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BBA - Proteins and Proteomics (v.1774, #10)

Editorial Board (pp. ii).

Proteomic analysis of cathepsin B and L-deficient mouse brain lysosomes by Sonja Stahl; Yvonne Reinders; Esther Asan; Walther Mothes; Ernst Conzelmann; Albert Sickmann; Ute Felbor (pp. 1237-1246).

Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ™) allowed us to simultaneously assess wildtype and cathepsin B^−/−L^−/− cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B^−/−L^−/− lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B^−/−L^−/− brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.

Keywords: Cerebrum; Cathepsin B; Cathepsin L; Lysosome; iTRAQ™; LC-MS/MS

Analysis of the structural consensus of the zinc coordination centers of metalloprotein structures by Kirti Patel; Anil Kumar; Susheel Durani (pp. 1247-1253).

In a recent sequence-analysis study it was concluded that up to 10% of the human proteome could be comprised of zinc proteins, quite varied in the functional spread. The native structures of only few of the proteins are actually established. The elucidation of rest of the sequences of not just human but even other actively investigated genomes may benefit from knowledge of the structural consensus of the zinc-binding centers of the currently known zinc proteins. Nearly four hundred X-ray and NMR structures in the database of zinc–protein structures available as of April 2007 were investigated for geometry and conformation in the zinc-binding centers; separately for the structural and catalytic proteins and individually in the zinc centers coordinated to three and four amino-acid ligands. Enhanced cysteine involvement in agreement with the observation in human proteome has been detected in contrast with previous reports. Deviations from ideal coordination geometries are detected, possible underlying reasons are investigated, and correlations of geometry and conformation in zinc-coordination centers with protein function are established, providing possible benchmarks for putative zinc-binding patterns of the burgeoning genome data.

Keywords: Abbreviations; H, His; Histidine; C, Cys; Cysteine; D, Asp; Aspartate; E, Glu; Glutamate; Zn; Zinc; PDB; Protein Data Bank; ZBP; Zinc Binding Patterns; C4; tetra coordinated zinc site; C5; penta coordinated zinc site; C6; hexa coordinated zinc site; Eq; Equatorial; Ax; Axial; O–Zn–O′; is angle between two bidentate oxygen atoms bonded to zinc. Structural proteins are those where zinc plays structural role whereas catalytic proteins are those where zinc is in catalytic roleMetalloprotein; Zinc–protein; Zinc binding pattern; Zinc coordination; Structural zinc-center; Catalytic zinc-center

Structure of the high-valent Fe^IIIFe^IV state in ribonucleotide reductase (RNR) of Chlamydia trachomatis—Combined EPR,⁵⁷Fe-,¹H-ENDOR and X-ray studies by Nina Voevodskaya; Marcus Galander; Martin Högbom; Pal Stenmark; Grant McClarty; Astrid Gräslund; Friedhelm Lendzian (pp. 1254-1263).

A recently discovered subgroup of class I ribonucleotide reductase (RNR) found in the infectious bacterium Chlamydia trachomatis ( C. trachomatis) was shown to exhibit a high-valent Fe^IIIFe^IV center instead of the tyrosyl radical observed normally in all class I RNRs. The X-ray structure showed that C. trachomatis WT RNR has a phenylalanine at the position of the active tyrosine in Escherichia coli RNR. In this paper the X-ray structure of variant F127Y is presented, where the tyrosine is restored. Using¹H- and⁵⁷Fe-ENDOR spectroscopy it is shown, that in WT and variants F127Y and Y129F of C. trachomatis RNR, the Fe^IIIFe^IV center is virtually identical with the short-lived intermediate X observed during the iron oxygen reconstitution reaction in class I RNR from E. coli. The experimental data are consistent with a recent theoretical model for X, proposing two bridging oxo ligands and one terminal water ligand. A surprising extension of the lifetime of the Fe^IIIFe^IV state in C. trachomatis from a few seconds to several hours at room temperature was observed under catalytic conditions in the presence of substrate. These findings suggest a possible new role for the Fe^IIIFe^IV state also in other class I RNR, during the catalytic radical transfer reaction, by which the substrate turnover is started.

Keywords: Ribonucleotide reductase class I; Chlamydia trachomatis; Structure of the high-valent Fe(III)Fe(IV)diiron state X; F127Y X-ray structure; 57Fe-electron nuclear double resonance ENDOR

Bowman–Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure by K.N. Rao; C.G. Suresh (pp. 1264-1273).

Different protease inhibitors including Bowman–Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Protease isoinhibitors of double-headed Bowman–Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata ( Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 Å resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman–Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function.

Keywords: Abbreviations; BBI-Bowman; Birk protease inhibitor; PIs; Protein Protease inhibitors; ES-MS; Electrospray ionization mass spectrometry; SDS-PAGE; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FPLC; Fast protein liquid chromatography; BT; Bovine Trypsin; NCS; Non-Crystallographic SymmetryBowman–Birk protease inhibitor; Vigna unguiculata; Plant protease inhibitor; Protein–protein interaction

Interaction of insulin with a triblock copolymer of PEG-(fumaric-sebacic acids)-PEG: Thermodynamic and spectroscopic studies by Pouneh S. Pourhosseini; Ali A. Saboury; Farhood Najafi; Mohammad N. Sarbolouki (pp. 1274-1280).

A comparative study on the interaction of (PEG-co-P(FA/SC)-co-PEG) triblock copolymer with bovine and human insulins was carried out using isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy. ITC data show that the copolymer has a low affinity for both proteins, with an association constant of about 7–9×10³ M−¹. Results also show that binding is enthalpically driven, and disfavored by conformational entropy. CD spectroscopy studies reveal a small increase in the helical content and a decrease in β-structure as well as random coil in both proteins. Acrylamide quenching experiments display reduced accessibility of tyrosines, while intrinsic fluorescence spectra show lower tyrosine emission. Furthermore, thermal unfolding experiments, studied by far-UV CD at 222 and 217 nm, demonstrate that upon interaction with the copolymer helix structure becomes less stable while the stability of β-structure remains unchanged. Altogether, these observations indicate that (PEG-co-P(FA/SC)-co-PEG) triblock copolymer has similar effect(s) on both proteins.

Keywords: Copolymers; Insulin; ITC; CD; Acrylamide quenching; Denaturation

Improving the activity and stability of thermolysin by site-directed mutagenesis by Kiyoshi Yasukawa; Kuniyo Inouye (pp. 1281-1288).

In previous site-directed mutagenesis study on thermolysin, mutations which increase the catalytic activity or the thermal stability have been identified. In this study, we attempted to generate highly active and stable thermolysin by combining the mutations so far revealed to be effective. Three mutant enzymes, L144S (Leu144 in the central α-helix located at the bottom of the active site cleft is replaced with Ser), G8C/N60C/S65P (Gly8, Asn60, and Ser65 in the N-terminal region are replaced with Cys, Cys, and Pro, respectively, to introduce a disulfide bridge between the positions 8 and 60), and G8C/N60C/S65P/L144S, were constructed by site-directed mutagenesis. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), the k_cat/ K_m values of L144S and G8C/N60C/S65P/L144S were 5- to 10-fold higher than that of the wild-type enzyme. The rate constants for thermal inactivation at 70 °C and 80 °C of G8C/N60C/S65P and G8C/N60C/S65P/L144S decreased to 50% of that of the wild-type enzyme. These results indicate that G8C/N60C/S65P/L144S is more active and stable than the wild-type thermolysin. Thermodynamic analysis suggests that the single mutation of Leu144→Ser and the triple mutation of Gly8→Cys, Asn60→Cys, and Ser65→Pro are independent.

Keywords: Abbreviations; FAGLA; N; -[3-(2-furyl)acryloyl]-glycyl-; l; -leucine amide; npr; neutral protease gene; ZDFM; N; -carbobenzoxy-; l; -aspartyl-; l; -phenylalanine methyl esterCatalytic activity; Metalloproteinase; Site-directed mutagenesis; Thermal stability; Thermolysin

Proteomic characterization of the site-dependent functional difference in the rat small intestine by Gotaro Iiizumi; Yuko Sadoya; Sayaka Hino; Naoki Shibuya; Hiroaki Kawabata (pp. 1289-1298).

To investigate the site-dependent functional difference in the small intestine, proteomic analysis was carried out on the three distinct parts of the rat small intestine. Male Wistar rats (7 weeks old) were fed a semi-purified diet ad libitum for 1 week. Intestinal tissues from the proximal, middle and distal regions of the small intestine were subjected to two-dimensional polyacrylamide gel electrophoresis, and the abundance of each spot was determined fluorometrically. MALDI-TOF/MS and LC-MS/MS analysis of the tryptic peptides were performed to identify the proteins. Many of the 180 identified proteins showed a distinctive distribution pattern along the small intestine. Glutathione S-transferase, Catechol O-methyltransferase and Villin 2 decreased gradually from the jejunum to the ileum, in contrast, non-specific dipeptidase and Keratin 19 increased gradually toward the ileum. The voltage-dependent anion channel 2 was most abundant in the duodenum while the L- and I-Fatty acid binding protein (FABP) and Cellular retinol binding protein (CRBP-II) were in the jejunum, and the Bile acid binding protein (BABP) was only observed in the ileum. The findings of these and of another proteins identified in this study may contribute to further understanding of the small intestinal function, and to clinical applications of small intestinal diseases.

Keywords: Proteome analysis; Small intestine; Regional function

Hb Santa Clara (β97His→Asn), a human haemoglobin variant: Functional characterization and structure modelling by Maria Cristina De Rosa; Cristiana Carelli Alinovi; Maria Eugenia Schininà; Maria Elisabetta Clementi; Antonio Amato; Maria Pia Cappabianca; Michela Pezzotti; Bruno Giardina (pp. 1299-1306).

This study examines the functional and structural effects of amino acid substitution at α₁β₂ interface of Hb Santa Clara (β97His→Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His β97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of βFG4 position has been further examined by computational models of known βFG4 variants, namely Hb Malmö (β97His→Gln), Hb Wood (β97His→Leu), Hb Nagoya (β97His→Pro) and Hb Moriguchi (β97His→Tyr). These findings demonstrate that, among the various residues at the α₁β₂ (and α₂β₁) intersubunit interface, His βFG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.

Keywords: Allosteric transition; Oxygen affinity; Cooperativity; Interface; Homology modelling

Oligomeric state of lipocalin-1 (LCN1) by multiangle laser light scattering and fluorescence anisotropy decay by Oktay K. Gasymov; Adil R. Abduragimov; Petra Merschak; Bernhard Redl; Ben J. Glasgow (pp. 1307-1315).

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5–68 μM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9–10 ns for TL (10 μM–1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes–Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.

Keywords: Tear lipocalin; Correlation time; Oligomeric state; Monomeric protein; Multiangle laser light scattering; Fluorescence anisotropy decay

Impact of cofactor on stability of bacterial (CopZ) and human (Atox1) copper chaperones by Faiza Hussain; Pernilla Wittung-Stafshede (pp. 1316-1322).

Here, we present the first characterization of in vitro unfolding and thermodynamic stability of two copper chaperone proteins: Bacillus subtilis CopZ and Homo sapiens Atox1 . We find that the unfolding reactions for apo- and Cu(I)-forms of CopZ and Atox1, induced by the chemical denaturant, guanidine hydrochloride (GuHCl), and by thermal perturbation are reversible two-state reactions. For both proteins, the unfolding midpoints shift to higher GuHCl concentrations and the thermodynamic stability is increased in the presence of Cu(I). Despite the same overall fold, apo-CopZ exhibits much lower thermal stability than apo-Atox1. Although the thermal stability of both proteins is increased in the presence of copper, the stabilizing effect is largest for the less stable variant. Divergent energetic properties of the apo- and holo-forms may be linked to conformational changes that facilitate copper transfer to the target.

Keywords: Abbreviations; CD; circular dichroism; T; _m; thermal midpoint; DTT; dithiothreitolProtein folding; Metallo-chaperone; Protein stability; Copper metabolism; Spectroscopy

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis by Yukiko Miyamoto; Takeharu Masaki; Shigeru Chohnan (pp. 1323-1330).

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K⁺, NH₄⁺, and Rb⁺, indicating that the Lactococcus enzyme is a K⁺-activated Type II enzyme. However, divalent cations including Mg²⁺ and Ca²⁺ significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K⁺ on its genome.

Keywords: N; -deoxyribosyltransferase; Trans-N; -deoxyribosylase; Lactococcus lactis; subsp.; lactis; K; ⁺; -activated enzyme; Nucleoside salvage pathway

Structural studies on Vibrio cholerae ToxR periplasmic and cytoplasmic domains by Tanaya Chatterjee; Rudra Prasad Saha; Pinak Chakrabarti (pp. 1331-1338).

The transcription activator ToxR controls the expression of cholera toxin, pilus colonization factor and outer membrane protein in Vibrio cholerae. It binds to the 5′-TTTTGAT-3′ tandemly repeated DNA sequence in the cholera toxin promoter region. ToxR is a membrane protein having distinct periplasmic and cytoplasmic domains. The two domains have been cloned, over-expressed and purified for structural studies. The cytoplasmic domain is more compact than the periplasmic domain. The periplasmic domain exists as dimer due to the presence of an interchain disulfide linkage, while the cytoplasmic domain is monomeric in solution implying the importance of the disulfide bond to homodimerize the native ToxR. By replacing one of the cysteines C293 with alanine, using site-directed mutagenesis, a C293A mutant was created at the periplasmic domain to elucidate the role of cysteine in dimerization of ToxR.

Keywords: Abbreviations; GdnHCl; guanidine hydrochloride; DTNB; 5,5′-dithio-bis(2-nitrobenzoic acid); IPTG; isopropyl-β-thiogalactopyranoside; DTT; dithiothreitol; PMSF; phenyl methyl sulfonyl fluoride; ESI-MS; electrospray ionization mass spectroscopy; NMWCO; net molecular weight cut-offToxR protein; Transcription regulation; Cholera toxin; Periplasmic domain; Cytoplasmic domain; Interchain disulfide bond; Site-directed mutagenesis

The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G by Annegret Ulke-Lemée; Laura Trinkle-Mulcahy; Steve Chaulk; Nina K. Bernstein; Nick Morrice; Mark Glover; Angus I. Lamond; Greg B.G. Moorhead (pp. 1339-1350).

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

Keywords: PP1; Phosphatase; Targeting; Nucleus; Polynucleotide kinase; Phosphohydrolase

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases by Donald E. Spratt; Valentina Taiakina; J. Guy Guillemette (pp. 1351-1358).

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca²⁺-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca²⁺ with mutations in the N-lobe (CaM₁₂), the C-lobe (CaM₃₄), or both lobes of CaM (CaM₁₂₃₄) to determine their effect on the binding and activation of the Ca²⁺-dependent neuronal (nNOS) and Ca²⁺-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein–protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca²⁺. Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.

Keywords: Abbreviations; Ca; ²⁺; calcium ion; CaM; calmodulin; CaM; ₁₂; CaM with aspartate residues 21 and 56 mutated to alanines; CaM; ₃₄; CaM with aspartate residues 93 and 131 mutated to alanines; CaM; ₁₂₃₄; CaM with aspartate residues 21, 56, 93, and 131 mutated to alanines; dansyl chloride; 5-dimethylaminonaphthalene-1-sulfonyl chloride; dansyl-CaM; ₁₂; dansyl chloride labeled CaM; ₁₂; dansyl-CaM; ₃₄; dansyl chloride labeled CaM; ₃₄; dansyl-CaM; ₁₂₃₄; dansyl chloride labeled CaM; ₁₂₃₄; NOS; nitric oxide synthase; eNOS; endothelial NOS (NOSIII); iNOS; inducible NOS (NOSII); nNOS; neuronal NOS (NOSI); cNOS; constitutive NOS; Heme; protoporphyrin IX; H; ₄; B; (6R,6S)-2-amino-4-hydroxy-6-(; l; -; erythro; -1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterindine; SOD; super oxide dismutase; PCR; polymerase chain reaction; ESI-MS; electrospray ionization-mass spectrometry; QTOF; quadrupole time-of-flight; DTT; dithiothreitol; EDTA; (ethylenedinitro)tetraacetic acid; FeCN; ferricyanide; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidCalmodulin; Calcium binding; Nitric oxide synthase; Nitric oxide; Protein–protein interaction; Electron transfer; Heme protein

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BBA - Proteins and Proteomics (v.1774, #10)