one approach, a series of oligopeptide conjugate prodrugs have been synthesised as protease-activatable substrates for either the extracellular matrix metalloproteinase MMP-9 or the lysosomal endoprotease legumain; each of which are overexpressed in the tumour environment and are effectors of tumour growth and metastasis. Specifically, a novel fluorogenic, oligopeptide FRET substrate prodrug of legumain HZ101 (Rho-Pro-Ala-Asn~GABA-spacer-AQ) has been characterised and shown to have theranostic potential. Proof of principle has been demonstrated using recombinant human legumain for which HZ101 is an efficient substrate and is latently quenched until cleaved.
    [Show full text]
  • Increases in GABA Concentrations During Cerebral Ischaemia: A
    J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.72.1.99 on 1 January 2002. Downloaded from 99 PAPER Increases in GABA concentrations during cerebral ischaemia: a microdialysis study of extracellular amino acids P J Hutchinson, M T O’Connell, P G Al-Rawi, C R Kett-White, A K Gupta, L B Maskell, J D Pickard, P J Kirkpatrick ............................................................................................................................. J Neurol Neurosurg Psychiatry 2002;72:99–105 Objectives: Increases in the extracellular concentration of the excitatory amino acids glutamate and aspartate during cerebral ischaemia in patients are well recognised. Less emphasis has been placed on the concentrations of the inhibitory amino acid neurotransmitters, notably γ-amino-butyric acid (GABA), despite evidence from animal studies that GABA may act as a neuroprotectant in models of See end of article for ischaemia. The objective of this study was to investigate the concentrations of various excitatory, inhibi- authors’ affiliations ....................... tory and non-transmitter amino acids under basal conditions and during periods of cerebral ischaemia in patients with head injury or a subarachnoid haemorrhage. Correspondence to: Methods: Cerebral microdialysis was established in 12 patients with head injury (n=7) or subarach- Mr PJ Hutchinson, Academic Department of noid haemorrhage (n=5). Analysis was performed using high performance liquid chromatography for Neurosurgery, University of a total of 19 (excitatory, inhibitory and non-transmitter) amino acids. Patients were monitored in neu- Cambridge, Box 167, rointensive care or during aneurysm clipping. Addenbrooke’s Hospital, Results: During stable periods of monitoring the concentrations of amino acids were relatively constant Cambridge CB2 2QQ, UK; enabling basal values to be established.
    [Show full text]
  • Analysis of Amino Acids by HPLC
    Analysis of Amino Acids by HPLC Rita Steed Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 Amino Acid Analysis - Agilent Restricted Page 1 June 24, 2010 Outline • Amino Acids – Structure, Chemistry • Separation Considerations • Challenges • Instrumentation • Derivatization – OPA, FMOC • Overview of Separations • Examples Amino Acid Analysis - Agilent Restricted Page 2 June 24, 2010 Amino Acids – Structure, Chemistry CH3 Alanine (()Ala) Glutamic Acid (()Glu) Amino Acid Analysis - Agilent Restricted Page 3 June 24, 2010 Amino Acids – Zwitterionic Amino Acid Analysis - Agilent Restricted Page 4 June 24, 2010 Separation Considerations • Zwitterions - poor solubility near iso- …electric point • Most have poor UV absorbance • Derivatization – OPA, FMOC •Reduce polarity – increases retention in reversed-phase chromatoggpyraphy •Improve sensitivity – UV, Fluorescence • Detector; DAD, FLD, MS, ELSD Amino Acid Analysis - Agilent Restricted Page 5 June 24, 2010 Ortho Phthalaldehyde (OPA) and Fluorenylmethoxy chloroformate (FMOC) Reactions with Amines OPA O SR’ R’SH H NR +RNH2 H Room Temperature O Fluorescence: Ex 340nm, Em 450nm Non-fluorescent DAD: 338 , 10nm; Ref . 390 , 20nm Does not absorb at 338nm FMOC RR’NH - HCl + or Room Temperature RNH2 NRR’ or NHR Fluorescence: Ex 260nm, Em 325nm Fluorescent DAD: 262, 16nm; Ref. 324,8nm Absorbs at 262nm and Fluorescences at 324nm Group/Presentation Title Agilent Restricted Month ##, 200X Names and Order of Elution for OPA and FMOC Derivatives of Amino Acids Peak # AA Name AA Abbreviation Derivative Type Peak # AA Name AA Abbreviation Derivative Type Group/Presentation Title Agilent Restricted Month ##, 200X Agilent AAA Methods - They’ve Evolved • Automated Amino Acid Analysis – AminoQuant I & II (1987) •1090 •1100, Pub.
    [Show full text]