Structural Insights Into Substrate Recognition and Processing by the 20S Proteasome
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I HIGH MASS ACCURACY COUPLED to SPATIALLY-DIRECTED
HIGH MASS ACCURACY COUPLED TO SPATIALLY-DIRECTED PROTEOMICS FOR IMPROVED PROTEIN IDENTIFICATIONS IN IMAGING MASS SPECTROMETRY EXPERIMENTS By David Geoffrey Rizzo Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemistry August, 2016 Nashville, Tennessee Approved: Richard M. Caprioli, Ph.D. Kevin L. Schey, Ph.D. John A. McLean, Ph.D. Michael P. Stone, Ph.D. i Copyright © 2016 by David Geoffrey Rizzo All Rights Reserved ii This work is dedicated to my family and friends, who have shown nothing but support for me in all of life’s endeavors. iii ACKNOWLEDGEMENTS “As we express our gratitude, we must never forget that the highest appreciation is not to utter words, but to live by them.” - John F. Kennedy – There are many people I must thank for showing kindness, encouragement, and support for me during my tenure as a graduate student. First and foremost, I would like to thank my research advisor, Richard Caprioli, for providing both ample resources and guidance that allowed me to grow as a scientist. Our discussions about my research and science in general have helped me become a much more focused and discerning analytical chemist. I must also thank my Ph.D. committee members, Drs. Kevin Schey, John McLean, and Michael Stone, who have brought valuable insight into my research and provided direction along the way. My undergraduate advisor, Dr. Facundo Fernández, encouraged me to begin research in his lab and introduced me to the world of mass spectrometry. -
THE THICKNESSES of HEMOGLOBIN and BOVINE SERUM ALBUMIN MOLECULES AS UNIMOLECULAR LA YERS AD- SORBED ONTO FILMS of BARIUM STEARATE* by ALBERT A
518 BIOCHEMISTRY: A. A. FISK PROC. N. A. S. THE THICKNESSES OF HEMOGLOBIN AND BOVINE SERUM ALBUMIN MOLECULES AS UNIMOLECULAR LA YERS AD- SORBED ONTO FILMS OF BARIUM STEARATE* By ALBERT A. FIsKt GATES AND CRELLIN LABORATORIES OF CHEMISTRY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA 4, CALIFORNIAt Communicated by Linus Pauling, July 17, 1950 The following work, which describes a method of measuring one dimen- sion of some protein molecules, is based on the determination of the apparent thickness of a unimolecular layer of globular protein molecules adsorbed from solution onto a metallic slide covered with an optical gauge of barium stearate. Langmuirl' 2 and Rothen3 have published a few results obtained by such a technique, but have not exploited the method thoroughly. A complete set of experimental data has been obtained by Clowes4 on insulin and protamine. He studied the effects of pH and time of exposure on the thickness of layers of protamine and insulin adsorbed onto slides covered with barium stearate and conditioned with uranyl acetate. He found that the pH was responsible for large variations in the thickness of the adsorbed layers and that the thickness of insulin layers adsorbed onto a protamine base was dependent on the concentration of the insulin. Since Clowes found thicknesses as high as 100 A. for protamine and 400 A. for insulin, he was without doubt usually dealing with multi- layers. Experimental.-The apparent thickness of a protein layer is measured with an optical instrument called the ellipsometer by Rothen,5-7 who has given a complete description of its design and optics and has calculated its sensitivity as 0.3 A. -
Flowering Buds of Globular Proteins: Transpiring Simplicity of Protein Organization
Comparative and Functional Genomics Comp Funct Genom 2002; 3: 525–534. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cfg.223 Conference Review Flowering buds of globular proteins: transpiring simplicity of protein organization Igor N. Berezovsky1 and Edward N. Trifonov2* 1 Department of Structural Biology, The Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel 2 Genome Diversity Centre, Institute of Evolution, University of Haifa, Haifa 31905, Israel *Correspondence to: Abstract Edward N. Trifonov, Genome Diversity Centre, Institute of Structural and functional complexity of proteins is dramatically reduced to a simple Evolution, University of Haifa, linear picture when the laws of polymer physics are considered. A basic unit of the Haifa 31905, Israel. protein structure is a nearly standard closed loop of 25–35 amino acid residues, and E-mail: every globular protein is built of consecutively connected closed loops. The physical [email protected] necessity of the closed loops had been apparently imposed on the early stages of protein evolution. Indeed, the most frequent prototype sequence motifs in prokaryotic proteins have the same sequence size, and their high match representatives are found as closed loops in crystallized proteins. Thus, the linear organization of the closed loop elements is a quintessence of protein evolution, structure and folding. Copyright 2002 John Wiley & Sons, Ltd. Received: 31 August 2002 Keywords: loop closure; prototype elements; protein structure; protein folding; Accepted: 14 October 2002 protein evolution; protein design; protein classification Introduction and the probability of the loop ends occurring in the vicinity of one another. The loop closure fortified One fundamental property of the polypeptide chain by the interactions between amino acid residues at is its ability to return to itself with the formation the ends of the loops provides an important degree of closed loops. -
Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins
International Journal of Molecular Sciences Article Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins M. Quadir Siddiqui 1,† , Maulik D. Badmalia 1,† and Trushar R. Patel 1,2,3,* 1 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; [email protected] (M.Q.S.); [email protected] (M.D.B.) 2 Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive, Calgary, AB T2N 4N1, Canada 3 Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada * Correspondence: [email protected] † These authors contributed equally to the work. Abstract: Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and func- tions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share Citation: Siddiqui, M.Q.; Badmalia, similarities with transcriptional regulators and have positively charged electrostatic patches, which M.D.; Patel, T.R. -
Proteasome 26S Subunit, Non Atpase 7 (PSMD7)
RPG282Hu01 10µg Recombinant Proteasome 26S Subunit, Non ATPase 7 (PSMD7) Organism Species: Homo sapiens (Human) Instruction manual FOR IN VITRO USE AND RESEARCH USE ONLY NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES 10th Edition (Revised in Jan, 2014) [ PROPERTIES ] Residues: Met1~Lys324 Tags: Two N-terminal Tags, His-tag and T7-tag Accession: P51665 Host: E. coli Purity: >90% Endotoxin Level: <1.0EU per 1μg (determined by the LAL method). Formulation: Supplied as lyophilized form in 20mM Tris, 150mM NaCl, pH8.0, containing 1mM EDTA, 1mM DTT, 0.01% sarcosyl, 5% trehalose, and preservative. Predicted isoelectric point: 6.7 Predicted Molecular Mass: 40.8kDa Applications: SDS-PAGE; WB; ELISA; IP. (May be suitable for use in other assays to be determined by the end user.) [ USAGE ] Reconstitute in sterile ddH2O. [ STORAGE AND STABILITY ] Storage: Avoid repeated freeze/thaw cycles. Store at 2-8oC for one month. Aliquot and store at -80oC for 12 months. Stability Test: The thermal stability is described by the loss rate of the target protein. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37oC for 48h, and no obvious degradation and precipitation were observed. (Referring from China Biological Products Standard, which was calculated by the Arrhenius equation.) The loss of this protein is less than 5% within the expiration date under appropriate storage condition. [ SEQUENCES ] The sequence of the target protein is listed below. MPELAVQKVV VHPLVLLSVV DHFNRIGKVG NQKRVVGVLL GSWQKKVLDV SNSFAVPFDE DDKDDSVWFL DHDYLENMYG MFKKVNARER IVGWYHTGPK LHKNDIAINE LMKRYCPNSV LVIIDVKPKD LGLPTEAYIS VEEVHDDGTP TSKTFEHVTS EIGAEEAEEV GVEHLLRDIK DTTVGTLSQR ITNQVHGLKG LNSKLLDIRS YLEKVATGKL PINHQIIYQL QDVFNLLPDV SLQEFVKAFY LKTNDQMVVV YLASLIRSVV ALHNLINNKI ANRDAEKKEG QEKEESKKDR KEDKEKDKDK EKSDVKKEEK KEKK [ REFERENCES ] 1. -
Phylogenomic Analysis of the Chlamydomonas Genome Unmasks Proteins Potentially Involved in Photosynthetic Function and Regulation
Photosynth Res DOI 10.1007/s11120-010-9555-7 REVIEW Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation Arthur R. Grossman • Steven J. Karpowicz • Mark Heinnickel • David Dewez • Blaise Hamel • Rachel Dent • Krishna K. Niyogi • Xenie Johnson • Jean Alric • Francis-Andre´ Wollman • Huiying Li • Sabeeha S. Merchant Received: 11 February 2010 / Accepted: 16 April 2010 Ó The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Chlamydomonas reinhardtii, a unicellular green performed to identify proteins encoded on the Chlamydo- alga, has been exploited as a reference organism for iden- monas genome which were likely involved in chloroplast tifying proteins and activities associated with the photo- functions (or specifically associated with the green algal synthetic apparatus and the functioning of chloroplasts. lineage); this set of proteins has been designated the Recently, the full genome sequence of Chlamydomonas GreenCut. Further analyses of those GreenCut proteins with was generated and a set of gene models, representing all uncharacterized functions and the generation of mutant genes on the genome, was developed. Using these gene strains aberrant for these proteins are beginning to unmask models, and gene models developed for the genomes of new layers of functionality/regulation that are integrated other organisms, a phylogenomic, comparative analysis was into the workings of the photosynthetic apparatus. Keywords Chlamydomonas Á GreenCut Á Chloroplast Á Phylogenomics Á Regulation A. R. Grossman (&) Á M. Heinnickel Á D. Dewez Á B. Hamel Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, CA 94305, USA Introduction e-mail: [email protected] Chlamydomonas reinhardtii as a reference organism S. -
Proteasome System of Protein Degradation and Processing
ISSN 0006-2979, Biochemistry (Moscow), 2009, Vol. 74, No. 13, pp. 1411-1442. © Pleiades Publishing, Ltd., 2009. Original Russian Text © A. V. Sorokin, E. R. Kim, L. P. Ovchinnikov, 2009, published in Uspekhi Biologicheskoi Khimii, 2009, Vol. 49, pp. 3-76. REVIEW Proteasome System of Protein Degradation and Processing A. V. Sorokin*, E. R. Kim, and L. P. Ovchinnikov Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; E-mail: [email protected]; [email protected] Received February 5, 2009 Abstract—In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for pro- teolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only pro- teins carrying a special “label” can get into it. A polyubiquitin chain plays the role of the “label”: degradation affects pro- teins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the “labeled” molecule, it is released and labels another molecule. This process has been named “Ub-dependent protein degradation”. In this review we systematize current data on the Ub–proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub- dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mech- anisms of proteasomal degradation and processing of proteins. -
1 Metabolic Dysfunction Is Restricted to the Sciatic Nerve in Experimental
Page 1 of 255 Diabetes Metabolic dysfunction is restricted to the sciatic nerve in experimental diabetic neuropathy Oliver J. Freeman1,2, Richard D. Unwin2,3, Andrew W. Dowsey2,3, Paul Begley2,3, Sumia Ali1, Katherine A. Hollywood2,3, Nitin Rustogi2,3, Rasmus S. Petersen1, Warwick B. Dunn2,3†, Garth J.S. Cooper2,3,4,5* & Natalie J. Gardiner1* 1 Faculty of Life Sciences, University of Manchester, UK 2 Centre for Advanced Discovery and Experimental Therapeutics (CADET), Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester, UK 3 Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, UK 4 School of Biological Sciences, University of Auckland, New Zealand 5 Department of Pharmacology, Medical Sciences Division, University of Oxford, UK † Present address: School of Biosciences, University of Birmingham, UK *Joint corresponding authors: Natalie J. Gardiner and Garth J.S. Cooper Email: [email protected]; [email protected] Address: University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, United Kingdom Telephone: +44 161 275 5768; +44 161 701 0240 Word count: 4,490 Number of tables: 1, Number of figures: 6 Running title: Metabolic dysfunction in diabetic neuropathy 1 Diabetes Publish Ahead of Print, published online October 15, 2015 Diabetes Page 2 of 255 Abstract High glucose levels in the peripheral nervous system (PNS) have been implicated in the pathogenesis of diabetic neuropathy (DN). However our understanding of the molecular mechanisms which cause the marked distal pathology is incomplete. Here we performed a comprehensive, system-wide analysis of the PNS of a rodent model of DN. -
An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale † ‡ ‡ ‡ § ‡ ∥ Upneet Kaur, He Meng, Fang Lui, Renze Ma, Ryenne N
Perspective Cite This: J. Proteome Res. 2018, 17, 3614−3627 pubs.acs.org/jpr Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale † ‡ ‡ ‡ § ‡ ∥ Upneet Kaur, He Meng, Fang Lui, Renze Ma, Ryenne N. Ogburn, , Julia H. R. Johnson, , ‡ † Michael C. Fitzgerald,*, and Lisa M. Jones*, ‡ Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, United States † Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, United States ABSTRACT: Over the past decade, a suite of new mass- spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein− ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL−MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states. -
Cellular and Molecular Signatures in the Disease Tissue of Early
Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of -
A Novel Proteasome Inhibitor NPI-0052 As an Anticancer Therapy
British Journal of Cancer (2006) 95, 961 – 965 & 2006 Cancer Research UK All rights reserved 0007 – 0920/06 $30.00 www.bjcancer.com Minireview A novel proteasome inhibitor NPI-0052 as an anticancer therapy 1 1 ,1 D Chauhan , T Hideshima and KC Anderson* 1Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, The Jerome Lipper Multiple Myeloma Center, Boston, MA 02115, USA Proteasome inhibitor Bortezomib/Velcade has emerged as an effective anticancer therapy for the treatment of relapsed and/or refractory multiple myeloma (MM), but prolonged treatment can be associated with toxicity and development of drug resistance. In this review, we discuss the recent discovery of a novel proteasome inhibitor, NPI-0052, that is distinct from Bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities; most importantly, it overcomes resistance to conventional and Bortezomib therapies. In vivo studies using human MM xenografts shows that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for Phase-I clinical trial of NPI-0052 in relapsed/ refractory MM patients. British Journal of Cancer (2006) 95, 961–965. doi:10.1038/sj.bjc.6603406 www.bjcancer.com & 2006 Cancer Research UK Keywords: protein degradation; proteasomes; multiple myeloma; novel therapy; apoptosis; drug resistance The systemic regulation of protein synthesis and protein degrada- inducible immunoproteasomes b-5i, b-1i, b-2i with different tion is essential -
Static Retention of the Lumenal Monotopic Membrane Protein Torsina in the Endoplasmic Reticulum
The EMBO Journal (2011) 30, 3217–3231 | & 2011 European Molecular Biology Organization | All Rights Reserved 0261-4189/11 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum Abigail B Vander Heyden1, despite the fact that it has been a decade since the protein was Teresa V Naismith1, Erik L Snapp2 and first described and linked to dystonia (Breakefield et al, Phyllis I Hanson1,* 2008). Based on its membership in the AAA þ family of ATPases (Ozelius et al, 1997; Hanson and Whiteheart, 2005), 1Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA and 2Department of Anatomy it is likely that torsinA disassembles or changes the confor- and Structural Biology, Albert Einstein College of Medicine, Bronx, mation of a protein or protein complex in the ER or NE. The NY, USA DE mutation is thought to compromise this function (Dang et al, 2005; Goodchild et al, 2005). TorsinA is a membrane-associated enzyme in the endo- TorsinA is targeted to the ER lumen by an N-terminal plasmic reticulum (ER) lumen that is mutated in DYT1 signal peptide. Analyses of torsinA’s subcellular localization, dystonia. How it remains in the ER has been unclear. We processing, and glycosylation show that the signal peptide is report that a hydrophobic N-terminal domain (NTD) di- cleaved and the mature protein resides in the lumen of the ER rects static retention of torsinA within the ER by excluding (Kustedjo et al, 2000; Hewett et al, 2003; Liu et al, 2003), it from ER exit sites, as has been previously reported for where it is a stable protein (Gordon and Gonzalez-Alegre, short transmembrane domains (TMDs).