Low-affinity Sites Research Articles

Mannosylated hemagglutinin peptides bind cyanovirin-N independent of disulfide-bonds in complementary binding sites.

Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various glycoproteins of enveloped viruses, such as influenza hemagglutinin (HA) and Ebola glycoprotein. Chemically synthesized dimannosylated HA peptides bound domain-swapped and dimeric CV-N with either four disulfide-bonds (Cys–Cys), or three Cys–Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant KD of 10 μM. Cys–Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by in silico structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites (KD ≈ 11 μM for one C58–C73 bond, and binding to dimannosylated peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E–C73R substitution at KD = 275 nM, and KD = 5 μM for two C58E–C73R substitutions. We were utilizing a triazole bioisostere linkage to form the respective mannosylated-derivative on the HA peptide sequence of residues glutamine, glycine, and glutamic acid. Thus, mono- and dimannosylated peptides with N-terminal cysteine facilitated site-specific interactions with HA peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.

Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI-mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

Autoregulation of RCO by Low-Affinity Binding Modulates Cytokinin Action and Shapes Leaf Diversity.

Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A.thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C.hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species.

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4−3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We developed SpiCee, a versatile and genetically-encoded chelator combining low and high affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically-controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Low affinity binding sites in an activating CRM mediate negative autoregulation of the Drosophila Hox gene Ultrabithorax.

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.

Convergence in LINE-1 nucleotide variations can benefit redundantly forming triplexes with lncRNA in mammalian X-chromosome inactivation

BackgroundAssociations between X-inactive transcript (Xist)–long noncoding RNA (lncRNA) and chromatin are critical intermolecular interactions in the X-chromosome inactivation (XCI) process. Despite high-resolution analyses of the Xist RNA-binding sites, specific interaction sequences are yet to be identified. Based on elusive features of the association between Xist RNA and chromatin and the possible existence of multiple low-affinity binding sites in Xist RNA, we defined short motifs (≥5 nucleotides), termed as redundant UC/TC (r-UC/TC) or AG (r-AG) motifs, which may help in the mediation of triplex formation between the lncRNAs and duplex DNA.ResultsThe study showed that r-UC motifs are densely dispersed throughout mouse and human Xist/XIST RNAs, whereas r-AG motifs are even more densely dispersed along opossum RNA-on-the-silent X (Rsx) RNA, and also along both full-length and truncated long interspersed nuclear elements (LINE-1s, L1s) of the three species. Predicted secondary structures of the lncRNAs showed that the length range of these sequence motifs available for forming triplexes was even shorter, mainly 5- to 9-nucleotides long. Quartz crystal microbalance (QCM) measurements and Monte Carlo (MC) simulations indicated that minimum-length motifs can reinforce the binding state by increasing the copy number of the motifs in the same RNA or DNA molecule. Further, r-AG motifs in L1s had a similar length-distribution pattern, regardless of the similarities in the length or sequence of L1s across the three species; this also applies to high-frequency mutations in r-AG motifs, which suggests convergence in L1 sequence variations.ConclusionsMultiple short motifs in both RNA and duplex DNA molecules could be brought together to form triplexes with either Hoogsteen or reverse Hoogsteen hydrogen bonding, by which their associations are cooperatively enhanced. This novel triplex interaction could be involved in associations between lncRNA and chromatin in XCI, particularly at the sites of L1s. Potential binding of Xist/XIST/Rsx RNAs specifically at L1s is most likely preserved through the r-AG motifs conserved in mammalian L1s through convergence in L1 nucleotide variations and by maintaining a particular r-UC/r-AG motif ratio in each of these lncRNAs, irrespective of their poorly conserved sequences.

Enhancing gene editing specificity by attenuating DNA cleavage kinetics.

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

In vivo PET Imaging of [11C]CIMBI-5, a 5-HT2AR Agonist Radiotracer in Nonhuman Primates.

5-HT2AR exists in high and low affinity states. Agonist PET tracers measure binding to the active high affinity site and thus provide a functionally relevant measure of the receptor. Limited in vivo data have been reported so far for a comparison of agonist versus antagonist tracers for 5-HT2AR used as a proof of principle for measurement of high and low affinity states of this receptor. We compared the in vivo binding of [11C]CIMBI-5, a 5-HT2AR agonist, and of the antagonist [11C]M100907, in monkeys and baboons. [11C]CIMBI-5 and [11C]M100907 baseline PET scans were performed in anesthetized male baboons (n=2) and male vervet monkeys (n=2) with an ECAT EXACT HR+ and GE 64-slice PET/CT Discovery VCT scanners. Blocking studies were performed in vervet monkeys by pretreatment with MDL100907 (0.5 mg/kg, i.v.) 60 minutes prior to the scan. Regional distribution volumes and binding potentials were calculated for each ROI using the likelihood estimation in graphical analysis and Logan plot, with either plasma input function or reference region as input, and simplified reference tissue model approaches. PET imaging of [11C]CIMBI-5 in baboons and monkeys showed the highest binding in 5-HT2AR-rich cortical regions, while the lowest binding was observed in cerebellum, consistent with the expected distribution of 5-HT2AR. Very low free fractions and rapid metabolism were observed for [11C]CIMBI-5 in baboon plasma. Binding potential values for [11C]CIMBI-5 were 25-33% lower than those for [11C]MDL100907 in the considered brain regions. The lower binding potential of [11C]CIMBI-5 in comparison to [11C]MDL100907 is likely due to the preferential binding of the former to the high affinity site in vivo in contrast to the antagonist, [11C]MDL100907, which binds to both high and low affinity sites.

Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.

Low-Affinity Binding Sites and the Transcription Factor Specificity Paradox in Eukaryotes.

Eukaryotic transcription factors (TFs) from the same structural family tend to bind similar DNA sequences, despite the ability of these TFs to execute distinct functions in vivo. The cell partly resolves this specificity paradox through combinatorial strategies and the use of low-affinity binding sites, which are better able to distinguish between similar TFs. However, because these sites have low affinity, it is challenging to understand how TFs recognize them in vivo. Here, we summarize recent findings and technological advancements that allow for the quantification and mechanistic interpretation of TF recognition across a wide range of affinities. We propose a model that integrates insights from the fields of genetics and cell biology to provide further conceptual understanding of TF binding specificity. We argue that in eukaryotes, target specificity is driven by an inhomogeneous 3D nuclear distribution of TFs and by variation in DNA binding affinity such that locally elevated TF concentration allows low-affinity binding sites to be functional.

DNA processing by the MOBH family relaxase TraI encoded within the gonococcal genetic island.

Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3′ of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.

Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites.

Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5’AWWSSYRMNNWWWTNNNWSGGNYWW3’. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities.

Mechanism of cognate sequence discrimination by the ETS-family transcription factor ETS-1

Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Sensitive lateral flow immunoassay for the detection of hepatitis B virus surface antigen

Bisphenol A molecular imprinted adsorbent (BMIA) was successfully synthesized by a sol-gel process and showed a good specific binding performance in the water. The further studies showed that the mass transfer process was controlled by in-diffusion, and the synthesis conditions would effect on the amount of imprinting sites. Scatchard model analysis evidenced that the high binding affinity sites and the low binding affinity sites were both on BMIA, and the high binding affinity sites played a key role in the specific binding process. Scatchard model analysis of temperature effect experiments and dosage effect experiments proved that the specific binding sites with high binding affinity and the unexpressed specific binding sites with low binding affinity were the two different states of the imprinting binding sites. The conversion between the two different states depended on the reaction driving force, and the increasing reaction driving force would increase the number of specific binding sites. Especially, the temperature showed a linear positive correlation with the amount of specific binding sites. Finally, a possible model was put forward to explain the two different states conversion mechanism of the imprinting sites.

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ

AbstractThe proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ–mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.

The ATPase mechanism of UvrA2 reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair.

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.

Characterization of tolazamide binding with glycated and normal human serum albumin by using high-performance affinity chromatography

Sulfonylurea drugs are antidiabetic drugs that are utilized in the treatment of type II diabetes and often have significant binding with human serum albumin (HSA). Immobilized samples of normal or glycated HSA in affinity microcolumns were used to investigate interactions of these proteins with the sulfonylurea drug tolazamide. HPLC and frontal analysis were used to first examine the overall binding of this drug with these samples of HSA. It was found that tolazamide had two general classes of binding sites (i.e., high and low affinity) for normal and glycated HSA. The higher affinity sites had binding constants of around 4.3–6.0 × 104 M−1 for these interactions at pH 7.4 and 37 °C, while the lower affinity sites had binding strengths of 4.9–9.1 × 103 M−1. Zonal competition studies between tolazamide and probes for Sudlow sites I and II on HSA were also performed and used to provide site-specific affinities for tolazamide at these sites. A decrease of 22% in affinity was observed for tolazamide at Sudlow site I and an increase up to 58% was seen at Sudlow site II when comparing glycated HSA with normal HSA. These observed changes were compared to those of other first-generation sulfonylurea drugs, providing information on how glycation can alter the total and local binding strength of tolazamide and related compounds with HSA under levels of glycation seen in patients with diabetes.

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation.

Two different states conversion mechanism of the imprinting sites

Bisphenol A molecular imprinted adsorbent (BMIA) was successfully synthesized by a sol-gel process and showed a good specific binding performance in the water. The further studies showed that the mass transfer process was controlled by in-diffusion, and the synthesis conditions would effect on the amount of imprinting sites. Scatchard model analysis evidenced that the high binding affinity sites and the low binding affinity sites were both on BMIA, and the high binding affinity sites played a key role in the specific binding process. Scatchard model analysis of temperature effect experiments and dosage effect experiments proved that the specific binding sites with high binding affinity and the unexpressed specific binding sites with low binding affinity were the two different states of the imprinting binding sites. The conversion between the two different states depended on the reaction driving force, and the increasing reaction driving force would increase the number of specific binding sites. Especially, the temperature showed a linear positive correlation with the amount of specific binding sites. Finally, a possible model was put forward to explain the two different states conversion mechanism of the imprinting sites.