Addition Of ATP Research Articles

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

FtsK is a hexameric DNA translocase that participates in the final stages of bacterial chromosome segregation. Here we investigate the DNA-binding and translocation activities of FtsK in real time by imaging fluorescently tagged proteins on nanofabricated curtains of DNA. We show that FtsK preferentially loads at 8-bp KOPS (FtsK Orienting Polar Sequences) sites and that loading is enhanced in the presence of ADP. We also demonstrate that FtsK locates KOPS through a mechanism that does not involve extensive 1D diffusion at the scale of our resolution. Upon addition of ATP, KOPS-bound FtsK translocates in the direction dictated by KOPS polarity, and once FtsK has begun translocating it does not rerecognize KOPS from either direction. However, FtsK can abruptly change directions while translocating along DNA independent of KOPS, suggesting that the ability to reorient on DNA does not arise from DNA sequence-specific effects. Taken together, our data support a model in which FtsK locates KOPS through random collisions, preferentially engages KOPS in the ADP-bound state, translocates in the direction dictated by the polar orientation of KOPS, and is incapable of recognizing KOPS while translocating along DNA.

ATP alters IKK‐2 inhibitor affinity for IKK2 enzyme and reveals non‐equivalent inhibitor binding sites

MethodInhibitor off‐rate was measured by two methods: (1) pre‐binding the enzyme with inhibitor followed by measuring the recovery of enzyme activity upon a 200‐fold dilution of the enzyme‐inhibitor complex in the presence of 1mM ATP and (2) measuring the shift in the compound potency (IC50) curves obtained by prebinding the enzyme and inhibitor in the absence of ATP followed by addition of either 1μM or 1mM ATP.ResultsThree literature inhibitors with potency (IC50) ranging from <1 to 35 nM were used for this study: PHA‐408 (Pfizer), GSK‐082 (GSK), and AVE‐002 (Aventis). The off‐rates (T1/2) measured by the dilution method are 124 and <15 min for PHA‐408 and GSK‐082, respectively. The off‐rate for AVE‐002 was biphasic with half of the enzyme‐inhibitor complex dissociating immediately (T1/2 < 2 min) and the other half bound tightly (T1/2 54 min). The IC50 curves at 1μM and 1mM ATP for PHA‐408 showed no shift using a short 2 min assay and is consistent with the slow off‐rate. GSK‐082 showed a > 10 fold loss of potency with 1mM ATP, as expected for a fast off‐rate inhibitor. AVE‐002 IC50 curve with 1μM ATP was monophasic but displayed a biphasic response with 1mM ATP.ConclusionATP can change the off‐rate of IKK‐2 inhibitor bound to the enzyme. Additionally, high ATP revealed that AVE‐002 bound in the two inhibitor binding sites of the IKK‐2 homodimer non‐equivalently.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

BackgroundPlasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.MethodsUsing the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.ResultsThe protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).ConclusionsThe data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Extracellular ATP induces P2X7-dependent nicotinamide phosphoribosyltransferase release in LPS-activated human monocytes

Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD biosynthesis, has recently been identified as a novel mediator of innate immunity. In the present study, we report that treatment of LPS-primed monocytes with ATP greatly enhanced the secretion of NAMPT in a time- and concentration-dependent manner without displaying any cytotoxic effect. NAMPT release was suppressed by pretreatment with the P2X(7) receptor (P2X(7)R) inhibitors oxidized ATP (oxATP) and KN-62, indicating the engagement of P2X(7)Rs. Furthermore, P2X(7)R was found to be involved in mediating cell permeability caused by the addition of ATP. To define a role of endogenous ATP in NAMPT secretion, LPS-primed monocytes were incubated in the presence of oxATP and KN-62, as well as the ATP-hydrolyzing enzymes apyrase and hexokinase. With the exception of oxATP, neither substance led to a decrease in NAMPT release, suggesting that autocrine/paracrine ATP is unlikely to be responsible for the LPS-induced release of NAMPT. In conclusion, the enhanced release of NAMPT by extracellular ATP described here indicates the requirement of a second stimulus for the efficient secretion of NAMPT. This mode of secretion, which also applies to IL-1β, might represent a general mechanism for the release of leaderless secretory proteins at locally restricted sites.

Intermediate progenitors are increased by lengthening of the cell cycle through calcium signaling and p53 expression in human neural progenitors

During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)(+)/BTG family member 2 (Btg2)(-), which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53-p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calcium-induced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis.

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation.

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson's disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.

A new perylene diimide-based fluorescent chemosensor for selective detection of ATP in aqueous solution

A new perylene diimide (PDI) ligand (1) functionalized with a Zn2+–dipicolylethylenediamine (Zn2+–DPEN) moiety was synthesized and first used as a fluorometric chemosensor to specifically detect the presence of ATP over other phosphate anions in aqueous solution. The solution of 1 upon addition of ATP displayed a remarkable absorption decrease compared with other anions, indicating the selectivity of 1 to ATP. Moreover, among these anions investigated, only ATP showed a considerable enhancement of fluorescent intensity of 1. The fluorescence molar ratio plot combined with the ESI-TOF mass measurement determined that binding stoichiometry between 1 and ATP is 1:1.

Short peptide tools for monitoring caspase and proteasome activities in embryonal and adult rat brain lysates: an approach for the differential identification of proteases

The numerous caspase-like activities present in nervous tissue can be investigated with labelled peptides. However, the cross-reactivities of peptides with both proteasomes and caspases complicate the analysis of protease activity. The pharmacological features of substrates and inhibitors specific for either caspases or proteasome caspase-like proteases in rat brain lysates were similar or identical to the profiles of commercially purified proteasome preparations. Caspase inhibitors bind directly to active proteasome centres, thus competing with selective antagonists of proteasomes. Separation of lysates by molecular weight does not separate active caspases from proteasomes because these enzymes co-localize under native electrophoresis. The addition of ATP or its analogues is associated with the differential modulation of proteasomal activity, which also leads to ambiguity in the data. However, induced caspase activity could be successfully differentiated from proteasome activity in embryonal brain lysates with the non-selective caspase inhibitors Z-VAD-FMK and Q-VD-OPh and the proteasome inhibitor AdaAhx(3)L(3)VS that are not cross-reactive. This strategy is proposed for the simultaneous examination of caspases and proteasomes using proteolysis experiments. The present study reveals that all of the caspase-like activities in the tissue lysates of non-injured adult rat brains were related to proteasomal caspase-like activities.

Freeing the Promoter Site: Mechanistic Insights into the Interaction of Mot1 with TBP using spFRET

Gene transcription is a central process of life and is highly regulated with a multitude of transcription factors. Recently, we could show that dynamics of the Tata-box Binding Protein (TBP) on the promoter site also plays a role in gene regulation. Using single-pair Forster Resonance Energy Transfer (spFRET) experiments, we have monitored the conformation of TBP bound to the H2B promoter site in the presence and absence of Mot1. Upon the binding of Mot1 to the TBP-DNA complex, a high FRET state between TBP and labeled DNA is formed. Contrary to what was expected, Mot1 bound to TBP-DNA complexes was unable to dissociate TBP from the DNA upon addition of ATP. Instead, the TBP-DNA complex returned to its original conformation even though Mot1 remain bound to the complex. Only when Mot1 was also present at nM concentrations in solution was dissociation of TBP from the promoter site observed upon addition of ATP. This suggests that a single Mot1 is either insufficient or inefficient at dissociating TBP alone.We also used spFRET to monitor the DNA conformation during the interaction of Mot1 with TBP-DNA. TBP-bound DNA was observed to fluctuate between three conformations. The FRET values of the conformation varied depending on the orientation of TBP on the promoter. Fluctuations between the same conformations were observed when Mot1 was bound to the TBP-DNA complex but with an altered kinetics. The results suggest that Mot1 primes incorrectly bound TBP for dissociation, preferably freeing promoter sites with an inappropriately bound TBP molecule.

Role of Polyhydroxybutyrate in Mitochondrial Calcium Transport

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to polyesters class and ubiquitously present in all living organisms. It has been demonstrated that mammalian mitochondrial membranes contain PHB polymer consisting of up to 120 butyrate residues. We found before that mitochondrial PHB complexed with Ca2+ and inorganic polyphosphate forms channels with properties similar to mitochondrial permeability transition pore. It is also experimentally proven that PHB in its free form possesses ionophoretic properties and mediates Ca2+ transport through the model phospholipid membranes. We hypothesized that PHB might significantly contribute to the process of mitochondrial Ca2+ transport. To test this idea we investigated Ca2+ transport in mitochondria with decreased levels of PHB. Mitochondrial PHB was reduced enzymatically by targeted expression of specific bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. Experiments were performed using transiently transfected cultured cells: HepG2, U87 and HeLa. PHB deficiency induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential (measured by TMRM) in HepG2 but not in U87 and HeLa cells. Kinetics of mitochondrial Ca2+ transport was measured using mitochondrial Ca2+ sensitive fluorescent probe - x-Rhod-1 and confocal microscopy. Mitochondrial Ca2+ signal was stimulated either by addition of ATP or Histamine in experiments with intact cells or by addition of increased concentrations of calcium to the recording solution in digitonin permeabilized cells. We found that over-expression of mitochondrially targeted PHB depolymeraze in mammalian cultured cells dramatically inhibits their Ca2+ uptake. Ca2+ uptake in these cells was restored by addition of mimic of calcium uniporter with electrogenic Ca2+ ionophore properties - ferutinin. Our data suggest that PHB is previously unrecognized essential component of mitochondrial Ca2+ uptake system.

An Optical Device Driven by Motor Protein

Here, we developed a motor-protein-driven optical device. To design the device, we focused on body color change of fish. This phenomenon is caused by melanophore cells, which contains thousands of pigment granules. In a melanophore, pigments are transported by motor proteins along radially arranged microtubules. Thus, the distribution of pigments in a melanophore (i.e. color of a melanophore) is changed. By mimicking this molecular system, we tried to make an artificial melanophore from minimal components: motor proteins, microtubules and pigment granules. Nuclei of microtubule assembly were attached to micro-fabricated dot patterns, and microtubules were grown from them, which produced a radial array of numerous microtubules (Figure A). When stained micro-particles with flagellar dynein were introduced, those particles were transported along radially arranged microtubules and were gathered by ATP addition, which resulted in changing color patterns of each artificial melanophore (Figure B, C). Furthermore, we succeeded in drawing desired pictures on “bio-display”, a display that one pixel is equivalent to one artificial melanophore, by adding ATP to particular pixels with caged ATP. This study has shown that a novel optical device can be constructed by arranging microtubules and motor protein molecules.

Phospholamban does not Interact with E2 Intermediates of the Cardiac Calcium Pump Stabilized by Metal Fluorides

The phospholamban (PLB) monomer binds in the groove between M2, M4, and M9 of the cardiac calcium pump (SERCA2a) and inhibits enzyme activity. We previously demonstrated that cross-linking of PLB with engineered Cys-residues to this region is prevented by micromolar Ca, but is stimulated by the nucleotides ATP or ADP. We proposed that PLB inhibits the catalytic activity of the enzyme by stabilizing a unique, nucleotide bound, Ca-free intermediate of SERCA2a (E2•ATP). Stabilization of E2•ATP prevents Ca binding at the high affinity Ca binding sites and formation of E1, thus blocking the kinetic cycle. To further test our model, we have now checked if PLB interacts with other E2 conformations, notably, SERCA2a with the bound metal fluorides (MgF42-, AlF4-, or BeF3-), which mimic the E2•Pi, E2P, and E2-P states, respectively. Interactions between PLB and SERCA2a were examined by cross-linking of N30C-PLB to K328 of WT-SERCA2a at the cytoplasmic extension of M4 and of V49C-PLB to V89C-SERCA2a at the C-terminal end of M2. Insect cell microsomes co-expressing N30C-PLB/SERCA2a or V49C-PLB/V89C-SERCA2a were first incubated with the metal fluorides in Ca-free buffer, and then subjected to chemical cross-linking. All these metal fluorides prevented cross-linking of PLB to SERCA2a at both the cytoplasmic and transmembrane regions, suggesting that PLB does not interact with E2•Pi, E2P, and E2-P formed in the absence of nucleotide. Remarkably, however, the cross-linking of PLB to SERCA2a was completely restored upon addition of 3mM ATP, allowing formation of E2•ATP. These results strongly support our model in which E2•ATP is the single conformation binding PLB, and further suggest that the binding interaction between E2•ATP and PLB is very strong, sufficient to prevent formation of E2•Pi, E2P, or E2-P, even when stabilized by metal fluorides.

Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10-100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.

Electron Tomography of MSP Filaments Derived from the Amoeboid Sperm of Ascaris Suum

Locomotion of nematode sperm is remarkably similar to that of most other crawling cells but is powered by a system of filaments composed of major sperm protein (MSP) instead of the actin-myosin machinery typically associated with amoeboid motility. The MSP motility apparatus has been reconstituted in vitro, and individual MSP filaments can be generated by the addition of ATP to detergent-treated Ascaris sperm cytosol. Filaments formed in this way have been examined using both electron cryo-tomography and also conventional tomography of negative-stained samples. This has allowed for the structural analysis of filaments formed in the presence of MSP accessory proteins. Subvolume averaging has been applied to individual filaments sampled along their lengths within complex filament meshworks, and the resulting physiological models were compared to earlier models derived using purified MSP, including a helical reconstruction of filaments polymerized in ethanol (King et al. 1992. JCS 101:847) and an x-ray crystal model of MSP subfilaments (Bullock et al. 1998. NSB 5:184). Comparisons suggest important differences between filaments formed under physiological and nonphysiological conditions. There are currently six Ascaris sperm proteins known to modulate MSP filament dynamics in sperm; these same proteins are absent from filaments prepared using purified MSP. Comparing MSP filament models with and without these accessory proteins allows us to map the binding sites of these accessory proteins and provides a greater understanding of how they effect MSP filament dynamics and influence motility. Supported by NIH Grant R37 GM29994 and by the American Heart Assoc.

Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37 °C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca 2+ addition, Ca 2+ chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tert-butyl hydroperoxide (TBHP) or H 2O 2 increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H 2O 2 oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.

Hsc70 Protein Interaction with Soluble and Fibrillar α-Synuclein

The aggregation of α-synuclein (α-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native α-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with α-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and α-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble α-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble α-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing α-Syn assembly inhibition by Hsc70. We show that Hsc70 binds α-Syn fibrils with a 5-fold tighter affinity compared with soluble α-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight α-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of α-Syn assemblies. We show a reduced cellular toxicity of α-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of α-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.

Dynamic Isolation and Unloading of Target Proteins by Aptamer-Modified Microtransporters

We describe here a new strategy for isolating target proteins from complex biological samples based on an aptamer-modified self-propelled microtube engine. For this purpose, a thiolated thrombin or a mixed thrombin-ATP aptamer (prehybridized with a thiolated short DNA) was coassembled with mercaptohexanol onto the gold surface of these microtube engines. The rapid movement of the aptamer-modified microtransporter resulted in highly selective and rapid capture of the target thrombin, with an effective discrimination against a large excess of nontarget proteins. Release of the captured thrombin can be triggered by the addition of ATP that can bind and displace the immobilized mixed thrombin-ATP aptamer in 20 min. The rapid loading and unloading abilities demonstrated by these selective microtransporters are illustrated in complex matrixes such as human serum and plasma. The new motion-driven protein isolation platform represents a new approach in bioanalytical chemistry based on active transport of proteins and offers considerable promise for diverse diagnostic applications.

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca(2+)/CaM but outlasts this initial Ca(2+)-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.