Alzheimer's-disease-associated conformation of intrinsically disordered tau protein studied by intrinsically disordered protein liquid-phase competitive enzyme-linked immunosorbent assay

Tau protein, the major constituent of paired helical filaments in Alzheimer's disease, belongs to the intrinsically disordered proteins (IDPs). IDPs are an emerging group in the protein kingdom characterized by the absence of a rigid three-dimensional structure. Disordered proteins usually acquire a "functional fold" upon binding to their interaction partner(s). This property of IDPs implies the need for innovative approaches to measure their binding affinity. We have mapped and measured the Alzheimer's-disease-associated epitope on intrinsically disordered tau protein with a novel two-step sandwich competitive enzyme-linked immunosorbent assay (ELISA). This approach allowed us to determine the binding affinity of disordered tau protein in liquid phase without any disturbance to the competitive equilibrium and without any need for covalent or noncovalent modification of tau protein. Furthermore, the global fitting method, used for the reconstruction of tau binding curves, significantly improved the assay readout. The proposed novel competitive ELISA allowed us to determine the changes in the standard Gibbs energy of binding, thus enabling measurement of tau protein conformation in the core of paired helical filaments. IDP competitive ELISA results showed, for the first time, that the tau protein C terminus of the Alzheimer's-disease-derived paired helical filaments core subunit adopts beta-turn type I' fold and is accessible from solution.     

C-terminal motif within Sec7 domain regulates guanine nucleotide exchange activity via tuning protein conformation

ADP-ribosylation factors (Arfs) play key roles in controlling membrane traffic and organelle structures. The activation of Arfs from GDP to GTP binding form is triggered by the guanine exchange factors (GEFs). There are six families of Arf-GEFs with a common guanine exchange catalytic domain (Sec7 domain) and various mechanisms of guanine exchange activity regulation. A loop region (loop>J motif) just following the helix J of Sec7 domain was found conserved and important for the catalytic activity regulation of Arf-GEFs. However, the molecular detail of the role the loop>J motif plays has been yet unclear. Here, we studied the catalytic domain of Sec7p, a yeast trans-Golgi network membrane localized Arf-GEFs, and found that the loop>J motif is indispensible for its GEF catalytic activity. Crystallographic, NMR spectrum and mutagenesis studies suggested that the loop>J motif with a key conserved residue Ile1010 modulates the fine conformation of Sec7 domain and thereby regulates its guanine exchange activity.     

Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation.

The histidine rich protein II (HRPII) from Plasmodium falciparum has been implicated as a heme polymerase which detoxifies free heme by its polymerization to inactive hemozoin. Histidine-iron center coordination is the dominant mechanism of interaction between the amino acid and heme. The protein also contains aspartate allowing for ionic/coordination interactions between the carboxylate side chain and the heme metal center. The pH profile of heme binding and polymerization shows the possibility of these two types of binding sites being differentiated by pH. Circular dichroism studies of the protein show that pH and heme binding cause a change in conformation above pH 6 implying the involvement of His-His+ transitions. Heme binding at pHs above 6 perturbs HRPII conformation, causing an increase in helicity.     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

Luteolin exhibits anti-inflammatory effects by blocking the activity of heat shock protein 90 in macrophages

Septic diseases represent the prevalent complications in intensive care units. Luteolin, a plant flavonoid, has potent anti-inflammatory properties; however, the molecular mechanism beneath luteolin mediated immune modulation remains unclear. Here in vitro investigations showed that luteolin dose-dependently inhibited LPS-triggered secretion and relocation of high mobility group B-1 (HMGB1) and LPS-induced production of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) in macrophages. The mechanism analysis demonstrated that luteolin reduced the release of HMGB1 through destabilizing c-Jun and suppressed HMGB1-induced aggravation of inflammatory cascade through reducing Akt protein level. As an inhibitor of Hsp90, luteolin destabilized Hsp90 client protein c-Jun and Akt. In vivo investigations showed that luteolin effectively protected mice from lipopolysaccharide (LPS)-induced lethality. In conclusion, the present study suggested that luteolin may act as a potential therapeutic reagent for treating septic diseases.     

Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.     

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-platelet activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.     

Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit

Rad53 is a conserved protein kinase with a central role in DNA damage response and nucleotide metabolism. We observed that the expression of a dominant-lethal form of RAD53 leads to significant expression changes for at least 16 genes, including the RNR3 and the HUG1 genes, both of which are involved in the control of nucleotide metabolism. We established by multiple biophysical and biochemical approaches that Hug1 is an intrinsically disordered protein that directly binds to the small RNR subunit Rnr2. We characterized the surface of interaction involved in Hug1 binding to Rnr2, and we thus defined a new binding region to Rnr2. Moreover, we show that Hug1 is deleterious to cell growth in the context of reduced RNR activity. This inhibitory effect of Hug1 on RNR activity depends on the binding of Hug1 to Rnr2. We propose a model in which Hug1 modulates Rnr2–Rnr1 association by binding Rnr2. We show that Hug1 accumulates under various physiological conditions of high RNR induction. Hence, both the regulation and the mode of action of Hug1 are different from those of the small protein inhibitors Dif1 and Sml1, and Hug1 can be considered as a regulator for fine-tuning of RNR activity.     

The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity

We previously reported that rice blast fungus or jasmonic acid induced the expression of rice pathogenesis-related class 10 (JIOsPR10) proteins (Kim et al. 2003, 2004). However, no further studies have been carried out to examine the expression, localization, and enzymatic activity of this protein in either developmental tissues or in tissues under abiotic stress conditions. In this study, rice JIOsPR10 was examined by Western blot analysis, immunolocalization, and biochemical assays. Western blots revealed that the JIOsPR10 protein was expressed in developmental tissues, including in flower and root. The protein was also expressed under abiotic stresses, such as occurs during senescence and wounding. Using immunohistochemical techniques, we determined that expression of JIOsPR10 was localized to the palea of flower, in the exodermis, and inner part of the endodermis of the root. In senescencing tissues of leaf and coleoptiles, its expression was localized in vascular bundles. The RNase activity using JIOsPR10 recombinant protein was determined and abolished after treatment with DTT in a native in-gel assay. To test this, we created JIOsPR10 mutant proteins containing serine substitutions of amino acids C81S, C83S, or both and examined their RNase activities. The activity of the C83S mutant was decreased in the agarose gel assay compared to the wild type. Taken together, we hypothesize that the JIOsPR10 protein possesses RNase activity that is sensitive to DTT, suggesting the importance of the disulfide bonding between cysteine residues and that it might play a role in constitutive self-defense mechanisms in plants against biotic and abiotic stresses.     

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.     

Calcium-sensitive activity and conformation of Caenorhabditis elegans gelsolin-like protein 1 are altered by mutations in the first gelsolin-like domain

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.     

The photosynthetic water oxidase: its proton pumping activity is short-circuited within the protein by DCCD

The photosynthetic water oxidase is composed of ˜15 polypeptides which are grouped around two functional parts: photosystem II and the catalytic manganese centre. Photochemically driven vectorial electron transfer between the manganese centre and bound plastoquinone causes deprotonation–protonation reactions at opposite sides of the thylakoid membrane. Thereby the water oxidase acts as a proton pump. Incubation of stacked thylakoids with N,N'-dicyclohexylcarbodiimide (DCCD) short-circuited its proton pumping activity. Under flashing light, the extent of both proton release into the lumen by water oxidation and of proton uptake from the medium by reduced quinone was diminished. Instead there was a rapid electrogenic backreaction with a strong H/D-isotope effect. Apparently protons which were produced by water oxidation were channelled across the transmembrane protein to the bound quinone. A more rapid protonation of the reduced quinone was evident from a shortening of the time lag for the reduction of photosystem I. These effects were paralleled by the preferential labelling with [¹⁴C]DCCD in stacked thylakoids of two polypeptides with 20 and 24 kd apparent molecular mass. These may be capping the oxidizing and the reducing terminus of the water oxidase to control proton extrusion and proton uptake respectively.     

Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 <Emphasis Type="Italic">in vitro</Emphasis>

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.     

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Mitotic activation of protein-tyrosine phosphatase alpha and regulation of its Src-mediated transforming activity by its sites of protein kinase C phosphorylation

During mitosis, the catalytic activity of protein-tyrosine phosphatase (PTP) alpha is enhanced, and its inhibitory binding to Grb2, which specifically blocks Src dephosphorylation, is decreased. These effects act synergistically to activate Src in mitosis. We show here that these effects are abrogated by mutation of Ser180 and/or Ser204, the sites of protein kinase C-mediated phosphorylation within PTPalpha. Moreover, either a Ser-to-Ala substitution or serine dephosphorylation specifically eliminated the ability of PTPalpha to dephosphorylate and activate Src even during interphase. This explains why the substitutions eliminated PTPalpha transforming activity, even though PTPalpha interphase dephosphorylation of nonspecific substrates was only slightly decreased. This occurred without change in the phosphorylation of PTPalpha at Tyr789, which is required for "phosphotyrosine displacement" during Src dephosphorylation. Thus, in addition to increasing PTPalpha nonspecific catalytic activity, Ser180 and Ser204 phosphorylation (along with Tyr789 phosphorylation) regulates PTPalpha substrate specificity. This involves serine phosphorylation-dependent differential modulation of the affinity of Tyr(P)789 for the Src and Grb2 SH2 domains. The results suggest that protein kinase C may participate in the mitotic activation of PTPalpha and Src and that there are intramolecular interactions between the PTPalpha C-terminal and membrane-proximal regions that are regulated, at least in part, by serine phosphorylation.     

The changes in conformation of (Na+ K+)-ATPase from rat brain membranes are accompanied by changes of protein segment movements in the nanosecond range

Differential polarized phase fluorometry of fluorescein-5-isothiocyanate (FITC) showed that the activation of (Na,K)-ATPase in crude plasma membranes from rat brain by 10 mmol.l-1 K+ and 100 mmol.l-1 Na+ significantly increased the rotational relaxational rate (R) of enzyme-bound FITC. This increase was blocked by both ouabain (0.1 mmol.l-1) and vanadate (0.1 mmol.l-1). In the absence of ATP, R was increased less after adding of 10 mmol.l-1 K+ to the membranes. The shifts in the nanosecond movements of the protein segments measured as R during the activation of (Na,K)-ATPase suggest that this type of movement might be of some functional importance.