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BBA - Proteins and Proteomics (v.1814, #2)

Editorial Board (pp. i).

Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey ( Macaca fascicularis) in vitro by Kuniko Akama; Tomoe Horikoshi; Takashi Nakayama; Masahiro Otsu; Noriaki Imaizumi; Megumi Nakamura; Tosifusa Toda; Michiko Inuma; Hisashi Hirano; Yasushi Kondo; Yutaka Suzuki; Nobuo Inoue (pp. 265-276).

Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation.► The proteins and mRNA of FABP7 and CRMP2 are upregulated from monkey ES cells to NS cells. ► CRABP1 protein is upregulated from monkey ES cells to NS cells but its mRNA is unchanged. ► CRMP2 protein is also upregulated from monkey NS cells to neurons but its mRNA is unchanged.

Keywords: Abbreviations; ES; embryonic stem; NS; neural stem; 2-DE; two-dimensional gel electrophoresis; PMF; peptide mass fingerprinting; LC-MS-MS; liquid chromatography- tandem mass spectrometry; NSS; neural stem sphere; ACM; astrocyte-conditioned medium; FGF; fibroblast growth factor; EGF; epidermal growth factor; MAP; microtubule-associated protein; GFAP; glial fibrillary acidic protein; IPG; immobilized pH gradient; IEF; isoelectric focusing; T-TBS; TBS containing 0.05% Tween; MS; mass spectrometry; ACN; acetonitrile; TFA; trifluoroacetic acid; FABP7; fatty acid binding protein 7; CRMP2; collapsin response mediator protein 2; CRABP1; cellular retinoic acid binding protein 1Embryonic stem cells; Differential expression; Neural stem cells; Neuronal differentiation; Monkey

Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3) by Helena Handrkova; Jiri Petrak; Petr Halada; Dagmar Pospisilova; Radek Cmejla (pp. 277-282).

Protein arginine methyltransferase 3 (PRMT3) is a cytosolic enzyme that catalyzes the formation of mono- and asymmetric dimethyl arginines, with ribosomal protein (RP) S2 as its main in vivo substrate. The interplay of PRMT3-RPS2 homologs in yeast is important for regulating the ribosomal subunit ratio and assembly. Prmt3–null mice display slower embryonic growth and development, although this phenotype is milder than in mouse RP gene knockouts. Defects in ribosome maturation are the hallmark of Diamond-Blackfan anemia (DBA). Sequencing of the PRMT3 gene in patients from the Czech DBA registry revealed a heterozygous mutation encoding the Tyr87Cys substitution. Although later analysis excluded this mutation as the cause of disease, we anticipated that this substitution might be important for PRMT3 function and decided to study it in detail. Tyr87 resides in a highly conserved substrate binding domain and has been predicted to be phosphorylated. To address the impact of putative Tyr87 phosphorylation on PRMT3 properties, we constructed two additional PRMT3 variants, Tyr87Phe and Tyr87Glu PRMT3, mimicking non-phosphorylated and phosphorylated Tyr87, respectively. The Tyr87Cys and Tyr87Glu-PRMT3 variants had markedly decreased affinity to RPS2 and, consequently, reduced enzymatic activity compared to the wild-type enzyme. The activity of the Tyr87Phe-PRMT3 mutant remained unaffected. No evidence of Tyr87 phosphorylation was found using mass spectrometric analysis of purified PRMT3, although phosphorylation of serines 25 and 27 was observed. In conclusion, Tyr87 is important for the interaction between PRMT3 and RPS2 and for its full enzymatic activity.► Protein arginine methyltransferase 3 methylates ribosomal protein (RP) S2. ► The PRMT3–RPS2 interaction is important for regulating the ribosomal subunit ratio and assembly. ► Mutation of conservative Tyr87 affects affinity and enzymatic activity of human PRMT3 towards RPS2.

Keywords: Abbreviations; PRMT3; protein arginine methyltransferase 3; RP; ribosomal protein; DBA; Diamond-Blackfan anemia; wt; wild-type; IP; immunoprecipitationPRMT3; RPS2; Arginine methylation

Cystine-mediated oligomerization of the Atlantic salmon serum C-type lectin by David M. Hudson; Neil R. Mattatall; Elke Uribe; Robert C. Richards; Huansheng Gong; K. Vanya Ewart (pp. 283-289).

The Atlantic salmon ( Salmo salar) serum lectin (SSL) is a C-type lectin that binds to bacteria including salmon pathogens. SSL has been shown to be oligomeric in salmon serum and it displays a stoichiometric band-laddering pattern when analyzed by SDS-PAGE under non-reducing conditions. In this study, a model was generated for SSL isoform 2 in silico in order to identify cysteines that are available to form intermolecular disulfide bonds facilitating oligomerization. Then, recombinant SSL was expressed in E. coli and mutants were produced at positions Cys72 and Cys149. The SSL preparations were purified by metal-affinity chromatography and shown to be functional by carbohydrate-affinity chromatography. The recombinant SSL formed oligomers, which were evident by non-reducing covalent cross-linking and non-reducing SDS-PAGE; however, the band patterns were different for the mutants, with the maximal and predominant multimer sizes distinct from the wild-type recombinant lectin. Further examination of oligomerization by size exclusion chromatography revealed a subunit number from 35 to at least 110 for the wild-type recombinant SSL and subunit numbers below 9 for each mutant SSL oligomer. Thus, both cysteines were found to contribute to oligomerization of SSL.► The salmon serum lectin (SSL) is modeled and surface cysteines are identified. ► Recombinant wild-type SSL and cysteine mutants are expressed in E. coli. ► Laboratory analyses reveal that both cysteines form intermolecular bonds. ► Analyses also show variation in the size of the covalent oligomers formed by SSL.

Keywords: Atlantic salmon; C-type lectin; Oligomerization; Disulfide bond

Proteomic changes in the gills of wild-type and transgenic radiosensitive medaka following exposure to direct irradiation and to X-ray induced bystander signals by Richard W. Smith; Jiaxi Wang; Carmel E. Mothersill; Thomas G. Hinton; Kouichi Aizawa; Colin B. Seymour (pp. 290-298).

The directly irradiated and bystander gill proteome was examined in wild-type and radiosensitive transgenic medaka. Direct irradiation increased the expression of annexin max 3, creatine kinase (CK), and lactate dehydrogenase (LDH) in both strains and reduced annexin A4 in wild-type medaka only. In bystander fish, same strain pairings increased CK and LDH in both strains and increased annexin max 3 and annexin A4 in radiosensitive medaka. Mixed strain pairings revealed that, in bystander fish, annexin max 3 was only increased by a bystander signal originating from a radiosensitive source, annexin A4 was increased in radiosensitive bystanders irrespective of the signal source, and CK and LDH were increased if either the bystander signal origin or the recipient bystander fish was radiosensitive. Warm-temperature acclimation related 65-kDa protein (Wap65) was increased in all bystander medaka, whether they were paired with the same or opposite strain and chromosome 5 SR-like CTD-associated factor (SR=serine–argenine-rich, CTD=C-terminal domain) (SCAF) protein was increased in radiosensitive bystander medaka only. Annexin A4, CK and LDH are associated with apoptosis and mirror the increase in apoptotic bodies previously reported in irradiated and bystander medaka, whereas increased Wap65 and LDH suggest a protective response. Thus the proteomic changes reported here could indicate both immediate protection and longer term adaptation to subsequent radiation exposure. In addition this investigation provides further evidence to show that the bystander signal can override the intrinsic genetically determined response and also that signal production and response can be modulated independently.► Radiation causes apoptosis-related proteomic changes in the medaka gill. ► The bystander effect also causes a protective response. ► These responses differ depending on transgenic sensitivity to radiation. ► The bystander signal can override the genetically determined response.

Keywords: Abbreviations; 2DE; 2-dimensional electrophoresis; ACN; acetonitrile; ANOVA; analysis of variance; CK; creatine kinase; IPG; immobilised pH gradient; LDH; lactate dehydrogenase; MS; mass spectrometry; MSDB; MS protein sequence database; nanoES; nano-electrospray; MS/MS; mass spectometry/mass spectrometry; NCBI; National Centre for Biotechnology and Information; PDH; pyruvate dehydrogenase; Q-TOF; quadrupole time of flight; rhoGDI; rho GDP dissociation inhibitor (GDP; =; guanosine diphosphate); ROS; reactive oxygen species; SCAF; SR-like CTD-associated factor (SR; =; serine–argenine-rich, CTD; =; C-terminal domain); SDS; sodium dodecyl sulphate; Wap65; Warm-temperature acclimation related 65-kDa proteinAnnexin; Apoptosis; Bystander effect; Warm-temperature acclimation related 65-kDa protein

Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis by Shun-ichi Tanaka; Yuichi Koga; Kazufumi Takano; Shigenori Kanaya (pp. 299-307).

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants ( k_ass) of 5.2×10³ M−¹ s−¹ at 40°C and 3.1×10⁵ M−¹ s−¹ at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k_ass values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3–7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.Tk‐serpin from the hyperthermophilic archaeon Thermococcus kodakaraensis was characterized. Tk‐serpin irreversibly inhibits Tk-subtilisin more strongly at 80°C than at 40°C. The inhibitory activity of Tk‐serpin increases as the temperature increases up to 100°C.Display Omitted► Tk-serpin from the hyperthermophilic archaeon Thermococcus kodakaraensis was characterized. ► Tk-serpin irreversibly inhibits Tk-subtilisin more strongly at 80°C than at 40°C. ► The inhibitory activity of Tk-serpin increases as the temperature increases up 100°C.

Keywords: Abbreviations; Serpin; serine proteinase inhibitor; Tk-serpin; a serpin homologue from; Thermococcus kodakaraensis; TKS; Tk-subtilisin from; T. kodakaraensis; CHT; chymotrypsin; SUC; subtilisin Carlsberg; PRK; proteinase K; RCL; reactive center loop; k; _ass; second-order association rate constant; SI; stoichiometry of inhibition; Suc-AAPF-; p; NA; succinyl-Ala-Ala-Pro-Phe-; p; -nitroanilide; EDTA; ethylenediaminetetraacetic acid; DTT; dithiothreitol; PMSF; phenyl methyl sulfonyl fluorideSerpin; Proteinase inhibitor; Thermococcus kodakaraensis; Hyperthermophilic archaeon; Subtilisin; Association rate constant

Posttranslational arginine methylation of lamin A/C during myoblast fusion by Su-Jin Kim; Byong Chul Yoo; Chang-Sub Uhm; Sang-Won Lee (pp. 308-317).

Protein arginine methylation is a major posttranslational modification that regulates various cellular functions, such as RNA processing and DNA repair. A recent report showed the involvement of protein arginine methyltransferase (PRMT) 4 in chromatin remodeling and gene expression during muscle differentiation in C2C12 cells. Because the fusion of myoblasts is a unique phenomenon observed in skeletal muscle differentiation, the present study focused on the expression and activities of PRMTs during myoblast fusion in primary rat skeletal muscle. N^G, N^G-asymmetric dimethylarginines (aDMA) and N^G, N′^G-symmetric dimethylarginines (sDMA) were both found consistently throughout myoblast fusion. However, PRMT1 exhibited the highest activity during myoblast fusion and maintained the elevated activity thereafter, whereas PRMT5 reached its highest activity only after myoblast fusion. To identify the proteins modified by such PRMTs, we conducted 2-dimensional electrophoresis (2-DE) of total proteins before and after myoblast fusion, and protein spots on the 2-DE gel immunoreactive for aDMA and sDMA were identified by mass spectrometric analysis. Among the proteins identified, lamin C2 was in particular observed to be dimethylated. Arginine methylation of lamin may therefore be important for muscle development and maintenance.Display Omitted► Expression of PRMTs during skeletal muscle differentiation. ► Change of PRMT catalytic activities during myoblast fusion. ► Alteration of proteins asymmetrically arginine-dimethylated during skeletal muscle differentiation. Identification of possible substrate proteins of PRMTs after myoblast fusion. ► Methylated lamin C2 peptide sequence.

Keywords: Abbreviations; Asym; asymmetrically dimethylated arginine; aDMA; N; ^G; , N; ^G; -asymmetric dimethylarginines; sDMA; N; ^G; , N′; ^G; -symmetric dimethylarginines; EGTA; ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; GAR; glycine arginine-rich; MMA; N; ^G; -monomethyl-L-arginine; PRMT; protein arginine methyltransferase; Sym; symmetrically dimethylated arginineSkeletal muscle differentiation; Lamin A/C; Mass spectrometry; Protein arginine methylation

FTIR 2D correlation spectroscopy of α₁ and α₂ fractions of an alkali-pretreated gelatin by Pieter Chys; Constant Gielens; Filip Meersman (pp. 318-325).

An alkali-pretreated gelatin (pI~4.9) was fractionated by means of alcohol coacervation and semi-preparative gel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatin were investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600–1700cm−¹). The gelation temperature was the same for all examined samples (24.5°C) while the melting temperature of the α₂ fraction was lower (30°C) than that of the other samples (32.5°C). The 2D COS plots indicate that on cooling (gelation) the core sequence of conformational changes is the same for all samples. On heating, however, the α₂ fraction deviates from the α₁-containing samples and shows an earlier disappearance of the triple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of the α₂ fraction thus results from a different conformational cascade of the α₂ chains upon melting. In all samples the initial conformational changes take place in the β-turns, providing further evidence for the models proposed previously.►An alkali-pretreated gelatin was fractionated. ►The melting temperature of the α₂ fraction was lower than for the other samples. ►The melting temperature of the α₂ fraction was lower than for the other samples. ►On heating the α₂ fraction deviates from the α₁-containing samples.►In all samples the initial conformational changes take place in the β-turns.

Keywords: Gelatin; α; fraction; FTIR spectroscopy; Alcohol coacervation; Fractionation; Sol–gel transition; 2D COS

Heme-binding characteristics of the isolated PAS-B domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms by Koya Hayasaka; Kenichi Kitanishi; Jotaro Igarashi; Toru Shimizu (pp. 326-333).

Mouse period homolog 2 (mPer2), an important transcriptional regulatory factor associated with circadian rhythms, is composed of two N-terminal PAS (PAS-A and PAS-B) domains and a C-terminal domain. The PAS-A domain of mPer2 binds the heme iron via a Cys axial ligand. A corresponding transcriptional regulatory factor, neuronal PAS 2 protein (NPAS2), also contains PAS-A and PAS-B domains at the N-terminus with heme-binding capability. In particular, the PAS-B domain appears important for protein–protein interactions critical for transcriptional regulation. In the present study, we examined the heme-binding characteristics of the isolated PAS-B domain of mPer2. Our experiments show that the Fe(III) heme binds the isolated PAS-B domain with a heme to protein stoichiometry of 1:1. The Fe(III) protein complex is suggested to consist of an admixture of 6-coordinated His-bound high-spin and low-spin complexes. Marked pH-dependent spectral changes were observed, in contrast to the spectrum of the Fe(III) bound PAS-A domain of mPer2, which appeared pH-resistant. Treatment with diethylpyrocarbonate abolished the heme-binding ability of this protein, supporting the proposal that His is the axial ligand. Heme dissociation was composed of two phases with rate constants of 4.3×10−⁴s−¹ (50%) and 4.0×10−³s−¹ (50%), which were markedly higher than that (1.5×10−⁷s−¹) of the prototype heme protein, myoglobin. The Soret CD band of the H454A PAS-B mutant was significantly different from those of wild-type and other His mutant proteins, strongly suggesting that His454 is one of the axial ligands for the Fe(III) complex.Display Omitted►The Fe(III) heme binds the isolated PAS-B domain of mPer2. ►The Fe(III) complex consists of an admixture of 6cHS and 6cLS complexes with His as the axial ligand. ►Results of chemical modification suggest that His is the axial ligand. ►From the Soret CD band, it is suggested that His454 is one of the axial ligands for the Fe(III) complex. ►The coordination structure of PAS-B is different from PAS-A.

Keywords: Abbreviations; PAS; acronyms of Per (; Drosophila; period clock protein)–Arnt (vertebrate aryl hydrocarbon receptor nuclear translocator)–Sim (; Drosophila; single-minded protein); PAS-B-mPer2; isolated PAS-B domain of mPer2; PAS-A-mPer2; isolated PAS-A domain of mPer2; BMAL1; mouse brain and muscle Arnt-like 1; NPAS2; neuronal PAS protein 2; PAS-A-NPAS2; isolated PAS-A domain of NPAS2; PAS-B-NPAS2; isolated PAS-B domain of NPAS2; Fe(III) heme; Fe(III) protoporphyrin IX complex or hemin; Fe(II) heme; Fe(II) protoporphyrin IX complex; DEPC; diethylpyrocarbonate; NEM; N; -ethylmaleimide; LS; low spin; HS; high spinHeme-binding protein; PAS domain; Circadian rhythm; Transcriptional regulation

Characterization of low-energy excited states in the native state ensemble of non-myristoylated and myristoylated neuronal calcium sensor-1 by Kousik Chandra; Yogendra Sharma; K.V.R. Chary (pp. 334-344).

Information on the low-energy excited states of a given protein is important as this controls the structural adaptability and various biological functions of proteins such as co-operativity, response towards various external perturbations. In this article, we characterized individual residues in both non-myristoylated (non-myr) and myristoylated (myr) neuronal calcium sensor-1 (NCS-1) that access alternate states by measuring nonlinear temperature dependence of the backbone amide-proton (¹H^N) chemical shifts. We found that ~20% of the residues in the protein access alternative conformations in non-myr case, which increases to ~28% for myr NCS-1. These residues are spread over the entire polypeptide stretch and include the edges of α-helices and β-strands, flexible loop regions, and the Ca²⁺-binding loops. Besides, residues responsible for the absence of Ca²⁺–myristoyl switch are also found accessing alternative states. The C-terminal domain is more populated with these residues compared to its N-terminal counterpart. Individual EF-hands in NCS-1 show significantly different number of alternate states. This observation prompts us to conclude that this may lead to differences in their individual conformational flexibility and has implications on the functionality. Theoretical simulations reveal that these low-energy excited states are within an energy band of 2–4kcal/mol with respect to the native state.► Twenty percent residues of non-myristoylated neuronal calcium sensor-1 access alternate states. ► Myristoylation increases population of residues accessing alternate states in NCS-1. ► The conformation fluctuations are non-uniformly spread over the entire sequence. ► C-terminal domain has more conformational fluctuations that N-terminal counterpart. ► Alternate states are within an energy band of 2–4kcal/mol with native state.

Keywords: Abbreviations; NCS-1; neuronal calcium sensor-1; HSQC; heteronuclear single quantum correlation; CaM; Calmodulin; CaBP; Ca; ²⁺; -binding protein; EF2; EF3 and EF4 represent the second, third, and fourth EF-hand motifs of NCS-1; non-myr NCS-1; non-myristoylated form of neuronal calcium sensor-1Nuclear magnetic resonance; Native state; Low-energy excited states; Alternate states; Calcium sensor proteins

Surface plasmon resonance study on functional significance of clustered organization of lectin-like oxidized LDL receptor (LOX-1) by Izuru Ohki; Hirokazu Amida; Risato Yamada; Mamoru Sugihara; Tomoko Ishigaki; Shin-ichi Tate (pp. 345-354).

Lectin-like oxidized low-density lipoprotein (OxLDL) receptor 1 (LOX-1) is the major OxLDL receptor of vascular endothelial cells and is involved in an early step of atherogenesis. LOX-1 exists as a disulfide-linked homodimer on the cell surface, which contains a pair of the ligand-binding domains (CTLD; C-type lectin-like domain). Recent research using living cells has suggested that the clustered state of LOX-1 dimer on the cell is functionally required. These results questioned how LOX-1 exists on the cell to achieve OxLDL binding. In this study, we revealed the functional significance of the clustered organization of the ligand-binding domain of LOX-1 with surface plasmon resonance. Biotinylated CTLD was immobilized on a streptavidin sensor chip to make CTLD clusters on the surface. In this state, the CTLD had high affinity for OxLDL with a dissociation constant ( K_D) in the nanomolar range. This value is comparable to the K_D measured for LOX-1 on the cell. In contrast, a single homodimeric LOX-1 extracellular domain had lower affinity for OxLDL in the supra-micromolar range of K_D. Monomeric CTLD showed marginal binding to OxLDL. In combination with the analyses on the loss-of-binding mutant W150A, we concluded that the clustered organization of the properly formed homodimeric CTLD is essential for the strong binding of LOX-1 to OxLDL.Display Omitted► Clustered CTLD showed affinity to OxLDL as found for LOX-1 on plasma membrane. ► LOX-1 binds to OxLDL in a multivalent manner. ► Properly formed CTLD dimer structure is required in the LOX-1 binding to OxLDL.

Keywords: Abbreviations; LOX-1; lectin-like oxidized low-density lipoprotein receptor 1; CTLD; C-type lectin-like domain; HAEC; human aortic endothelial cell; ECD; extracellular domain; SPR; surface plasmon resonance; RU; resonance unit; OxLDL; oxidized low-density lipoprotein; AcLDL; acetylated low-density lipoprotein; βME; β-mercaptoethanol or 2-mercaptoethanol; NMR; nuclear magnetic resonance; CD; circular dichroism; HSQC; heteronuclear single quantum coherence spectroscopy; apoB; apolipoprotein B; apoE; apolipoprotein E; HSQC; heteronuclear single-quantum coherence spectroscopy; DC-SIGN; dendritic cell-specific ICAM-3 grabbing non-integrinLOX-1; Oxidized LDL; Surface plasmon resonance; Atherosclerosis

The M2-type isoenzyme of pyruvate kinase phosphorylates prothymosin α in proliferating lymphocytes by Diaz-Jullien Cristina Díaz-Jullien; David Moreira; Concepción Sofía Sarandeses; Guillermo Covelo; Pablo Barbeito; Manuel Freire (pp. 355-365).

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.►M2 isoform of pyruvate kinase phosphorylates prothymosin α. ►Phosphorylation of pyruvate kinase is responsible for its bifunctionality. ► Only 5–10% of M2 pyruvate kinase shows prothymosin α kinase activity.

Keywords: Abbreviations; ProTα; prothymosin α; Tα; ₁; thymosin α; ₁; CK-2; casein kinase-2; PMSF; phenylmethylsulfonyl fluoride; T3; 3′-5′-triiodo-; l; -thyronine; FBP; fructose 1,6-bisphosphate; PEP; phosphoenolpyruvate; LDH; lactate dehydrogenase; PK; pyruvate kinase; ConA; concanavalin A; IL-2; interleukin-2; AS; ammonium sulfateProthymosin; M2 pyruvate kinase; Phosphorylation; Proliferation

A screen for potential ferredoxin electron transfer partners uncovers new, redox dependent interactions by G.T. Hanke; Y. Satomi; K. Shinmura; T. Takao; T. Hase (pp. 366-374).

Ferredoxin (Fd) is the primary soluble acceptor at the end of the photosynthetic electron transport chain, and is known to directly transfer electrons to a wide range of proteins for use in metabolism and regulatory processes. We have conducted a screen to identify new putative Fd interaction partners in the cyanobacteria Synechocystis sp. PCC 6803 using Fd-chromatography in combination with MALDI-TOF mass spectrometry. Many novel interactions were detected, including several redox enzymes, which are now candidates for further experiments to investigate electron transfer with Fd. In addition, some proteins with regulatory activity related to photosynthesis were identified. We cloned and expressed one such protein, known as RpaA, which is a specific regulator of energy transfer between phycobilisomes and PSI. Using the recombinant protein we confirmed direct interaction with Fd, and discovered that this was dependent on redox state. The screen for putative Fd-binding proteins was repeated, comparing oxidizing and reducing conditions, identifying many proteins whose interaction with Fd is redox dependent. These include several additional signaling molecules, among them the LexA repressor, Ycf53 and NII, which are all involved in interpreting the redox state of the cell.►A screen for ferredoxin binding proteins identifies new putative electron transfer partners. ►RpaA binds to Fd in a redox dependent manner. ►Binding to ferredoxin of sulfite reductase, but not nitrite reductase, depends on redox state. ►Ferredoxin differentially binds many other novel proteins in oxidizing and reducing conditions.

Keywords: Abbreviations; Fd; ferredoxin; PSI; photosystem 1; FNR; ferredoxin:NADP; ⁺; reductase; MALDI; matrix-assisted laser-desorption; MALDI-TOF; MADLI-time of flight; FTR; Fd:thioredoxin reductase; SiR; sulfite reductase; NiR; nitrite reductase; FMN; flavin; Fd-GOGAT; Fd-dependent glutamine oxoglutarate aminotransferaseFerredoxin; Photosynthesis; Electron transfer; Synechocystis; Redox regulation; RpaA

Conformational and thermal stability of mature dimeric human myeloperoxidase and a recombinant monomeric form from CHO cells by Srijib Banerjee; Johanna Stampler; Furtmuller Paul G. Furtmüller; Christian Obinger (pp. 375-387).

Myeloperoxidase (MPO) is a lysosomal heme enzyme present in the azurophilic granules of human neutrophils and monocytes. It is a critical element of the human innate immune system by exerting antimicrobial effects. It is a disulfide bridged dimer with each monomer containing a light and a heavy polypeptide and its biosynthesis and intracellular transport includes several posttranslational processing steps. By contrast, MPO recombinantly produced in Chinese hamster ovary cell lines is monomeric, partially unprocessed and contains a N-terminal propeptide (proMPO). It mirrors a second form of MPO constitutively secreted from normal bone marrow myeloid precursors. In order to clarify the impact of posttranslational modifications on the structural integrity and enzymology of these two forms of human myeloperoxidase, we have undertaken an investigation on the conformational and thermal stability of leukocyte MPO and recombinant proMPO by using complementary biophysical techniques including UV-Vis, circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Mature leucocyte MPO exhibits a peculiar high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of dithiothreitol. Unfolding of secondary and tertiary structure occurs concomitantly with denaturation of the heme cavity, reflecting the role of three MPO-typical heme to protein linkages and of six intra-chain disulfides for structural integrity by bridging N- and C-terminal regions of the protein. Recombinant monomeric proMPO follows a similar unfolding pattern but has a lower conformational and thermal stability. Spectroscopic and thermodynamic data of unfolding are discussed with respect to the known three-dimensional structure of leukocyte MPO as well as to known physiological roles.►Mature leucocyte MPO exhibits peculiar high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of dithiothreitol. ►Protein unfolding occurs concomitantly with denaturation of the heme cavity, reflecting the role of heme to protein linkages and intra-chain disulfides in bridging N- and C-terminal regions. ►Recombinant monomeric proMPO follows a similar unfolding pattern but has a lower conformational and thermal stability.

Keywords: Abbreviations; MPO; mature dimeric leukocyte myeloperoxidase; proMPO; partially unprocessed monomeric MPO that contains the N-terminal propeptide; CHO; Chinese hamster ovary; GdnHCl; guanidinium hydrochloride; DTT; dl; -dithiothreithol; ATR-FTIR; attenuated total reflection Fourier transform infrared spectroscopy; ECD; electronic circular dichroism spectroscopy; [; θ; ]; molecular ellipticity; λ; _max; emission wavelength maxima; T; temperature; T; _m; midpoint of the thermal unfolding curve; Δ; G; °; _H2O; conformational stability at 25°; Δ; G; °; change in standard free enthalpy; m; efficacy of denaturant in unfolding; C; _m; midpoint of denaturation curve; Δ; H; change in enthalpy; Δ; S; change in entropy; Δ; H; _m; change in enthalpy at; T; _m; Δ; S; _m; change in entropy at; T; _m; Δ; C; _p; change in heat capacityMyeloperoxidase; proMPO; Innate immune system; Proteolytic processing; Thermal unfolding; Conformational stability

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics by Edward Chang; Sohee Kim; Kelsey N. Schafer; Jeff Kuret (pp. 388-395).

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.► Pseudophosphorylation of tau protein at residue Thr212 promotes tau aggregation. ► Aggregation rate rises owing to increased filament nucleation. ► Extent of aggregation increases owing to a decreased rate of filament disaggregation. ► Aberrant tau phosphorylation may directly promote neurofibrillary lesion formation.

Keywords: Abbreviations; ODS; Sodium octadecyl sulfateAggregation; Alzheimer's disease; Tau protein; Phosphorylation; Kinetics

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BBA - Proteins and Proteomics (v.1814, #2)

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BBA - Proteins and Proteomics (v.1814, #2)