Aversive stimulus-tuned responses in the CA1 of the dorsal hippocampus.

Throughout life animals inevitably encounter unforeseen threatening events. Activity of principal cells in the hippocampus is tuned for locations and for salient stimuli in the animals' environment thus forming a map known to be pivotal for guiding behavior. Here, we explored if a code of threatening stimuli exists in the CA1 region of the dorsal hippocampus of mice by recording neuronal response to aversive stimuli delivered at changing locations. We have discovered a rapidly emerging, location independent response to innoxious aversive stimuli composed of the coordinated activation of subgroups of pyramidal cells and connected interneurons. Activated pyramidal cells had higher basal firing rate, more probably participated in ripples, targeted more interneurons than place cells and many of them lacked place fields. We also detected aversive stimulus-coupled assemblies dominated by the activated neurons. Notably, these assemblies could be observed even before the delivery of the first aversive event. Finally, we uncovered the systematic shift of the spatial code from the aversive to, surprisingly, the reward location during the fearful stimulus. Our results uncovered components of the dorsal CA1 circuit possibly key for re-sculpting the spatial map in response to abrupt aversive events.

Antimicrobial screening involving Helicobacter pylori of nano-therapeutic compounds based on the amoxicillin antibiotic drug.

Nano-structure Cu(II) complex [Cu(AMAB)2 ]Cl2 with Schiff base (AMAB) derived from the condensation between 4-(dimethylamino)benzaldehyde and amoxicillin trihydrate was prepared. (AMAB) Schiff base and its Cu(II) complex were identified and confirmed by different physicochemical techniques. The Schiff base (AMAB) was coordinated to copper ion through carbonyl oxygen and imine nitrogen donor sites. X-ray powder diffraction shows a cubic crystal system of the Cu(II) complex. The density functional theory was used to optimize the structure geometries of the investigated compounds. The molecular docking of the active amino acids of the investigated proteins' interactions with the tested compounds was evaluated. The bactericidal or bacteriostatic effect of the compounds was screened against some bacterial strains. The activity of Cu-chelate against Gram-negative bacteria was mainly more effective than its (AMAB) ligand and vice versa in the case of Gram-positive bacteria. The biological activity of the prepared compounds with biomolecules calf thymus DNA (CT-DNA) was determined by electronic absorption spectra and DNA gel electrophoresis technique. All studies revealed that the Cu-chelate derivative exhibited better binding affinity to both CT-DNA than the AMAB and amoxicillin itself. The anti-inflammatory effect of the designed compounds was determined by testing their protein denaturation inhibitory activity spectrophotometrically. All obtained data supported that the designed nano-Cu(II) complex with Schiff base (AMAB) is a potent bactericide against H. pylori, and exhibits anti-inflammatory activity. The dual inhibition effects of the designed compound represent a modern therapeutic approach with extended spectrum of action. Therefore, it can act as good drug target in antimicrobial and anti-inflammtory therapies. Finally, H. pylori resistance to amoxicillin is absent or rare in many countries, thus amoxicillin nanoparticles may be beneficial for countries where amoxicillin resistance is reported.

Hsp110 mitigates α-synuclein pathology in vivo.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein and its deposition into pathologic Lewy bodies. While extensive research has been carried out on mediators of α-synuclein aggregation, molecular facilitators of α-synuclein disaggregation are still generally unknown. We investigated the role of molecular chaperones in both preventing and disaggregating α-synuclein oligomers and fibrils, with a focus on the mammalian disaggregase complex. Here, we show that overexpression of the chaperone Hsp110 is sufficient to reduce α-synuclein aggregation in a mammalian cell culture model. Additionally, we demonstrate that Hsp110 effectively mitigates α-synuclein pathology in vivo through the characterization of transgenic Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed that overexpression of Hsp110 can override the protein changes driven by the α-synuclein transgene. Furthermore, overexpression of Hsp110 is sufficient to prevent endogenous α-synuclein templating and spread following injection of aggregated α-synuclein seeds into brain, supporting a role for Hsp110 in the prevention and/or disaggregation of α-synuclein pathology.

[Lipid-lowering therapy of patients suffering from acute coronary syndrome in a Hungarian county hospital in 2015]. / Akut coronariaszindróma miatt 2015-ben kezelt betegeink lipidcsökkento terápiája.

INTRODUCTION: The actual guidelines of cardiovascular prevention lay special emphasis on the lipid-lowering therapy of patients suffering from acute coronary syndrome (ACS). AIM: To evaluate the occurrence of high-intensity statin therapy, recommended by guidelines, at discharge in a Hungarian county hospital with hemodynamic laboratory in patients who underwent percutaneous intervention, furthermore the LDL-cholesterol (LDL-C) levels and goal attainment rate in the first year. METHOD: Retrospective data collection from the hospital database regarding the therapy at discharge and the lipid levels in the year following the intervention due to ACS in 2015. RESULTS: Due to ACS event, 454 patients had coronary intervention in 2015, at discharge more than 90% of them received high-intensity statin (more than 80% rosuvastatin, 40 mg) or corresponding combination therapy. In 154 cases we found half-year lipid results; the median of LDL-C was 1.9 mmol/L, the 1.8 mmol/L target value attainment rate was 48.7%. Results after one year were found in 292 cases (73% without the deceased and foreign patients); the LDL-C median proved to be 2.0 mmol/L, the target level attainment rate was 37.3%. There was no significant difference between the results of patients from the three different ACS forms and between those of men and women. CONCLUSIONS: The lipid lowering therapy of the revascularized patients who come back for medical visits is acceptable, but greater emphasis has to be laid on increasing the rate of controlled patients compared to the present two-thirds. Orv Hetil. 2018; 159(12): 478-484.

Reduced high-frequency motor neuron firing, EMG fractionation, and gait variability in awake walking ALS mice.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease prominently featuring motor neuron (MN) loss and paralysis. A recent study using whole-cell patch clamp recording of MNs in acute spinal cord slices from symptomatic adult ALS mice showed that the fastest firing MNs are preferentially lost. To measure the in vivo effects of such loss, awake symptomatic-stage ALS mice performing self-initiated walking on a wheel were studied. Both single-unit extracellular recordings within spinal cord MN pools for lower leg flexor and extensor muscles and the electromyograms (EMGs) of the corresponding muscles were recorded. In the ALS mice, we observed absent or truncated high-frequency firing of MNs at the appropriate time in the step cycle and step-to-step variability of the EMG, as well as flexor-extensor coactivation. In turn, kinematic analysis of walking showed step-to-step variability of gait. At the MN level, the higher frequencies absent from recordings from mutant mice corresponded with the upper range of frequencies observed for fast-firing MNs in earlier slice measurements. These results suggest that, in SOD1-linked ALS mice, symptoms are a product of abnormal MN firing due at least in part to loss of neurons that fire at high frequency, associated with altered EMG patterns and hindlimb kinematics during gait.

Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110.

Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoter-driven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ∼5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery.

Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.

In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~10(6) copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt's lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~10(6) copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.

Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS.

Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) affects motor neurons (MNs) in the brain and spinal cord. Understanding the pathophysiology of this condition seems crucial for therapeutic design, yet few electrophysiological studies in actively degenerating animal models have been reported. Here, we report a novel preparation of acute slices from adult mouse spinal cord, allowing visualized whole cell patch-clamp recordings of fluorescent lumbar MN cell bodies from ChAT-eGFP or superoxide dismutase 1-yellow fluorescent protein (SOD1YFP) transgenic animals up to 6 mo of age. We examined 11 intrinsic electrophysiologic properties of adult ChAT-eGFP mouse MNs and classified them into four subtypes based on these parameters. The subtypes could be principally correlated with instantaneous (initial) and steady-state firing rates. We used retrograde tracing using fluorescent dye injected into fast or slow twitch lower extremity muscle with slice recordings from the fluorescent-labeled lumbar MN cell bodies to establish that fast and slow firing MNs are connected with fast and slow twitch muscle, respectively. In a G85R SOD1YFP transgenic mouse model of ALS, which becomes paralyzed by 5-6 mo, where MN cell bodies are fluorescent, enabling the same type of recording from spinal cord tissue slices, we observed that all four MN subtypes were present at 2 mo of age. At 4 mo, by which time substantial neuronal SOD1YFP aggregation and cell loss has occurred and symptoms have developed, one of the fast firing subtypes that innvervates fast twitch muscle was lost. These results begin to describe an order of the pathophysiologic events in ALS.

Absence of lipofuscin in motor neurons of SOD1-linked ALS mice.

Lipofuscin, or aging pigment, is accreted as red autofluorescence in the lysosomes of motor neuron cell bodies in the ventral horn of WT mice by 3 mo of age. Strikingly, in two presymptomatic ALS mouse strains transgenic for mutant human Cu/Zn superoxide dismutase (SOD1), G85R SOD1YFP and G93A SOD1, little or no lipofuscin was detected in motor neuron cell bodies. Two markers of autophagy, sequestosome 1 (SQSTM1/p62) and microtubule-associated protein 1 light chain 3 (LC3), were examined in the motor neuron cell bodies of G85R SOD1YFP mice and found to be reduced relative to WT SOD1YFP transgenic mice. To elucidate whether the autophagy/lysosome pathway was either impaired or hyperactive in motor neurons, chloroquine was administered to 3-mo-old G85R SOD1YFP mice to block lysosomal hydrolysis. After 2 wk, lipofuscin was now observed in motor neurons, and SQSTM1 and LC3 levels approached those of WT SOD1YFP mice, suggesting that the autophagy/lysosome pathway is hyperactive in motor neurons of SOD1-linked ALS mice. This seems to be mediated at least in part through the mammalian target of rapamycin complex 1 (MTORC1) pathway, because levels of Ser757-phosphorylated Unc-51-like kinase 1 (ULK1), an MTORC1 target, were greatly reduced in the G85R SOD1YFP motor neurons, correspondent to an activated state of ULK1 that initiates autophagy.

Production of RNA for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

RNA-Seq profiling of spinal cord motor neurons from a presymptomatic SOD1 ALS mouse.

Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ~10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5-6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.

Flexible connection of the N-terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency.

ClpB reactivates aggregated proteins in cooperation with DnaK/J. The ClpB monomer contains two nucleotide-binding domains (D1, D2), a coiled-coil domain, and an N-terminal domain attached to D1 with a 17-residue-long unstructured linker containing a Gly-Gly motif. The ClpB-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB, but the events preceding the translocation are poorly understood. The N-terminal domains form a ring surrounding the entrance to the channel and contribute to the aggregate binding. It was suggested that the N-terminal domain's mobility that is maintained by the unstructured linker might control the efficiency of aggregate reactivation. We produced seven variants of ClpB with modified sequence of the N-terminal linker. To increase the linker's conformational flexibility, we inserted up to four Gly next to the GG motif. To decrease the linker's flexibility, we deleted the GG motif and converted it into GP and PP. We found that none of the linker modifications inhibited the basal ClpB ATPase activity or its capability to form oligomers. However, the modified linker ClpB variants showed lower reactivation rates for aggregated glucose-6-phosphate dehydrogenase and firefly luciferase and a lower aggregate-binding efficiency than wt ClpB. We conclude that the linker does not merely connect the N-terminal domain, but it supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. Moreover, our results suggest that selective pressure on the linker sequence may be crucial for maintaining the optimal efficiency of aggregate reactivation by ClpB.

Delimiting sub-areas in water bodies using multivariate data analysis on the example of Lake Balaton (W Hungary).

The main aim of the European Water Framework Directive (WFD, 2000) is to commit European Union Member states to the achievement of good qualitative and quantitative status for all water bodies by 2015. To achieve this, a reference state has to be determined and appropriate monitoring has to be carried out. Based on the fact that the WFD classifies Lake Balaton, the largest shallow freshwater lake in Central Europe, as one water body, and due to the lack of funds, the number of sampling locations on the lake was decreased. The aim of this study was to determine how many sub-areas with different WFD-related attributes (in this case, parameters) can be delimited in the so-called one water body of Lake Balaton, so that a number of representative sampling locations might be retained. To determine Lake Balaton's different water quality areas (i.e. sub-areas of water body) 23 parameters (inorganic compounds) were examined from 10 sampling locations for the time interval 1985-2004 using cluster- and discriminant analysis, and Wilks' lambda distribution. With cluster analysis we were able to determine two time intervals (1985-1997 and 1998-2004) with three patterns of sub-areas, two from the first and one from the latter interval. These patterns pointed to the fact that for the whole investigated time interval (1985-2004) a total of five sub-areas were present, changing in number and alignment. Then the results were verified using discriminant analysis, and the parameters which influenced the sub-areas the most were determined using Wilks' lambda distribution. The conclusion was that to be able to follow the changes in alignment of the sub-areas and to get a comprehensive picture of Lake Balaton, a minimum of five sampling locations should be retained, one in each sub-area. Based on this study the Water Authorities chose to keep five out of ten sampling locations so that the sub-areas could be described. We consider this a great success and the methodology as an example for setting up sub-areas in a water body.

Aggregate reactivation mediated by the Hsp100 chaperones.

Hsp100 family of molecular chaperones shows a unique capability to resolubilize and reactivate aggregated proteins. The Hsp100-mediated protein disaggregation is linked to the activity of other chaperones from the Hsp70 and Hsp40 families. The best-studied members of the Hsp100 family are the bacterial ClpB and Hsp104 from yeast. Hsp100 chaperones are members of a large super-family of energy-driven conformational "machines" known as AAA+ ATPases. This review describes the current mechanistic model of the chaperone-induced protein disaggregation and explains how the structural architecture of Hsp100 supports disaggregation and how the co-chaperones may participate in the Hsp100-mediated reactions.

Efficient regioselective three-component domino synthesis of 3-(1,2,4-Triazol-5-yl)-1,3-thiazolidin-4-ones as potent antifungal and antituberculosis agents.

In research for promising antibacterial and antifungal compounds, a series of 2-aryl 3-[1,2,4]triazol-5-yl 4-thiazolidinones 1 were synthesized by a domino reaction of 5-amino-1H-[1,2,4]triazoles 3, aromatic aldehydes, and α-mercaptoacids in boiling toluene in the presence of molecular sieves 4 Å. Of the twenty novel 3-[1,2,4]triazol-5-yl 4-thiazolidinone derivatives, four compounds 2-benzo[d][1,3]dioxol-6-yl-3-[(3-morpholin-4-yl)-1H-1,2,4-triazol-5-yl)]-1,3-thiazolidin-4-one (1i), 2-(4-chlorophenyl)-5-methyl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1p), 2-benzo[d][1,3]dioxol-6-yl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1s), 2-benzo[d][1,3]dioxol-6-yl-5-methyl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1t) exhibited MICs of 4 µg/mL or less versus Mycobacterium tuberculosis. Moreover, these compounds were screened against Candida albicans. Compounds 1p, 1s gave MICs of 1 µg/mL or less, and were fungicidal. Finally, compound 1s was evaluated against an expanded fungal panel and showed good activity against Cryptococcus neoformans. In addition, compound 1s also appeared to be fungicidal against Aspergillus arrhizus, with MIC <1 µg/mL.

Synergistic cooperation between two ClpB isoforms in aggregate reactivation.

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB.

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's gamma-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

Domain stability in the AAA+ ATPase ClpB from Escherichia coli.

ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.

The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.

Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.