purification-cystic-fibrosis-transmembrane-conductance-regulator

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in <em>Saccharomyces cerevisiae</em>

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

University of Manchester

Sekii Ladies Clinic. Furukawa, Miyagi, Japan.

P-glicoproteina (ABCB1) &egrave; una proteina di ATP-binding cassette che &egrave; associata con l&#39;acquisizione di resistenza multipla in cancro e il fallimento della chemioterapia negli esseri umani. Intuizioni strutturali di questa proteina sono descritti usando una combinazione di piccolo angolo x-ray scattering dati e cristallografia di cryo-elettrone. Ci hanno paragonato le strutture con omologhi batterici e discutere lo sviluppo di modelli di omologia per P-glicoproteina sulla base della struttura di Sav1866 batterica.

Intuizioni strutturali P-glicoproteina (ABCB1) da piccolo angolo di dispersione di raggi x e cristallografia di elettroni.

P-glycoprot&#xE9;ine (ABCB1) est une prot&#xE9;ine ATP-binding cassette qui est associ&#xE9; &#xE0; l&#x26;#39;acquisition de multi-r&#xE9;sistance aux m&#xE9;dicaments dans le cancer et l&#x26;#39;&#xE9;chec d&#x26;#39;une chimioth&#xE9;rapie chez les humains. Aper&#xE7;u structurels dans cette prot&#xE9;ine sont d&#xE9;crites en utilisant une combinaison de donn&#xE9;es d&#x26;#39;angle de petites diffusion des rayons X et des donn&#xE9;es de cristallographie cryo-&#xE9;lectronique. Nous avons compar&#xE9; les structures avec des homologues bact&#xE9;riens, et de discuter de l&#x26;#39;&#xE9;laboration de mod&#xE8;les d&#x26;#39;homologie de la P-glycoprot&#xE9;ine sur la base de la structure bact&#xE9;rienne Sav1866.

Aper&#xE7;us structurels dans la P-glycoprot&#xE9;ine (ABCB1) par l&#x26;#39;angle X-ray Scattering petites et cristallographie &#xE9;lectronique

P-Glykoprotein (ABCB1) ist ein Protein der ATP-Bindung-Kassette, die mit dem Erwerb der Multiresistenz bei Krebs und das Versagen der Chemotherapie beim Menschen ist. Strukturelle Einblicke in dieses Protein werden mit einer Kombination aus kleinen Winkel R&ouml;ntgen Streuung und Cryo-Elektron Kristallographie Daten beschrieben. Wir haben die Strukturen mit bakterielle homologe verglichen, und die Entwicklung der Homologie-Modelle f&uuml;r P-Glykoprotein auf Basis der bakteriellen Sav1866 Struktur zu diskutieren.

Strukturelle Einblicke in P-Glykoprotein (ABCB1) durch Kleine Gekr&ouml;pfte Kristallstrukturanalyse Streuung und Elektron.

P-&#x7CD6;&#x86CB;&#x767D; (ABCB1) &#x662F; ATP &#x7ED3;&#x5408;&#x76D2;&#x5F0F;&#x86CB;&#x767D;&#xFF0C;&#x83B7;&#x5F97;&#x5728;&#x80BF;&#x7624;&#x591A;&#x836F;&#x8010;&#x836F;&#x548C;&#x5316;&#x7597;&#x5728;&#x4EBA;&#x7C7B;&#x4E2D;&#x7684;&#x5931;&#x8D25;&#x4E0E;&#x76F8;&#x5173;&#x8054;&#x3002;&#x8FD9;&#x79CD;&#x86CB;&#x767D;&#x8D28;&#x7684;&#x7ED3;&#x6784;&#x6DF1;&#x5165;&#x63CF;&#x8FF0;&#x4E86;&#x4F7F;&#x7528;&#x5C0F;&#x89D2;&#x5EA6; x &#x5C04;&#x7EBF;&#x6563;&#x5C04;&#x6570;&#x636E;&#x548C;&#x4F4E;&#x6E29;&#x7535;&#x5B50;&#x6676;&#x4F53;&#x5B66;&#x6570;&#x636E;&#x7684;&#x7EC4;&#x5408;&#x3002;&#x6211;&#x4EEC;&#x6BD4;&#x8F83;&#x4E86;&#x4E0E;&#x7EC6;&#x83CC;&#x7684;&#x540C;&#x7CFB;&#x7269;&#xFF0C;&#x7ED3;&#x6784;&#xFF0C;&#x5E76;&#x8BA8;&#x8BBA;&#x57FA;&#x4E8E;&#x7EC6;&#x83CC;&#x7684; Sav1866 &#x7ED3;&#x6784;&#x7684; P &#x7CD6;&#x86CB;&#x767D;&#x7684;&#x540C;&#x6E90;&#x6027;&#x6A21;&#x578B;&#x5F00;&#x53D1;&#x3002;

&#x7ED3;&#x6784;&#x6D1E;&#x5BDF; P-&#x7CD6;&#x86CB;&#x767D; (ABCB1) &#x7684;&#x5C0F;&#x89D2; x &#x5C04;&#x7EBF;&#x6563;&#x5C04;&#x548C;&#x7535;&#x5B50;&#x6676;&#x4F53;&#x5B66;&#x3002;

P-glycoprotein (ABCB1) is an ATP-binding cassette protein that is associated with the acquisition of multi-drug resistance in cancer and the failure of chemotherapy in humans. Structural insights into this protein are described using a combination of small angle X-ray scattering data and cryo-electron crystallography data. We have compared the structures with bacterial homologues, and discuss the development of homology models for P-glycoprotein based on the bacterial Sav1866 structure.

Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography.

P-&#x7CD6;&#x86CB;&#x767D;&#x8CEA;&#xFF08;ABCB1&#xFF09;&#x306F;&#x3001;&#x764C;&#x306E;&#x591A;&#x5264;&#x8010;&#x6027;&#x306E;&#x7372;&#x5F97;&#x304A;&#x3088;&#x3073;&#x30D2;&#x30C8;&#x306B;&#x304A;&#x3051;&#x308B;&#x5316;&#x5B66;&#x7642;&#x6CD5;&#x306E;&#x5931;&#x6557;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;ATP-&#x7D50;&#x5408;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x86CB;&#x767D;&#x8CEA;&#x3067;&#x3042;&#x308B;&#x3002;&#x3053;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x69CB;&#x9020;&#x7684;&#x6D1E;&#x5BDF;&#x306F;&#x3001;&#x5C0F;&#x89D2;X&#x7DDA;&#x6563;&#x4E71;&#x30C7;&#x30FC;&#x30BF;&#x3068;&#x30AF;&#x30E9;&#x30A4;&#x30AA;&#x96FB;&#x5B50;&#x7DDA;&#x7D50;&#x6676;&#x5B66;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x8A18;&#x8FF0;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x79C1;&#x305F;&#x3061;&#x306F;&#x3001;&#x7D30;&#x83CC;&#x306E;&#x30DB;&#x30E2;&#x30ED;&#x30B0;&#x3092;&#x6301;&#x3064;&#x69CB;&#x9020;&#x4F53;&#x3092;&#x6BD4;&#x8F03;&#x3057;&#x3001;&#x7D30;&#x83CC;&#x306E;Sav1866&#x69CB;&#x9020;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;P-&#x7CD6;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30DB;&#x30E2;&#x30ED;&#x30B8;&#x30FC;&#x30E2;&#x30C7;&#x30EB;&#x306E;&#x958B;&#x767A;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x5C0F;&#x89D2;X&#x7DDA;&#x6563;&#x4E71;&#x6CD5;&#x3068;&#x96FB;&#x5B50;&#x7DDA;&#x7D50;&#x6676;&#x5B66;&#x306B;&#x3088;&#x308B;P-&#x7CD6;&#x86CB;&#x767D;&#x8CEA;&#xFF08;ABCB1&#xFF09;&#x3078;&#x306E;&#x69CB;&#x9020;&#x7684;&#x77E5;&#x898B;

P-&#xB2F9;&#xB2E8;&#xBC31;&#xC9C8; (ABCB1) &#xC774;&#xB77C;&#xBA74; &#xC554; &#xD6C4;&#xCC9C;&#xC131; &#xC800;&#xD56D; &#xC218;&#xC9D1;&#xACFC; &#xC778; &#xAC04;&#xC5D0; &#xB300; &#xD55C; &#xD654;&#xD559; &#xC694;&#xBC95;&#xC758; &#xC2E4;&#xD328;&#xC640; &#xAD00;&#xB828; &#xB41C; ATP &#xBC14;&#xC778;&#xB529; &#xCE74;&#xC138;&#xD2B8; &#xB2E8;&#xBC31;&#xC9C8;. &#xC774; &#xB2E8;&#xBC31;&#xC9C8; &#xAD6C;&#xC870; &#xD1B5;&#xCC30;&#xB825; &#xC791;&#xC740; &#xAC01; x &#xC120; &#xC0B0;&#xB780; &#xB370;&#xC774;&#xD130; &#xBC0F; &#xACF3;&#xC744; &#xC54C;&#xC544;&#xB0B4;&#xB294;-&#xC804;&#xC790; &#xACB0;&#xC815;&#xD559; &#xB370;&#xC774;&#xD130;&#xC758; &#xC870;&#xD569;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC138;&#xADE0; homologues &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAD6C;&#xC870;&#xB97C; &#xBE44;&#xAD50; &#xD558; &#xACE0; P-&#xB2F9;&#xB2E8;&#xBC31;&#xC9C8; &#xC138;&#xADE0; Sav1866 &#xAD6C;&#xC870;&#xB97C; &#xAE30;&#xBC18;&#xC73C;&#xB85C; homology &#xBAA8;&#xB378; &#xAC1C;&#xBC1C; &#xB17C;&#xC758;.

P-&#xB2F9;&#xB2E8;&#xBC31;&#xC9C8; (ABCB1)&#xC73C;&#xB85C; &#xC791;&#xC740; &#xAC01; x &#xC120; &#xC0B0;&#xB780; &#xBC0F; &#xC804;&#xC790; &#xACB0;&#xC815;&#xD559;&#xC5D0; &#xC758;&#xD574; &#xAD6C;&#xC870; &#xD1B5;&#xCC30;&#xB825;.

P-glicoprote&#xED;na (ABCB1) es una prote&#xED;na de uni&#xF3;n del ATP de cassette que se asocia con la adquisici&#xF3;n de resistencia a m&#xFA;ltiples f&#xE1;rmacos en el c&#xE1;ncer y el fracaso de la quimioterapia en los seres humanos. Conocimientos estructurales en esta prote&#xED;na se describen mediante una combinaci&#xF3;n de datos peque&#xF1;os &#xE1;ngulo de dispersi&#xF3;n de rayos X y la crio-electr&#xF3;nica de datos de cristalograf&#xED;a. Hemos comparado las estructuras bacterianas con hom&#xF3;logos, y discutir el desarrollo de modelos de homolog&#xED;a de la glicoprote&#xED;na-P sobre la base de la estructura bacteriana Sav1866.

Perspectivas estructurales en la P-glicoprote&#xED;na (ABCB1) de &#xE1;ngulo peque&#xF1;o dispersi&#xF3;n de rayos X y cristalograf&#xED;a de electrones

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state (13)C-labeling experiments using [1-(13)C]-, [2-(13)C]-, and [U-(13)C(6)]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.

Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling.

IWI Orthodontics. Tokyo.

Mutations to members of the A subfamily of ATP binding cassette (ABC) proteins are responsible for a number of diseases; typically they are associated with aberrant cellular lipid transport processes. Mutations to the ABCA4 protein are linked to a number of visual disorders including Stargardt's disease and retinitis pigmentosa. Over 500 disease-associated mutations in ABCA4 have been demonstrated; however, the genotype-phenotype link has not been firmly established. This shortfall is primarily because the function of ABCA4 in the visual cycle is not yet fully understood. One hypothesis suggests that ABCA4 mediates the trans-bilayer translocation of retinal-phosphatidylethanolamine conjugates to facilitate the retinal regeneration process in the visual cycle. This review examines the evidence to support, or refute, this working hypothesis on the function of this clinically important protein.

The lipid translocase, ABCA4: seeing is believing.

ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein.

Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.

CFTR (cystic fibrosis transmembrane conductance regulator) is a member of the ATP-binding cassette family of membrane proteins. Although almost all members of this family are transporters, CFTR functions as a channel with specificity for anions, in particular chloride and bicarbonate. In this review we look at what is known about CFTR structure and function within the context of the ATP-binding cassette family. We also review current strategies aimed at obtaining the high resolution structure of the protein. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

Article	Year
Sekii Ladies Clinic. Furukawa, Miyagi, Japan. Architectural record\| PubMed ID: 12120014	2002
Structural insights into P-glycoprotein (ABCB1) by small angle X-ray scattering and electron crystallography. FEBS letters\| PubMed ID: 18657537	2008
Subcellular flux analysis of central metabolism in a heterotrophic Arabidopsis cell suspension using steady-state stable isotope labeling. Plant physiology\| PubMed ID: 19939942	2010
IWI Orthodontics. Tokyo. Architectural record\| PubMed ID: 20873406	2010
The lipid translocase, ABCA4: seeing is believing. The FEBS journal\| PubMed ID: 21554544	2011
Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids. Biochimica et biophysica acta\| PubMed ID: 24036079	2014
CFTR structure and cystic fibrosis. The international journal of biochemistry & cell biology\| PubMed ID: 24534272	2014

Naomi Pollock

Biography

JoVE Journal Publications

Other Publications