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BBA - Proteins and Proteomics (v.1774, #4)

Editorial Board (pp. ii).

The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells by Silke Henrich; Stuart J. Cordwell; Ben Crossett; Mark S. Baker; Richard I. Christopherson (pp. 413-432).

Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular ‘housekeeping’ metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt's lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% ( n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% ( n=90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.

Keywords: Abbreviations; 2-DE; two-dimensional gel electrophoresis; 2-DLC; two-dimensional liquid chromatography; IPG; immobilized pH gradient; MALDI-TOF; matrix-assisted laser desorption-ionization time-of-flight; MS; mass spectrometry; MS-MS; tandem mass spectrometry; pI; iso-electric point; PMF; peptide mass fingerprinting; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPITC; 4-sulfophenyl isothiocyanateDNA-binding protein; Burkitt's lymphoma; Nuclear proteome; Two-dimensional gel electrophoresis

Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC) by Jessica L. Reynolds; Supriya D. Mahajan; Donald E. Sykes; Stanley A. Schwartz; Madhavan P.N. Nair (pp. 433-442).

In the US, the increase in methamphetamine (METH) use has been associated with increased human immunodeficiency virus (HIV-1) infection. Dendritic cells (DC) are the first line of defense against HIV-1. DC play a critical role in harboring HIV-1 and facilitate the infection of neighboring T cells. However, the role of METH on HIV-1 infectivity and the expression of the proteome of immature dendritic cells (IDC) has not been elucidated. We hypothesize that METH modulates the expression of a number of proteins by IDC that foster the immunopathogenesis of HIV-1 infection. We utilized LTR amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to analyze the effects of METH on HIV-1 infectivity (HIV-1 IIIB; CXCR4-tropic, X4 strain) and the proteomic profile of IDC. Our results demonstrate that METH potentiates HIV-1 replication in IDC. Furthermore, METH significantly differentially regulates the expression of several proteins including CXCR3, protein disulfide isomerase, procathepsin B, peroxiredoxin and galectin-1. Identification of unique, METH-induced proteins may help to develop novel markers for diagnostic, preventive and therapeutic targeting in METH using subjects.

Keywords: Abbreviations; HSPA8; Hsp70, protein 8 isoform 1; IDC; immature dendritic cells; MDC; mature dendritic cells; PDI; protein disulfide isomerase; HSPA5; dnaK-type molecular chaperoneMethamphetamine; Immature dendritic cell (IDC); Difference Gel Electrophoresis (DIGE); HPLC-MS/MS

Val326 of Thermoactinomyces vulgaris R-47 amylase II modulates the preference for alpha-(1,4)- and alpha-(1,6)-glycosidic linkages by Keisuke Ito; Sohei Ito; Kousuke Ishino; Akiko Shimizu-Ibuka; Hiroshi Sakai (pp. 443-449).

Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) catalyzes not only the hydrolysis of alpha-(1 ,4)- and alpha-(1 ,6)-glycosidic linkages but also transglycosylation. The subsite +1 structure of alpha-amylase family enzymes plays important roles in substrate specificity and transglycosylation activity. We focused on the amino acid residue at the 326th position based on information on the primary structure and crystal structure, and replaced Val with Ala, Ile, or Thr. The V326A mutant favored hydrolysis of the alpha-(1 ,4)-glycosidic linkage compared to the wild-type enzyme. In contrast, the V326I mutant favored hydrolysis of the alpha-(1 ,6)-glycosidic linkage and exhibited low transglycosylation activity. In the case of the V326T mutant, the hydrolytic activity was almost identical to that of the wild-type TVA II, and the transglycosylation activity was poor. These results suggest that the volume and the hydrophobicity of the amino acid residue at the 326th position modulate both the preference for glycosidic linkages and the transglycosylation activity.

Keywords: Alpha-amylase; Subsite +; 1; Transglycosylation; Substrate specificity; Site-directed mutagenesis; Oligosaccharide

Mutational analysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system by Frantz Liszewska; Małgorzata Lewandowska; Danuta Płochocka; Agnieszka Sirko (pp. 450-455).

Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein–protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein–protein interactions and the enzymatic activity of OASTL.

Keywords: Abbreviations; AD-GAL4; transcription activation domain of GAL4; BD-GAL4; DNA-binding domain of GAL4; CS; cysteine synthase; NPCS1; O; -acetylserine (thiol) lyase from; Nicotiana plumbaginifolia; (Accession nr of cDNA sequence- AY450295); OAS; O; -acetylserine; OASTL; O; -acetylserine (thiol) lyase (EC 2.5.1.47); OASTL-A; O; -acetylserine (thiol) lyase A from; Escherichia coli; (the product of; cysK; gene); OAS/OAHTL; O; -acetyl serine/; O; -acetylhomoserine (thiol) lyase (EC 2.5.1.47/2.5.1.49); ONPG; o-nitrophenyl-β-; d; -galactopyranoside; SAT; serine acetyltransferase (EC 2.3.1.30); Y2H; yeast two-hybridCysteine synthase; Site-directed mutagenesis; Yeast two-hybrid system

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: The role of the C- and N-terminal extensions by Md. Ziaul Amin; Tetsuya Bando; Razia Ruksana; Frederick Anokye-Danso; Yasuo Takashima; Yasuji Sakube; Hiroaki Kagawa (pp. 456-465).

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.

Keywords: Abbreviations; TN; troponin; TNI; troponin I; TNC; troponin C; TNT; troponin T; TM; tropomyosinTroponin I; Caenorhabditis elegans; Troponin T; Troponin C; Tropomyosin; C-terminal extension

Fluorescence spectroscopic analysis of the proximity changes between the central helix of troponin C and the C-terminus of troponin T from chicken skeletal muscle by Ying-Ming Liou; Huan-Lu Chao (pp. 466-473).

Recent structural studies of the troponin (Tn) core complex have shown that the regulatory head containing the N-lobe of TnC is connected to the IT arm by a flexible linker of TnC. The IT arm is a long coiled-coil formed by α-helices of TnI and TnT, plus the C-lobe of TnC. The TnT is thought to play a pivotal role in the linking of Ca²⁺-triggered conformational changes in thin filament regulatory proteins to the activation of cross-bridge cycling. However, a functional domain at the C-terminus of TnT is missing from the Tn core complex. In this study, we intended to determine the proximity relationship between the central helix of TnC and the TnT C-terminus in the binary and the ternary complex with and without Ca²⁺ by using pyrene excimer fluorescence spectroscopy and fluorescence resonance energy transfer. Chicken fast skeletal TnC contains a Cys102 at the E helix, while TnT has a Cys264 at its C-terminus. These two cysteines were specifically labeled with sulfhydryl-reactive fluorescence probes. The measured distance in the binary complex was about 19 Å and slightly increased when they formed the ternary complex with TnI (20 Å). Upon Ca²⁺ binding the distance was not affected in the binary complex but increased by ∼4 Å in the ternary complex. These results suggest that TnI plays an essential role in the Ca²⁺-mediated change in the spatial relationship between the C-lobe of TnC and the C-terminus of TnT.

Keywords: Troponin; Pyrene excimer fluorescence; Fluorescence resonance energy transfer; Proximity change

Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activity by Teresa Tiago; Paulo Martel; Carlos Gutiérrez-Merino; Manuel Aureliano (pp. 474-480).

Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V₁₀O₂₈⁶⁻) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V₁₀ binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V₁₀ oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V₁₀ to bind at the back-door, but only on the “open” structures where there is access to the phosphate binding-loop. It is suggested that V₁₀ acts as a “back-door stop” blocking the closure of the 50-kDa cleft necessary to carry out ATP-γ-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V₁₀ and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.

Keywords: Back-door; Decavanadate; Myosin; Molecular Docking

Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2 by Maja Christiansen; Charlotte S. Jørgensen; Inga Laursen; Daniel Hirschberg; Peter Højrup; Gunnar Houen (pp. 481-492).

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.

Keywords: Abbreviations; BCIP/NBT; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium; CIE; crossed immunoelectrophoresis; DBP; vitamin D-binding protein; DTT; dithiothreitol; ESI; electro spray ionization; FPLC; fast protein liquid chromatography; Gc; group-specific component; HCCA; alpha-cyano-4-hydroxycinnamic acid; HILIC; hydrophilic interaction chromatography; HPLC; high performance liquid chromatography; IEF; isoelectric focusing; LC; liquid chromatography; MAF; macrophage-activating factor; MALDI; matrix assisted laser desorption ionization; MS; mass spectrometry; RP-HPLC; reversed phase HPLC; TFA; trifluoroacetic acid; TNBP; tri-N-butylphosphate; TOF; time of flight; TTN; 50 mM Tris, 0.3 M NaCl, 1% Tween 20, pH 7.5Gc globulin; Vitamin D-binding protein; Isoforms; Glycosylation; Mass spectrometry

A fluorescence study of type I and type II receptors of bone morphogenetic proteins with bis-ANS (4, 4′-dianilino-1, 1′-bisnaphthyl-5, 5′ disulfonic acid) by Huiran Yin; Qing Zhou; Markandeswar Panda; Lee-Chuan C. Yeh; Michelle C. Zavala; John C. Lee (pp. 493-501).

Crystallography studies on several members of the bone morphogenetic protein (BMP) receptors suggested that hydrophobic regions in these proteins play an important role in their structure and function. In the present study, the environment sensitive fluorescent probe 4, 4′-dianilino-1, 1′-bisnaphthyl-5, 5′ disulfonic acid (bis-ANS) was used to study the hydrophobic regions of the extracellular domain of the type I and II receptors for bone morphogenetic proteins (ecBMPR-IB and ecBMPR-II). A single bis-ANS binding site per receptor molecule was found for both receptors, but the two receptors interacted with bis-ANS with distinctive characteristics. A significant shift in the emission maximum from 498 to 510 nm was detected when bis-ANS binds ecBMPR-IB, but a negligible change in the emission maximum was observed when the dye binds ecBMPR-II. Under identical reaction conditions, the maximum fluorescence intensities of the probe ( I_max) for the ecBMPR-IB and -II are 4.0 and 6.2×10⁴ arbitrary units, respectively. The probe binds to ecBMPR-IB and -II with K_d=11.0 and 17.5 μM, respectively. The bis-ANS modified site on both receptor types was not readily accessible to acrylamide quenching. Fluorescence energy transfer experiments further revealed close proximity between the tyrosine (in ecBMPR-IB) and the tryptophan residue (in ecBMPR-II) and the respective bis-ANS binding site in these receptors. The binding of bis-ANS did not alter the ligand binding activity of ecBMPR-IB, but enhanced that of ecBMPR-II. These results show that the bis-ANS-modified hydrophobic site on the ecBMPR-IB and -II molecules plays a different functional role.

Keywords: BMP receptors; bis-ANS; Hydrophobic surface; Protein structure; Fluorescence

Identification of residues participating in the interaction between an intraluminal loop of inositol 1,4,5-trisphosphate receptor and a conserved N-terminal region of chromogranin B by Jinho Kang; Sunmi Kang; Seung Hyun Yoo; Sunghyouk Park (pp. 502-509).

The inositol 1,4,5-trisphosphate receptor (IP3R) is a membrane channel that conducts calcium ions from the intracellular calcium stores. Despite a wealth of information on the cytoplasmic regulation of the IP3R, little is known about its regulation on the luminal side of the calcium stores. Here, we report studies on the IP3R intraluminal loop L3-2 and a conserved N-terminal region of chromogranin B. The IP3R loop is an important part of the channel's pore-forming region, and the chromogranin peptide has been shown to competitively inhibit calcium signaling by IP3R. Using the NMR titration approach, we showed that a part of the L3-2 is involved in a specific interaction with the chromogranin B peptide. Further NMR resonance assignments revealed that the 14th–20th residues of L3-2 are the keys to the binding to the chromogranin B peptide. Through detailed analysis of the data, we suggest a mechanism of IP3R regulation by chromogranin B involving conformational exchanges of the L3-2 region. Our report presents the findings of the first study on the interaction between the luminal loop of the IP3 receptor and its regulator at residue-resolution. The approaches described here should help to guide further studies on the interactions between the IP3R and other luminal side regulators.

Keywords: IP3R; Chromogranin; Interaction; NMR; Regulation; Assignment

Isolation and characterization of a thermostable β-xylosidase in the thermophilic bacterium Geobacillus pallidus by Denny Quintero; Zoraida Velasco; Estefanía Hurtado-Gómez; José L. Neira; Lellys M. Contreras (pp. 510-518).

The isolation, purification, biochemical and biophysical characterization of the first reported β-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 °C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 °C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of α-helix (44%) and β-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of β-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.

Keywords: Abbreviations; β-xylosidase; ^gp; β-xylosidase from; Geobacillus pallidus; GH; glycoside hydrolase; o-NPX; o-nitrophenyl-β-; d; -xylanopyranoside; p-NPX; p-nitrophenyl-β-; d; -xylanopyranoside; SEC; size-exclusion chromatographyβ-xylosidase; FTIR; Thermostability; GH52 family; Circular dichroism; Activity

The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding by Jia Zeng; Meimei Geng; Yuandong Liu; Lexian Xia; Jianshe Liu; Guanzhou Qiu (pp. 519-525).

Rusticyanin is a small blue copper protein isolated from Acidithiobacillus ferrooxidans with extreme acid stability and redox potential. The protein is thought to be a principal component in the iron respiratory electron transport chain in this microorganism, but its exact role in electron transfer remains controversial. The gene of rusticyanin was cloned then overexpressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. It was reported that Cys138, His85 and His143 were important residues for copper binding, but the significance of Cys138 was not verified so far. We constructed the mutant expression plasmids of these three residues using site-directed mutagenesis. Mutant proteins were expressed in E. coli and purified with a nickel metal affinity column. The EPR and atomic absorption spectroscopy results confirmed that Cys138 was crucial for copper binding. Removal of the sulfhydryl group of Cys138 resulted in copper loss. Mutations of His85 and His143 showed little effect on copper binding.

Keywords: Abbreviations; Amino acids; A, Ala; alanine; C, Cys; cysteine; D, Asp; aspartic acid; H, His; histidine; S, Ser; serine; RUS; rusticyanin; A. ferrooxidans; Acidithiobacillus ferrooxidans; HiPIP; high redox potential iron sulfur proteins; IPTG; isopropyl-; d; -thiogalactopyranoside; PAGE; polyacrylamide gel electrophoresis; PCR; polymerase chain reaction; RMSD; root mean square deviation; SDS; sodium dodecylsulfate; UV/Vis; ultraviolet-visible spectroscopy; EPR; electronic paramagnetic resonanceRusticyanin; Acidithiobacillus ferrooxidans; Cysteine; Sulfhydryl group; Expression; Purification; His-tag

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