Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners

In mammals, protein kinase CK2 has two isozymic forms of its catalytic subunit, designated CK2gr; and CK2. CK2 and CK2 exhibit extensive similarity within their catalytic domains but have completely unrelated C-terminal sequences. To systematically examine the cellular functions of each CK2 isoform in mammalian cells, we have generated human osteosarcoma U2-OS cell lines with the expression of active or inactive versions of each CK2 isoform under the control of an inducible promoter [22]. Examination of these cell lines provides evidence for functional specialization of CK2 isoforms at the cellular level in mammals with indications that CK2 is involved in the control of proliferation and/or cell survival. To understand the molecular basis for functional differences between CK2 and CK2, we have undertaken studies to identify proteins that interact specifically with each isoform of CK2 and could contribute to the regulation of their independent functions. A novel pleckstrin-homology domain containing protein, designated CK2-interacting protein 1 (i.e. CKIP-1) was isolated using the yeast two hybrid system as a protein that interacts with CK2 but not CK2 [23]. When expressed in cells as a fusion with green fluorescent protein, CKIP-1 localizes to the cell membrane and to the nucleus. In this study, we present evidence from deletion analysis of CKIP-1 suggesting that a C-terminal region containing a putative leucine zipper has a role in regulating its nuclear localization. Collectively, our data supports a model whereby CKIP-1 is a non-enzymatic regulator of CK2 that regulates the cellular functions of CK2 by targeting or anchoring CK2 to specific cellular localization or by functioning as an adapter to integrate CK2-mediated signaling events with components of other signal transduction pathways.     

Dynamic transition of α-helix to β-sheet structure in linear surfactin correlating to critical micelle concentration

Summary Linear heptapeptide surfactin was prepared by alkaline cleavage of the lactone ring of cyclic surfactin. The structure of linear surfactin was characterised and confirmed by FAB-mass-spectroscopy, FT-IR and HPLC analysis. It was found that linear surfactin easily forms micelles in aqueous solutions by coordinating -sheet formation from -helical monomolecules, and the cmc value found to be 1.28×10^–5 M. The CD spectra indicates conformational change of linear surfactin from -helical below cmc to -sheet above cmc.     

Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil

The prothymosin a kinase (ProTK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin (ProT), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTK is more effectively activated by Mn²⁺ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin ₁ and thymosin ₁₁, derived from the amino terminus of ProT, despite the fact that the sites of phosphorylation of ProT are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProT and ProTK activity. ProTK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTK, which is therefore presumably phosphorylated by another kinase.     

Antioxidant properties of alpha-crystallin

-Crystallin is a major chaperone lens protein to which has been ascribed antioxidant functions. In the present work we have evaluated the antioxidant and free radical scavenging properties of bovine -crystallin in a series of in vitro models: zimosan-induced, luminol-enhanced chemiluminescence response of polymorphonuclear leukocytes, the autoxidation of brain homogenate, bleaching of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-derived radical cations, trapping of peroxyl radicals, and reactivity toward hypochloric acid. In all these systems, the reactivity of -crystallin is higher than or similar to that of bovine serum albumin. It is concluded that, given the high concentrations of -crystallin in the lenses, its capacity to interact with free radicals and to remove hypochlorous acid could contribute to the maintenance of the lens functionality.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or -chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of -globulin hydrolyzed by trypsin or -chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed -globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with -globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of -globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary 2D ¹H NMR spectroscopy of two -helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from –10 to –6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the -helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in -helices in H₂O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in -helices. Supplementary Material is available upon request, comprising seven pages with listings of experimental details and the NMR shift data for the two peptides.     

Limited Proteolysis of Bacillus thuringiensis CryIG and CryIVB δ-Endotoxins Leads to Formation of Active Fragments That Do Not Coincide with the Structural Domains

Bacillus thuringiensis true toxins consist of three domains: the N-terminal, -helical domain followed by two -structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of true toxins. The first pattern, observed for CryIA and CryIVD -endotoxins, results in the proteolysis of the loops connecting -strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth -helixes of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB -endotoxin fragments indicates that only two -helixes, the sixth and the seventh within the first domain, followed by the two -structural domains are sufficient for the insecticidal activity.     

Syntheses of 1-O-benzyl-α-glucoside and 1-O-benzyl-α-maltoside by transglycosylation of α- amylase from soluble starch in aqueous solution

Glycosides, 1-O-benzyl--glucoside (BG) and 1- O-benzyl--maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an -amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had -configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by -glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the -amylase used for its synthesis but it was not hydrolyzed above pH 8.0.     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

NMR structure of the ribosomal protein L23 from Thermus thermophilus

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel -strands and three -helices connected as ------, and a large and flexible loop inserted between the third and fourth -strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Chemoattractant activity of Staphylococcus aureus serine proteinase modified human plasma α-1-proteinase inhibitor

S. aureus serine proteinase inactivates human -1-proteinase inhibitor (-1-PI) by attacking a single peptide bond between Glu³⁵⁴ and Ala³⁵⁵ giving a modified inhibitor which is a tight complex of Mr=4,000 and 48,000 fragments. In the present paper we show that this proteolytically inactivated -1-PI is a potent chemotactic factor for human neutrophiles at a nanomolar concentration, and we discuss its potential involvement in the inflammatory reaction due to S. aureus infections.     

Cytochemical evidence for the presence of actin in the nucleus of the voodoo lily appendix

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against -smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti--smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC–phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear -smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against -tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti--tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50kDa. This is the first cytochemical evidence for the presence of -smooth muscle-actin-like protein in the plant nucleus.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

The stereospecific bioconversion of α-aminopropionitrile to L-alanine by an immobilised bacterium isolated from soil

Summary Bacterial isolate APN grew on -aminoacetonitrile and -amino propionitrile (-APN) converting the -aminonitriles to glycine and alanine, respectively. The nitrilase had an apparent Km for -APN of 34mM in whole cells and 21mM when immobilised. The alanine formed from APN was 87% in the L-form and was produced for 45 days after immobilisation in alginate.     

The effect of modification and fragmentation of α-lactalbumin on lactose and lactosamine synthase reactions

In the presence of the modifier protein -lactalbumin, bovine milk galactosyltransferase transfers galactose to glucose forming lactose instead of transferring toN-acetylglucosamine formingN-acetyllactosamine. At low concentrations of -lact-albumin, the lactosamine synthase activity is stimulated by -lactalbumin and decreases when the lactose synthase activity develops along a sigmoidal curve. The observation suggests that different interactions between -lactalbumin and enzyme were responsible for the modulating effect of the -lactalbumin in the lactose and lactosamine synthase reactions.To study the nature of the protein-protein interactions, -lactalbumin was both modified and cleaved chemically. Reduction and alkylation with iodoacetic acid, iodoacetamide or 4-vinylpyridine abolished the ability of the -lactalbumin to induce lactose synthase activity but stimulated lactosamine synthase activity 7-to 12-fold.A peptide fragment corresponding to residues 26–60 of -lactalbumin isolated from a 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene (BNPS-skatole) fragmentation of the molecule was active in the lactosamine but not lactose synthase reaction. We concluded that, whereas lactose synthase required -lactalbumin, in the native conformation, lactosamine synthase activity was stimulated by a linear sequence of amino acids in peptide 26–60.Abbreviations MES 4-N-morpholinoethanesulfonic acid - TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP-Gal uridinediphosphogalactose - BNPS-skatole 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene - EDTA ethylene diamine tetra acetic acid