Whereas increasing evidence has linked the adverse roles of PM2.5 with cardiovascular and respiratory diseases, limited but growing emerging evidence suggests that PM2.5 exposure can affect the nervous system, causing neuroinflammation, synaptic dysfunction and cognitive deterioration. However, the molecular mechanisms underlying the synaptic and cognitive deficits elicited by PM2.5 exposure are largely unknown.PM2.5 (1 and 5 mg/kg bw) every other day for 4 weeks. The mice were also stereotaxically injected with β-site amyloid precursor protein cleaving enzyme 1  shRNA or LV-miR-574-5p lentiviral constructs in the absence or presence of PM2.5 aspiration at 5 mg/kg bw every other day for 4 weeks. Spatial learning and memory were assessed with the Morris water maze test, and synaptic function integrity was evaluated with electrophysiological recordings of long-term potentiation (LTP) and immunoblot analyses of glutamate receptor subunit expression. The expression of α-secretase (ADAM10), BACE1, and γ-secretase (nicastrin) and the synthesis and accumulation of amyloid β (Aβ) were measured by immunoblot and enzyme-linked immunosorbent assay (ELISA). MicroRNA (miRNA) expression was screened with a microRNA microarray analysis and confirmed by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. Dual-luciferase reporter gene and chromatin immunoprecipitation (ChIP) analyses were used to detect the binding of miR-574-5p in the 3’UTR of BACE1 and NF-κB p65 in the promoter of miR-574-5p, respectively.C57BL/6 mice received oropharyngeal aspiration of PM2.5 aspiration caused neuroinflammation and deteriorated synaptic function integrity and spatial learning and memory, and the effects were associated with the induction of BACE1. The action was mediated by NF-κB p65-regulated downregulation of miR-574-5p, which targets BACE1. Overexpression of miR-574-5p in the hippocampal region decreased BACE1 expression, restored synaptic function, and improved spatial memory and learning following PM2.5 exposure.PM2.5, suggesting that miR-574-5p is a potential intervention target for the prevention and treatment of PM2.5-induced neurological disorders.Taken together, our findings reveal a novel molecular mechanism underlying impaired synaptic and cognitive function following exposure to PMThe online version of this article (10.1186/s12989-017-0215-3) contains supplementary material, which is available to authorized users. Air pol disease . While ifunction –9. This function –19. Chanfunction  and are function , 22. Synfunction . Glutamad memory . Individd memory  and may d memory . Importaathology , 27, 28.2.5 exposure alters the miRNA profile in the brain and whether and how the dysregulated miRNAs mediate PM exposure-induced synaptic and cognitive deficits.MicroRNAs (miRNAs), a group of 18- to 25-nucleotide-long noncoding RNA molecules, are increasingly being recognized as key regulators that target proteins at the translational level . Importa2.5 aspiration impaired synaptic and cognitive function, and the effects were associated with the induction of BACE1. Furthermore, we provide evidence that this action was mediated by NF-κB p65-induced downregulation of miR-574-5p, which targets BACE1. Importantly, overexpression of miR-574-5p in the hippocampal region decreased BACE1 expression, restored synaptic function, and improved spatial memory and learning following PM2.5 aspiration. Our results reveal a novel miRNA-modulated mechanism responsible for synaptic and cognitive impairments caused by PM2.5 exposure.β-Site amyloid precursor protein (APP) cleaving enzyme 1  is a critical enzyme catalyzing amyloid β (Aβ) peptide generation by cleaving APP, and BACE1 activation is a hallmark of early stage cognitive deficits and plays an important role in progressive conversion to AD. Previously, several miRNAs have been demonstrated to be involved in post-transcriptional regulation of BACE1, including miR-9, miR-29, miR-107, miR-186, miR-188, miR-298, and miR-328 , 41. Alt2.5 aspiration.During the experiment, none of the animals died, and food intake and body weight remained unchanged following PM2.5 and an inert particle (black carbon) at the same level of exposure. As shown in Fig. 2.5 aspiration dose-dependently increased IL-1β and TNF-α levels in the lungs, blood and brain, and significant changes were observed at 1 mg/kg bw for IL-1β in the lungs and TNF-α in the blood and at 5 mg/kg bw for both IL-1β and TNF-α in the lungs, blood and brain. The results suggest the possibility that PM2.5 exposure affects the nervous system through systemic inflammation.Neuroinflammation is one of the main pathogenic mechanisms leading to synaptic and cognitive deficits, and PM can invade the brain through inflammatory mediators released from primary entry organs or secondary deposition sites . To clar2.5 has been reported to alter the brain inflammatory milieu and accelerate AD progression, mainly through BACE1-catalyzed APP cleavage [2.5 aspiration might promote BACE1- and γ-secretase- but not α-secretase-mediated cleavage of APP, leading to Aβ synthesis and accumulation and synaptic and cognitive impairment. To test this idea, we treated animals with PM2.5 at two dosing regimens (1 and 5 mg/kg bw) and first measured the levels of hippocampal Aβ and APP, BACE1, γ-secretase (nicastrin) and α-secretase (ADAM10) expression. Unexpectedly, PM2.5 aspiration did not increase production or deposition of Aβ42  of the hippocampus. As shown in Fig. 2.5 exposure. Considering that the glutamate receptor is responsible for excitatory synaptic integrity, we further detected the expression of NMDA and AMPA receptor subunits . As shown in Fig. 2.5 at 1 and 5 mg/kg bw for GluA2 and at 5 mg/kg bw for GluA1, GluN1, GRIN2A, and GRIN2B. Additionally, we found that expression of postsynaptic density protein 95 (PSD-95), a postsynaptic marker, was statistically significantly reduced . To clarify whether PMced Fig. , and the2.5 exposure contributes to impairments in functional synaptic integrity and spatial learning and memory, then BACE1 inhibition should eliminate or attenuate these impairments. To test this hypothesis, we silenced BACE1 expression by stereotaxically injecting a lentivirus vector (LV) expressing BACE1 shRNA into the hippocampal area . Among these altered miRNAs, miR-574-5p was homologous to the corresponding human miRNA, and the significantly downregulated expression was validated by miRNA real-time qPCR  to predict potential binding sites between NF-κB p65 and miR-574-5p, and the results showed that there was at least one NF-κB binding site. Importantly, by designing PCR amplicons, the binding site was validated via chromatin immunoprecipitation (ChIP) analysis. As presented in Fig. 2.5 treatment. Additionally, through the dual-luciferase reporter system in HEK293T cells, we found that PM2.5 directly stimulated NF-κB activity  and the P3 fragment. In the amyloidogenic pathway, APP is cleaved by β-secretase and γ-secretase to produce the soluble fragment sAPP-β, Aβ40, Aβ42 and AICD. To clarify the above results, we further detected the expression of α-secretase (ADAM10) and γ-secretase (nicastrin) and did not observe significant alterations. Aβ deposition is slow and protracted, and the slow rate of Aβ deposition indicates a wide time window for altering Aβ accumulation [2.5 exposure for 4 weeks; however, we cannot exclude the possibility that the time point was too early to see significant alterations. Previous studies have shown that BACE1-catalyzed APP cleavage exerted positive effects on cognitive function, and AICD, which is correlated with the promotion of synaptic transmission and memory performance, is a potential mediator during this process [2.5 exposure resulted in decreased expression of glutamate AMPA and NMDA receptor subunits and LTP and spatial learning and memory impairment in normal wild-type animals. Similar to our results, other studies have documented that PM2.5 altered synaptic gene expression and caused synaptic and cognitive dysfunction [2.5-induced attenuation of AMPA and NMDA receptor subunit expression and impairment of LTP and spatial learning and memory. Although we did not clarify the polypeptide produced by BACE1-mediated cleavage of APP, these findings confirm that BACE1 plays an important role in PM2.5-mediated synaptic and cognitive impairment and suggest the possibility that further study of BACE1 regulation may provide insight into the underlying signaling pathways mediating harmful outcomes.BACE1 is not only a dominant enzyme in Aβ synthesis from APP but also functions as an aspartic protease with altered enzymatic activity and protein expression in the brain early in the development of mild cognitive impairment . Other slevation . In the mulation . Klunk emulation . In the  process . Our prefunction , 27, 28.function . Changesfunction , 69. Imp2.5 exposure. To address this question, we performed a miRNA microarray analysis and found that miR-574-5p, which is homologous in humans and targets BACE1, was downregulated in a dose-dependent manner following PM2.5 exposure, suggesting that BACE1 elevation following PM2.5 exposure is likely the result of reduced miR-574-5p expression. If this hypothesis is correct, restoring or reversing these downregulated miRNAs in animal models following PM2.5 exposure should ameliorate synaptic and cognitive impairment by reducing BACE1 expression. Indeed, our results showed that overexpression of miR-574-5p significantly reduced BACE1 elevation, which supported this speculation. Importantly, miR-574-5p overexpression significantly restored the attenuation of glutamate receptor expression and the deterioration of LTP and spatial learning and memory in response to PM2.5 aspiration. This information provides further evidence that the adverse effects of BACE1-mediated synaptic and cognitive deterioration resulting from PM2.5 aspiration are associated with downregulation of miR-574-5p through its binding to the 3’UTR of BACE1.Considering the vital role of miRNAs in post-transcriptional regulation, their widespread expression in different brain regions and their regulation by multiple mechanisms, miRNAs are increasingly considered to be central players in synaptic plasticity, a critical mechanism that is thought to underlie many complex brain functions, including learning and memory . Indeed,2.5 exposure, we used ChIP analyses to show that NF-κB p65 was able to bind to the promoter region of the miR-574-5p gene. Additionally, PM2.5 increased NF-κB reporter activity, indicating that NF-κB activation following PM2.5 exposure amplifies NF-κB-mediated inhibition of miR-574-5p transcription. Importantly, pharmacological and genetic silencing of NF-κB p65 prevented the miR-574-5p reduction and BACE1 elevation following PM2.5 exposure, supporting our hypothesis. The PM2.5-induced increase in BACE1 expression not fully rescued by NF-κB shRNA may be due to several transcription factors have interactions with BACE1 except NF-κB. For instance, specificity protein 1 (Sp1) plays an important role in regulation of BACE1 to process APP generating Aβ in AD [2.5 aspiration suppresses miR-574-5p expression by augmenting NF-κB activity, which then facilitates BACE1 activation and results in synaptic and cognitive impairment.Numerous investigations have reported a role of miRNAs in physiological functions, such as immune responses, cell proliferation, cell death, and inflammation, which are also known to be regulated by NF-κB . Recent Aβ in AD . In addi2.5 exposure leads to impaired synaptic and cognitive function, and this action is associated with a previously unrecognized role for BACE1 induction through NF-κB signaling-mediated miR-574-5p downregulation. The results suggest a novel mechanism for PM2.5-produced adverse effects on the nervous system and present a potential intervention target for prevention.The present study confirms that PM2.5 sampling point was located in Taiyuan  of Shanxi Province. The samples were collected onto quartz filters  with PM middle-volume air samplers  from November 2012 to February 2013 according to our previously reported conditions [2.5 was transferred into aqueous suspension by soaking the PM2.5-loaded filters in Milli-Q deionized water for 30 min, followed by vortexing (5 min) and sonication (30 min). Prior to use, the dried sample was diluted with sterilized saline and then swirled and sonicated for 10 min.The PMnditions , and thenditions . The col2/g. The particles were suspended in sterilized saline and then swirled and sonicated for 10 min before oropharyngeal aspiration.Printex u was purchased from Degussa Ltd.  and utilized as black carbon powder. The size of its particle was 30 nm under the electron microscope and the specific surface area was 97.63 m2.5 in mice. According to Grade II PM2.5 in China, the amount of PM2.5 exposure at 0.075 mg/m3 for 2 days is 0.019 mg. As previously mentioned, the respiratory volume of the mice was 90 mL/min  and the respiratory volume was 0.259 m3 for 2 days. Thus, the PM2.5 exposure dose in mice should be 0.95 mg/kg bw every 2 days. However, the average concentration of PM2.5 in northern China with non-haze weather was 0.161 mg/m3 [3 [2.5 dose used was 1 mg/kg bw, within the range of the second standard in Chinese ambient air quality regulation, and the higher PM2.5 exposure dose used for mice was 5 mg/kg bw, which was 5-fold higher than that in Grade II PM2.5 in China but still in the range of the reported maximum PM2.5 levels.Male C57BL/6 mice, approximately 8 weeks old, were obtained from the Experimental Animal Center at the Academy of Military Medical Sciences of Chinese PLA  and housed under standard conditions. Considering the actual population exposure dose, we chose the administration dose of 1 and 5 mg/kg bw PM61 mg/m3 , and themg/m3 [3 . In our 2.5 treatment groups, animals received oropharyngeal aspiration of PM2.5 (1 and 5 mg/kg bw) every other day for 4 weeks, with each group administered 20 or 100 μg of PM2.5 in 20 μL of saline. In the vehicle control group, mice were treated with the same amount of saline prepared by ultrasonic oscillation of the control membrane filter using the same protocol. In the control group, mice received oropharyngeal aspiration of saline every other day for 4 weeks. In the black carbon treatment groups, animals received oropharyngeal aspiration of black carbon (5 mg/kg bw) every other day for 4 weeks, with each group administered 100 μg of black carbon in 20 μL of saline. To provide direct evidence that BACE1 and miR-574-5p were involved in the neurological effects observed after PM2.5 treatment, animals were stereotaxically injected with lentiviral constructs encoding BACE1 shRNA and control shRNA or LV-miR-574-5p and LV-scramble control for interference measurement. In detail, mice received a bilateral microinjection (5 μL at 0.2 μL/min/side) into the hippocampus . After 6 weeks, four groups  received an oropharyngeal aspiration of 5 mg/kg PM2.5 or saline, which was processed by ultrasonic oscillation of the control membrane filter, every other day for 4 weeks. Before PM2.5 or saline administration, the mice were anesthetized with isoflurane . When not being treated, the animals had free access to water and standard feed. Mice were sacrificed 18 h after the final exposure. The hippocampal tissues, lungs and blood (with no interference treatment) were collected, quickly frozen in liquid nitrogen, and stored at −80 °C. The mice were treated humanely according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Shanxi University .In the PM6) were divided randomly into different groups. The control group was incubated only in Neurobasal/B27 medium, and the other groups were treated with 10 μg/ml PM2.5 for 24 h in the absence or presence of the NF-κB inhibitor SC-514 (100 μM) or were subjected to NF-κB silencing using small hairpin RNA (shRNA).Primary cultured hippocampal neurons were prepared according to a protocol previously reported . BrieflyAgilent Mouse miRNA V19.0 was used for miRNA microarray analysis. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through GEO Series accession number GSE93967. Briefly, total RNA was extracted and purified, and the integration was inspected according to previously described methods . Then, mhttp://www.targetscan.org), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), mirwalk  and miranda (http://www.microrna.org/microrna/home.do) websites were used to predict miRNA targets. The pLVX-IRES-TDtomato LV (Clontech) was used to insert mature miR-574-5p driven by the CMV promoter. pHelper 1.0 and pHelper 2.0 vectors were used for viral envelope production. pLVX-IRES-TDtomato LV was generated and packaged in HEK293T cells, and the transfection rate was measured by observing the expression of enhanced red fluorescent protein (RFP) under a fluorescence microscope. The average LV titer was 1.0 × 108 infectious units/mL. pLVX-IRES-TDtomato LV was also used for BACE1 shRNA in C57BL/6 mice. The oligos for the BACE1 shRNA construct (5′-GACGCTCAACATCCTGGTG-3′) and scramble construct (5′-TTGGCTTTGCTGTCAGCGC-3′) were used for BACE1 knockdown experiments.The TargetScan (pLVX-shRNA2 LV (Clontech) was used for NF-κB p65 shRNA in primary cultured hippocampal neurons. The transfection rate was measured by observing the expression of enhanced green fluorescent protein (GFP) under a fluorescence microscope. Oligos for the NF-κB p65 shRNA construct (5′-AGGACCTATGAGACCTTCAAG-3′) and control shRNA (empty vectors) were used for knockdown of NF-κB p65. All of the constructs were verified by sequencing.2O, 2 μL of 10× miScript Universal Primer, and 2 μL of 5 μmol/L specific primers (Invitrogen). The reaction conditions for mmu-miR-574-5p and mmu-miR-202 were as follows: after 15 min at 95 °C, 40 cycles were performed at 95 °C for 20 s with respective annealing temperatures of 58.6 °C (mmu-miR-574-5p) and 55 °C (mmu-miR-202) for 20 s and then 72 °C for 30 s. The primer sequences were 5′-TGAGTGTGTGTGTGTGAGTGTGT-3′ for mmu-miR-574-5p and 5′-TTGAACCCTTTTCCATCTGA-3′ for mmu-miR-202.Total RNA was extracted according to the manufacturer’s protocol using an miRNeasy Mini Kit  and then synthesized to complementary DNA (cDNA) using a reverse transcription kit . RT-PCR with specific primers was performed on a qTOWER 2.2 Real-Time PCR machine  according to the protocol for the miScript SYBR Green PCR Kit . Briefly, each 20-μL PCR reaction contained 2 μL of cDNA (2.5 ng/μL), 10 μL of 10× QuantiTect SYBR Green, 4 μL of RNase-free H4/well) in DMEM containing 10% FBS 24 h before transfection. Using Lipofectamine 2000 (Invitrogen), cells were transfected with 100 ng/mL of either the wild-type or mutant 3′-UTR vector and 50 nM miR-574-5p mimic or 100 nM miR-574-5p inhibitor. At 48 h after transfection, luciferase activity was measured in cell lysates using a dual-luciferase reporter kit . HEK 293 T cells are a very useful tool for luciferase reporter assays and for lentiviral production and amplification [2.5 on NF-κB promoter activity were determined in HEK293T cells transfected with the pGL3-NF-κB promoter luciferase construct. At 24 h after transfection, cells were exposed to 10 μg/ml PM2.5 for 24 h, and then, luciferase activity was measured in cell lysates using a dual-luciferase reporter kit .To assess the interaction between miR-574-5p and BACE1 mRNA, the BACE1 3′-UTR was amplified by PCR using genomic DNA from mouse brain tissues and the following primers: 5′-CCGCTCGAGGGAGGCCCGTGGGCAGATGATGG-3′ (forward) and 5′-ATAAGAATGCGGCCGCCAGTGGAGATAGGTCAGTCATTTTTC-3′ (reverse). A construct possessing mutations disrupting the putative mir-574-5p binding site in the 3′-UTR of BACE1 was prepared using KOD Plus neo DNA Polymerase (ToYoBo) with the following primers: 5′-TGGTTCTTGGGCTAGGTTGTGGGGGGGTGTGAGACCTCTTCCCTGCCAGTTCTAACAC -3′ (forward) and 5′-GTGTTAGAACTGGCAGGGAAGAGGTCTCACACCCCCCCACAACCTAGCCCAAGAACCA-3′ (reverse). Both the wild-type and mutated plasmid clones were verified by DNA sequencing. The PCR products were cloned into the psiCHECK-2 vector (Promega) downstream of the luciferase gene. HEK293T cells were plated onto 24-well plates  was used for observation.According to a previously reported protocol , approxihttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) was applied to find the NF-κB binding site within the promoter region. The primers for ChIP were as follows (amplicon size: 134 bp): 5′-AATCACATCCCACTCCCA-3′ (forward) and 5′-CCCTGCCTTCCATTACGA-3′ (reverse) for the binding site. Briefly, the hippocampal tissues were fixed with formaldehyde and washed twice with PBS containing protease inhibitors. The tissues were subsequently homogenized in PBS and centrifuged ; then, the precipitates were resuspended in SDS lysis buffer and sonicated to obtain DNA lysate. The samples were centrifuged, and the supernatants were diluted with ChIP dilution buffer. The samples were subsequently precleared to reduce the nonspecific background and then incubated with primary antibody (anti-NF-κB) overnight. The antibody/histone complex was collected via the addition of Salmon Sperm DNA/Protein A Agarose Slurry, washed with different wash buffers, and eluted from the antibody; the protein and DNA cross-linking was reversed with NaCl. Finally, DNA was recovered via phenol/chloroform extraction and ethanol precipitation, and a PCR reaction and DNA agarose electrophoresis were conducted to obtain the final results.ChIP analysis was performed according to the manufacturer’s instructions  to test the binding of NF-κB p65 to the miR-574-5p promoter. PROMO  was filled with water rendered opaque by the addition of white, non-toxic paint. A circular platform with a diameter of 15 cm was placed 1 cm below the water surface in the center of a specific quadrant. If an animal failed to reach the platform within a fixed period of 60 s, it was gently guided to the location and was allowed to remain on it for 10 s. The animals were given training sessions using visible and invisible platforms for a period of 8 consecutive days . In the non-spatial water maze, the mice received 3 consecutive days of training to find the same platform elevated above the water surface. Invisible platform training was carried out for 5 continuous days (5 sessions), and each session consisted of 4 trials. For each trial, the mouse was released from the wall of the tank and allowed to search, find, and stand on the platform for 10 s within the 60-s trial period. For each training session, the sequence designed for the animals’ training was determined in a random manner that varied each day so that it differed in the separate sessions for each animal and was different for each individual animal. An EthoVision video tracking device  was used to record the latency of reaching the target as a measure of task performance. The average latencies on each hidden platform were recorded and compared. Memory tests were conducted in a probe trial performed 24 h after the last training trial. A probe trial was performed in which the platform was removed from the tank, and the animals were allowed to swim for 60 s. The number of target annulus crossovers and the time spent in the target quadrant were measured.2 and 5% CO2-saturated ACSF at 32–34 °C. Field excitatory postsynaptic potential (fEPSP) recordings were made in response to stimulation of the perforant path in the DG region of the hippocampus at a frequency 0 of 0.05 Hz using an Axoclamp-2B patch-clamp amplifier  in bridge mode. Hippocampal LTP was induced by theta burst stimulation (TBS) consisting of a series of 10 bursts of 5 pulses at 100 Hz.After the animal was sacrificed and the brain was dissected and kept in cold oxygenated artificial cerebrospinal fluid (ACSF), the slices (350–400 μm) were cut, moved to a holding chamber containing oxygenated ACSF at 34 °C for 0.5–1 h, and then kept in an incubator containing oxygenated ACSF at room temperature for recovery for more than 1.5 h. Before recording, each slice was transferred to a recording chamber continuously perfused with 95% OProteins were extracted from hippocampal tissues and quantified according to our previously described method . After SLevels of Aβ42 in hippocampal tissues and the concentrations of IL-1β and TNF-α in the lung, blood and hippocampal tissues were measured according to the manufacturer’s instructions . Approximately 0.04 g of tissue was homogenized with 400 μL 0.9% NaCl on ice. The homogenates were centrifuged for 20 min at 3000 rpm at 4 °C. The supernatant was then transferred to a new tube for analysis according to the ELISA reagent kit.2.5 treatment and control/vehicle groups, and two-way ANOVA followed by Student’s t-test for rescue experiments. Differences in all tests were considered significant when P < 0.05.The data are presented as the means ± S.E. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by LSD’s post hoc analysis for PMAdditional file 1: Figure S1.2.5 exposure. Figure S2. BACE1 inhibition rescues morphological alterations of synapse. Figure S3. miR-574-5p overexpression recovers morphological alterations of synapse. Figure S4. A dual-luciferase analysis of NF-κB reporter activity was detected in HEK293T cells in response to PM2.5 stimulation. Table S1. Contents of PAHs, inorganic ions, carbon and elements in PM2.5 samples. (DOC 1544 kb)TEM observation of morphological alterations of synapse following PM


Changes in global and regional wall motion between rest and stress are important prognostic markers, uncommonly observed with vasodilator stress radionuclide myocardial perfusion imaging (MPI). Cardiac MRI (CMR) with tagging has the potential to more precisely quantify normal and abnormal ventricular wall motion responses. We herein report CMR findings in 5 very low-likelihood patients for CAD undergoing rest/regadenoson stress with tagging.Five females (54 ± 10 yrs) were consented to participate in this Saint Luke's IRB approved research protocol. All had a prior normal rest/regadenoson stress Rubidium-82 MPI , rest and peak stress wall motion, and coronary calcium score of 0), within 60 days of participation. CMR images were acquired on a 1.5 Tesla Signa HDxt  and 8 channel coil during a single visit with rest imaging preceding stress. Late Gadolinium Enhancement (LGE) images were acquired last to determine if infarcted tissue contributed to any local changes in ventricular motion.An FRGE-based SPAMM sequence was acquired along the short axis of the left ventricle (LV) at basal, mid, and apical locations at both rest and stress in place of MPI. Typical imaging parameters included a 256 × 160 matrix, 20 phases, a flip angle of 5° and views per segment adjusted to heartrate. Stress imaging began at the peak heart rate response, approximately 30 seconds after manual administration of regadenoson (0.4 mg) and saline flush. An average increase of 34 ± 9 BPM was observed.Tag line intersections were identified and recorded at both end-diastole (ED) and end systole (ES) with ImageJ (NIH) software. The angle of twist was defined as the angular change of a ray projected between the identified tag intersection and the center of the LV cavity from ED to ES, and calculated for each slice location at both rest and stress. A two-sided students t-test with paired data was used to determine significance (a = 0.05).p = 0.000); mid-ventricular 1.7 ± 1.8° v 0.3 ± 2.9° ; and apical 8.4 ± 4.0° v 6.1 ± 5.2 ° . Rest images were in agreement with previously reported values (4). Significant differences were observed between all combination of slice locations at both rest and stress, within the slice location at the base, and a trend towards the apex  or Counter clock-wise (CCW) average twist at rest and stress for each slice were as follows: basal 5.2 ± 2.1° v 7.6 ± 2.1° (CW, This study establishes a baseline for regadenoson-induced changes in ventricular twist in low-likelihood for CAD subjects suggesting the usefulness of SPAMM derived indices in evaluating ischemic populations.


Carbonic anhydrases—metalloenzymes that have been found in all domains of life—enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2). Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata. Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi.Adaptation to alternating CO 2 concentrations as CO2 levels range from very low in the atmosphere (0.04%) to high (5% and more) in other niches, including the human body. In fungi, CO2 is sensed via two pathways. The first regulates virulence in pathogenic yeast by direct activation of adenylyl cyclase. The second pathway, although playing a fundamental role in fungal metabolism, is much less understood. Here the transcription factor Cst6/Rca1 controls carbon homeostasis by regulating carbonic anhydrase expression. Upstream signaling in this pathway remains elusive. We identify Sch9 as the kinase controlling Cst6/Rca1 activity in yeast and demonstrate that this pathway is conserved in pathogenic yeast species, which highlights identified key players as potential pharmacological targets. Furthermore, we provide a direct link between adaptation to changing CO2 conditions and lipid/Pkh1/2 signaling in yeast, thus establishing a new signaling cascade central to metabolic adaptation.All living organisms have to cope with alternating CO In the first, HCO3−, which is in equilibrium with membrane-permeable CO2 via action of the catalytic enzyme carbonic anhydrase (CA), directly activates adenylyl cyclases to signal via downstream protein kinase A (PKA)  . This paathogens , 10.2 fixation and HCO3− homeostasis and thus central metabolism. One readout of this pathway is regulation of expression of the β-class fungal CA Nce103p in response to environmental CO2 . Loss of Rca1 in the pathogenic yeasts C. albicans and C. glabrata and loss of the Rca1 orthologue Cst6 in S. cerevisiae led to a common phenotype displaying lack of CA induction in low CO2  to low-CO2 conditions (NCE103air), reaching maximum induction levels at 60 min (23.3 ± 4.9-fold induction)  as the wild type (WT) were considered putative kinase candidates. Of the 155 strains screened, 5 mutants met these criteria: the tpd3Δ, ptp1Δ, bud32Δ, sch9Δ, and ptk2Δ strains (sch9Δ mutant showed the highest upregulation of ScNCE103CO2 expression (3.55 ± 1.55-fold), whereas ScNCE103air expression (6.12 ± 2.98-fold) was similar to that of the WT. This deregulation pattern resulted in a low fold change between ScNCE103air and ScNCE103CO2 of 1.91 ± 0.7-fold  and ScCST6 (B) was measured by qRT-PCR and normalized to expression at time point 0 in 5% CO2. ScNCE103 mRNA levels increase significantly in air; the highest expression was measured after 60 min. In contrast, ScCST6 expression is independent of CO2 levels. The graphs show means ± standard deviations. Significance against expression in 5% CO2 was defined as P ≤ 0.05 (*) and P ≤ 0.01 (**). n = 3. Download FIG S1, PDF file, 0.1 MB.COCopyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02211-16.2TABLE S1 TABLE S1, PDF file, 0.2 MB.Strains, plasmids, and primers used in the study. Download Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02211-16.3TABLE S2 NCE103 expression and standard deviation (SD) of S. cerevisiae kinase/phosphatase mutants in a changing CO2 environment. Download TABLE S2, PDF file, 0.2 MB.Mean ScCopyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02211-16.4FIG S2 S. cerevisiae wild-type and deletion mutant strains were grown in 5% CO2 to exponential phase and were either maintained at 5% CO2 or transferred to air for 60 min. RNA was extracted, and ScNCE103 expression was analyzed by qRT-PCR. Data were normalized to WT expression in 5% CO2. Putative Cst6 kinase deletion mutants were identified according to elevated ScNCE103 expression in 5% CO2 compared to the WT. Download FIG S2, PDF file, 0.04 MB.Workflow of mutant library screening for Cst6 kinase identification. Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02211-16.5TABLE S3 TABLE S3, PDF file, 0.1 MB.Kinase candidate genes identified in mutant library screening. Download Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. cerevisiae CA, we measured protein levels in the sch9Δ mutant cultivated under different CO2 conditions. In contrast to the WT, Nce103 levels in the sch9Δ strain were elevated regardless of the strain being exposed to low or high CO2   . The perifferent , the GFPTo demonstrate physical interaction between Cst6 and Sch9, immunoprecipitation experiments were performed. Endogenous ScCst6 was precipitated from WT lysate using a specific anti-Cst6 antibody. Immunocomplexes were bound to Sepharose beads and incubated with recombinantly expressed Sch9-His. After extensive washing, Western blot analysis of bound proteins was performed . Beads wS. cerevisiae under control of the inducible ScGAL1 promoter. Sch9 expression was induced by shifting the cells from glucose- to galactose-containing medium. S. cerevisiae-overexpressing cells were cultivated in 5% CO2, assuming that the kinase should be most active under this condition. ScSch9-His was purified from cell lysate via immunoprecipitation. Successful purification was followed by Western blot analysis resulting in a strong single band at ~120 kDa in the bead-bound fraction  and subjected to 4 to 20% SDS-PAGE. Total protein was Coomassie stained, corresponding bands were excised, and trypsin-LysC, AspN, LysargiNase, or GluC digestion was performed to achieve high sequence coverage. In addition, an in-solution digestion with trypsin-LysC and GluC was done to also detect peptides of extended length. Phosphopeptides were enriched using TiO2 and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results clearly showed that multiple sites of the Cst6 protein can be subject of phosphorylation. In the WT, we detected 19 different phosphorylation sites in at least two independent experiments -X-S (CST6 into the cst6Δ mutant to prove influence on ScNCE103 expression. The cst6Δ mutant reverted with WT CST6 showed ScNCE103 expression comparable to that of the WT (NCE103CO2 (2.73 ± 0.43-fold), while ScNCE103air (6.52 ± 2.12-fold) expression was unaltered. Interestingly, the pattern of expression of the cst6Δ + CST6S266A strain corresponds to the expression pattern of the sch9Δ mutant. This confirms our hypothesis and emphasizes S266 to be phosphorylated in 5% CO2 in order to inhibit NCE103 expression. Phosphomimetic mutation of CST6 (CST6S266D) led to slightly decreased ScNCE103air expression in comparison to WT CST6, but differences were not statistically significant. This might point to alternative regulation mechanisms regulating NCE103 expression, especially under the air condition.S266 is the only residue within ScR/K)-X-S . Hence, f the WT . Phospho2-dependent regulation mediated via Cst6/Rca1 is conserved in some yeast species  on CgNCE103 expression under changing CO2 conditions. Expression of CgNCE103 in 5% CO2 and air after 60 min was quantified by qRT-PCR according to the previously described protocol for S. cerevisiae  compared to the control. Noteworthy and in contrast to the pattern found in S. cerevisiae, CgNCE103air expression was also increased. However, despite these differences, Sch9 clearly affects NCE103 regulation in C. glabrata.Our findings and previous work suggest that CO species , 18. Duerevisiae . The C. SCH9 on C. albicans NCE103 (CaNCE103) using similar protocols , similar to the effects of SCH9 deletion in S. cerevisiae and C. glabrata. Remarkably, also CaNCE103air levels were elevated, which coincides with what we found for C. glabrata, but not for S. cerevisiae. Taken together, these findings clearly indicate a conserved mechanism of CO2 adaptation in pathogenic and nonpathogenic yeast species and emphasize Sch9 is key to CO2 sensing in all three species.We also verified the influence of rotocols . The C. t strain  showed sS. cerevisiae Sch9 is regulated by target-of-rapamycin (US approved name [USAN] sirolimus)-complex 1 (TORC1) and homologues of the mammalian 3-phosphoinositide-dependent protein kinase (mPDK1), Pkh1 and Pkh2, which phosphorylate Sch9 at specific sites  to be phosphorylated in 5% CO2 . Notably, the tor1Δ mutant had been included in our initial qRT-PCR screening and showed slightly but not significantly increased CA expression in high CO2   . Howeverpression , probablrevisiae . As a corevisiae , we usedrevisiae  to inves10.1128/mBio.02211-16.6TABLE S4 NCE103 expression and standard deviation (SD) of S. cerevisiae SCH9 regulator mutants and S. cerevisiae under rapamycin (USAN sirolimus) treatment. Download TABLE S4, PDF file, 0.1 MB.Mean ScCopyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the NCE103 expression of strains with site-specific mutations of known Sch9 phosphorylation sites addressed by TORC1 and Pkh1/2. As a control, the sch9Δ mutant reverted with WT SCH9 showed NCE103 expression levels in 5% CO2 and air comparable to those of the WT strain. Surprisingly, also phosphoablative mutation of all 6 TORC1 sites , referred to as SCH96A, resulted in WT-like NCE103 expression levels (NCE103CO2 expression (2.7 ± 0.59), while ScNCE103air expression was not changed. This is comparable with the deregulation pattern of the sch9Δ mutant. Additional S/T-to-A mutation of the 6 TORC1 sites within SCH9  showed no additive effect on ScNCE103 expression -X-S -X-S .NCE103 in S. cerevisiae, C. glabrata, and C. albicans is conserved. Deletion of ScSCH9, CgSCH9, and CaSCH9 affected NCE103 expression in comparable ways as they lead to a significant upregulation of NCE103CO2  and C. glabrata Rca1 (S387). We now show proof that S266, but not other candidate residues, is phosphorylated in CST6 under elevated CO2 and thus provide a molecular explanation for the conservation of CO2 signaling in yeast  and hypoxia (<10%) but not under either condition alone  of proteins. It is conserved in minearum . In mammminearum , althougminearum , which aminearum , 42. HIFgulation . Indeed,CE103CO2 , but theCE103CO2 . Insteadr 5% CO2 . It shoua Pkh1/2 . An inflon alone .TPD3 and deletion of SCH9 result in comparable phenotypes  were cultivated at 37°C as duplicates in 5% CO2 and transferred to air for 10, 30, 60, 90, 120, or 180 min to induce ScNCE103 expression. Pellets were harvested, and RNA was extracted using a hot phenol-chloroform method . Exponential cultures were transferred to air for 60 min to induce the highest ScNCE103 expression. ScNCE103 expression of mutants was normalized to WT expression in 5% CO2  and cloned into E. coli expression vector p41. NCE103 and SCH9 were expressed in E. coli strain BL21(DE3) . Bacteria were grown in LB medium  with 50 µg/ml ampicillin at 37°C. ScNCE103 expression was induced with 1 mM IPTG  at an optical density at 600 nm (OD600) of 0.2 and cultivated overnight until harvesting. Expression of ScSCH9 was induced at an OD600 of 0.8 for 4 h at room temperature. ScCST6 expression was performed using E. coli Rosetta. Cells were cultivated in LB medium with 50 µg/ml ampicillin and 34 µg/ml chloramphenicol at 37°C until they reached an OD600 of 0.4. Expression was induced with 1 mM IPTG for 4 h at 37°C. Bacteria were harvested, resuspended in purification buffer , and disrupted by freezing and thawing for a total of 3 rounds. Recombinant proteins were purified by IMAC using Ni-nitrilotriacetic acid (NTA) Sepharose . His-tagged proteins were eluted with purification buffer containing 250 mM imidazole. For further purification of ScCst6-His, size exclusion chromatography using an Äkta with a Sephadex 200 GL column  was performed. Protein purity was determined by silver staining, and quantity was measured with a bicinchoninic acid (BCA) assay. At least 1 mg purified protein was used to produce specific rabbit antibodies against ScCst6-His and ScNce103-His (Thermo Fisher Scientific).S. cerevisiae exponential cultures was cultivated in 5% CO2 or transferred to air for 90 min. Cells were harvested, resuspended in 500 µl yeast lysis buffer , and lysed with glass beads  five times for 1 min each with incubation on ice between lyses. Lysates were centrifuged , and total protein was measured using a BCA protein assay kit (Thermo Fisher Scientific). After addition of 5× SDS loading dye and heating at 95°C for 5 min, samples  were subjected to 4 to 20% SDS-PAGE. Transfer to polyvinylidene difluoride (PVDF) membrane was performed using the Bio-Rad Wet/Tank blotting system for 1 h. Three percent nonfat dry milk in Tris-buffered saline (TBS) was used for membrane blocking and antibody binding. Membranes were incubated with custom-made rabbit anti-ScNce103  and mouse anti-β-actin  overnight at 4°C with gentle rotation. After incubation with fluorophore-labeled secondary antibodies  for 2 h at 4°C, signals were detected with the FluorChem Q Imager . Quantitative analysis was done using the AlphaView software (ProteinSimple).For detection of ScNce103 levels, 10 ml of S. cerevisiae WT, cst6Δ, and sch9Δ strains were transformed with pRS316 plasmid, containing GFP under control of the ScNCE103 promoter using the lithium-acetate (LiAc) method according to Gietz et al.  at the time point of the switch from CO2 to air and after 60 and 120 min of incubation. GFP fluorescence of 50 cells per time point was measured using ZEN software (Zeiss), and mean fluorescence was calculated.The z et al. . In brieCST6-His and ScSCH9-His in S. cerevisiae was achieved using the ScGAL1 promoter. Yeast cells were cultivated in minimal medium, supplemented with histidine, methionine, and leucine at 30°C overnight. Overexpression of ScCST6 and ScSCH9 was induced by a shift from minimal medium to YEP medium  containing 2% raffinose and after overnight culture to YEP with 2% galactose for an additional 6 h. Cells were harvested, resuspended in yeast lysis buffer, and lysed with glass beads as described. Cell extracts were tested by Western blotting for overexpression.Overexpression of ScS. cerevisiae WT or ScSCH9-His-overexpressing cultures (50 ml) were harvested, resuspended in 500 µl immunoprecipitation (IP) buffer , and lysed with glass beads as described. Extracts were incubated with either custom-made rabbit anti-Cst6, saturated with 5 µg 6×His tag peptide , or anti-6×His antibody  overnight at 4°C. Fifty microliters of protein-G Sepharose beads  was washed with IP wash buffer (IP buffer plus 500 mM NaCl) and incubated with immunocomplexes for 1 h at 4°C. Beads were centrifuged at 2,500 rpm for 2 min, supernatant was taken off with a syringe and a 25-gauge needle, and beads were resuspended in wash buffer and incubated 1 min with shaking. This washing procedure was repeated four times. For coimmunoprecipitation (co-IP), beads were furthermore incubated with 20 µg recombinant ScSch9-His lysate for 3 h at 4°C. After extensive washing, beads were boiled in SDS sample buffer, subjected to 4 to 20% SDS-PAGE, and analyzed by Western blotting. Rabbit anti-ScCst6  was used for detection of endogenous ScCst6 and fluorescein isothiocyanate (FITC)-labeled mouse anti-6×His  for detection of 6×His-tagged ScSch9.Exponential S. cerevisiae ScSCH9-His-overexpressing culture. After washing of bead-bound immunocomplexes with IP wash buffer, an additional washing step with 1× kinase buffer  was performed. One microgram of bacterially expressed and purified ScCst6-His was added to immunocomplexes in 50 µl kinase buffer. After addition of 100 µM ATP and 25 µCi [γ-32P]ATP , reaction mixtures were incubated at 30°C for 30 min. The kinase reaction was stopped by heating at 95°C for 5 min in 4× SDS loading dye, and samples were subjected to 4 to 20% SDS-PAGE. Gels were dried, and radioactive incorporation was detected by autoradiography.ScSch9-His was immunoprecipitated from 2-dependent phosphorylation in ScCst6, ScCST6-overexpressing S. cerevisiae cultures were harvested, and ScCst6-His was purified using IMAC. For mass spectrometry analysis, in-gel digestion with trypsin-LysC, AspN, and GluC  or LysargiNase , and the remaining peptides were purified with C18 minicolumns . Dried peptides were solubilized in MS buffer (0.05% trifluoroacetic acid in 2% acetonitrile–98% H2O) and applied to LC-MS/MS analysis, carried out on an Ultimate 3000 nano-rapid-separation liquid chromatography (nano-RSLC) system coupled to a QExactive Plus mass spectrometer (both from Thermo Fisher Scientific). Thermo raw files were processed via the Proteome Discoverer (PD) v1.4.0.288. Tandem mass spectra were searched against the NCBI protein database of S. cerevisiae using the algorithms of Mascot v2.4.1 , Sequest HT, and MS Amanda. For further information, see To detect COargiNase  and in-s10.1128/mBio.02211-16.7TEXT S1 TEXT S1, PDF file, 0.1 MB.Supplemental methods. Download Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the CST6 (GeneArt Gene Synthesis) were inserted in the pRS316 plasmid using PacI/AscI restriction sites. The ScCST6 promoter was cloned in pRS316 using SacI/PacI. After transformation of the S. cerevisiae cst6Δ mutant, ScNCE103 expression in 5% and 0.04% CO2 was measured as described and normalized to WT expression in 5% CO2. For construction of ScSCH9 with S/T-to-A mutation of all 6 TORC1 sites (SCH96A), the sequence of SCH9T570A, 6A was used as the template. A570 was remutated to T570 using QuikChange II site-directed mutagenesis kit (Agilent Technologies) with mutation-specific primers. The plasmid with the corrected sequence was checked via sequencing and used for transformation of the sch9Δ mutant.Phosphoablative (S266A) and phosphomimetic (S266D) sequences of ScSCH9 was achieved by replacement of the CgSCH9 open reading frame (ORF) with a NAT1 cassette, which mediates resistance against nourseothricin. NAT1 was amplified by PCR with EcoRI and SacI restriction sites and cloned into pBluescript. A homologous region upstream of the CgSCH9 ORF with a length of 1,000 bp was amplified by colony PCR and cloned into pBluescript using KpnI/XhoI. The same was done for a 1,000-bp fragment located downstream of CgSCH9 ORF using restriction sites SacII and SacI. The whole construct, the NAT1 cassette flanked by homologous regions, was restricted with KpnI/SacI and purified in order to transform C. glabrata. AFG1, a strain lacking LIG4, was used for transformation because of its increased efficiency of homologous recombination  Insertion of NAT1 and deletion of CgSCH9 were analyzed by colony PCR of the untransformed parental strain (AFG1) and sch9 mutant. For amplification of NAT1, internal primers G1-CgSCH9 and X2-NAT1 were used for amplification of CgSCH9 G1-CgSCH9 and I2-CgSCH9. The strain AFG1 showed no signal for NAT1 but did show signal for SCH9. In contrast, the sch9 deletion mutant exhibited a PCR product for NAT1 but not for SCH9. As a marker, the GeneRuler 1-kb DNA ladder (Thermo Fisher Scientific) was used in lane 1. (B) For verification of the correct position of NAT1 cassette insertion, PCR with external primers G1-CgSCH9 and G4-CgSCH9 was used; these primers bind outside the inserted cassette. If the SCH9 ORF is present, a PCR product of 4,385 bp occurs. If the SCH9 ORF is replaced by NAT1, a shift to 3,463 bp is visible. As a marker, the GeneRuler 1-kb DNA ladder (Thermo Fisher Scientific) was used in lane 1. Download FIG S3, PDF file, 0.2 MB.Verification of Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02211-16.9TEXT S2 TEXT S2, PDF file, 0.1 MB.Supplemental references. Download Copyright © 2017 Pohlers et al.2017Pohlers et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the NCE103 and CaNCE103 expression was performed according to the protocol for ScNCE103 expression described in the first section. As housekeeping genes, CgACT1 for measurement of CgNCE103 and CaACT1 for measurement of CaNCE103 were used.Measurement of CgP < 0.05) was calculated using a two-sided t test for unpaired samples.Data are presented as arithmetic means ± standard deviation, and statistical significance (


Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5–TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains. TRAF6 is a RING E3 ligase that builds Lys63-linked ubiquitin chains. Here, the authors present the crystal structure of TRAF6 bound to the Ubc13~Ub conjugate, which, together with biochemical assays, reveals the role of the zinc finger domains and why RING dimerisation is required for TRAF6 activity. This is because ubiquitin chains that are synthesised by TRAF6 serve as a platform for the activation of downstream kinases such as TAK1 and Akt5.Many immune signalling pathways rely on the synthesis of ubiquitin chains. Non-degradative ubiquitin chains have important roles in determining the strength, duration and type of inflammatory response by functioning as molecular glue to stabilise signalling complexes. The extent of ubiquitin chain synthesis following cytokine engagement determines whether downstream effector molecules are recruited and activated. One E3 ligase with a critical role in many immune signalling pathways is TNF receptor-associated factor 6 (TRAF6). TRAF6 was initially identified because of its requirement for interleukin1-receptor (IL1-R)-mediated activation of NF-κB6, and poor prognosis for head and neck cancers7. Overexpression of TRAF6 predicts a poor response to chemotherapy and radiotherapy for colorectal cancer patients, with molecular studies suggesting this is because TRAF6 regulates mitochondrial translocation of p538. It has also been suggested that chronic activation of TRAF6 is associated with aberrant splicing in haemopoietic cells in myelodysplastic syndromes9.Befitting its central role in signalling, disruption of TRAF6 function has been linked to cancer and inflammatory disorders. Amplification of TRAF6 is associated with lung carcinoma10. These domains are responsible for trimerisation and receptor binding11. At their N-termini TRAF2–7 have a RING domain, followed by a series of zinc finger (ZF) domains10  E2 complex following engagement of receptors15. For other TRAFs, the E2 partners are still to be defined, and in fact TRAF2 contains an insertion in its RING domain that appears to abrogate E2 binding16.TRAF6 belongs to the TRAF family of proteins (TRAF1–7), which are defined by the presence of a TRAF-C/MATH domain and a TRAF-N or coiled coil (CC) domains10 Fig. . The RIN18. Recent studies have shown RING E3s achieve this by binding the E2 conjugated ubiquitin so that the flexible linker between ubiquitin and the E2 is bound to a groove on the E224. While some RING-ubiquitin contacts are conserved14, the additional interactions that prime the conjugate for catalysis differ25. Like TRAF6, the activity of many RING domains depends on their dimerisation because one RING domain binds the E2, while residues from the C terminus of the other RING protomer make essential contacts with ubiquitin2126. However, TRAF RING domains do not contain a structural equivalent to the C-terminal tail that is essential for many dimeric RING domains21. Therefore, the mechanism by which TRAFs stabilise the E2~Ub conjugate in the active (closed) conformation remains uncertain. Furthermore, many receptor complexes contain at least two different TRAF proteins. This raises the possibility that TRAF heterodimers could be functional—as observed for other RING E3 ligases such as MDM2/MDMX and BRCA1/BARD128.The RING domain not only recruits the E2~ubiquitin (E2~Ub) conjugate, but also enhances the rate of ubiquitin transfer from the E2 to the substrate29. To identify the essential features that regulate ubiquitin chain assembly by TRAF6 we have determined structures of the RING and ZF domains of TRAF6 bound to the Ubc13~Ub conjugate in two crystal forms at 3.4 and 3.9 Å. Both structures show that the Ubc13~Ub conjugate contacts both protomers of the TRAF6 dimer. Importantly, ZF1 and the helix that connects it to the RING domain contact ubiquitin and enhance the rate of ubiquitin transfer. The structure and biochemical assays explain why RING dimerisation is required for assembly of ubiquitin chains by TRAF6, and account for the importance of the ZF domains for activity. Furthermore, we also show that the conserved RING dimer interface facilitates formation of TRAF heterodimers that are functional E3 ubiquitin ligases.In TRAF6, the RING together with ZF1 is required for E3 ligase activity12 and we initially used a comparable human construct, referred to here as RZ3 (HsRZ3). Using GST-pulldown assays and analytical size-exclusion chromatography, we established that HsRZ3 preferentially binds to the Ubc13~Ub conjugate . The sequence of the RING domain and first ZF are highly conserved between DrRZ3 and HsRZ3  has been reported previouslyins Fig. . Additioate Fig.  that couate Fig. .Table 1D3 dimer bound to two Ubc13~Ub conjugate molecules  has a main-chain RMSD of 1.0 Å. The contacts between the conjugate and TRAF6 are comparable on both sides of the dimer, but the structure is asymmetric because there is no density for ZF2 and ZF3 in one molecule of TRAF6. There was no evidence of any proteolysis, and a large solvent channel occupies the space where ZF2 and ZF3 are expected, suggesting that the two missing ZFs are highly mobile in the absence of any crystal contacts. We therefore expressed and purified a truncated construct that contained the RING and just ZF1 of D. rerio TRAF6 (denoted DrRZ1). RZ1 and RZ3 had a comparable level of activity   Fig. . In addi26 and TRIM25-Ubc13~Ub24 complexes and is compatible with the Ubc13~Ub/Mms2 complex31 . Single turnover assays where wild-type Ubc13 was first charged with Cy3-labelled ubiquitin containing a K63R mutation (*UbK63R) were used to assess activity. The purified conjugate was incubated with Uev1A and ubiquitin-D77 in the presence or absence of TRAF6 variants. Fluorescence imaging was used to monitor and quantify the appearance of diubiquitin (*UbK63R-Ub) over time  mutant appeared comparable to the no E3 control  also resulted in a substantial decrease in conjugate discharge, while mutation of Ser129 on the next turn of the linker-helix, and Glu144 did not impede activity. Similar results were obtained with multi-turnover chain-building assays  mutant of HsRZ3 that does not interact with Ubc13 because the E2 binding interface is disrupted12. We hypothesised that each mutant would be inactive alone, but if the two TRAF6 RING species associate, the Ubc13-binding and ubiquitin-binding sites from different mutants could co-operate to form a complete binding site for the E2~Ub conjugate.To further investigate the importance of ZF1 and RING dimerisation, we assessed the ability of inactive TRAF6 mutants to recover activity when mixed. In addition to the ArgZF1) activity was considerably increased  revealed an extended conserved region that comprises the RING dimer interface and the ubiquitin binding site on the linker-helix Fig. . The hig3 from TRAF5 and analysed its ability to interact with TRAF6 using size-exclusion chromatography  activity was not recovered in an equivalent mixing experiment. This is probably because, like the equivalent TRAF6 mutant, the R101A mutation destabilises dimer formation  was mixed with the TRAF6 ArgZF1 activity was not recovered to the same extent as observed when WT TRAF5 was included.TRAF5 RZays Fig. , right. Arg Fig. . We reasTogether these data indicate that the linker-helix and ZF1 in TRAF5 are important for activity. In addition, these results provide evidence that TRAF RING heterodimers form and promote ubiquitin transfer.TRAF proteins are adaptor molecules that have a trimeric ‘TRAF’ domain, which recognises the intracellular tails of membrane-embedded cytokine receptors, and also a dimeric RING domain that assembles ubiquitin chains in response to cytokine signalling. Here we account for the importance of TRAF RING dimers and show that the ZF1 domain of TRAF6 interacts with ubiquitin to stabilise the closed conformation of the Ubc13~Ub conjugate. Thus, ZF1 in TRAF6 plays a critical role in accelerating K63 chain synthesis by Ubc13-Uev1A. We also demonstrate that the RING domains of TRAF5 and TRAF6 form a stable heterodimer that is an active E3 ligase, supporting the notion that TRAF heterodimers can regulate ubiquitylation.26. However, even though the RING-E2~Ub interactions are conserved . In this way, basic residues C-terminal to the TRAF6 RING domain appear to fulfil the role of the well-established locking aromatic residue in other dimeric RINGs, such as RNF4 and ML-IAP26.The conformation of the Ubc13~Ub conjugate bound to TRAF6 resembles that reported for the RNF4 and TRIM25 complexes with Ubc13~Ub, and is compatible with the assembly of K63-linked ubiquitin chains by TRAF6ved Fig. , the RINved Fig.  so that 24. In fact, the RING domains of TRIM25 and TRAF6 overlay remarkably well, and this similarity extends beyond the RING domain to include the TRAF6 linker-helix and the α-helix that links the TRIM25 RING to its B-box domain. A helix C-terminal to the RING domain is also found in the structures of other TRIMs, and in RNF125 and LNX2 , Toll-, NOD- and RIG-I-like receptors36. Furthermore, the coiled coil/TRAF-C domains of TRAF1 and TRAF2 are known to form a heterotrimer comprising one copy of TRAF1 and two copies of TRAF237. TRAF1 does not possess a RING domain and these 1:2 TRAF1/2 heterodimers likely favour TRAF2 RING dimerisation. However, this raises the potential that other TRAF heterotrimers might form. Irrespective of whether heterotrimers form, there remains a crucial symmetry mismatch in that trimeric TRAFs interact with trimeric receptors, but RING domain dimers assemble ubiquitin chains. This symmetry mismatch is likely to be exacerbated in cells because many cytokine receptors signal via receptor clustering38, where several TRAF trimers would be expected to come into close proximity. As a result, even if TRAF homotrimers form there is considerable potential for TRAF RING heterodimers to bridge trimeric TRAF complexes at the cell membrane. For example, in the simplest case it would be anticipated that if TRAF homotrimers form and a RING dimer is favoured, one RING domain would be free to interact with the RING of a neighbouring trimer as in Fig. 39.TRAF RING heterodimers have not been reported before, but multiple TRAFs are frequently found associated with signalling complexesRegulation of TRAF proteins is complex, and much work remains to establish how trimeric TRAFs couple with dimeric RING domains to transduce signals. However, our data not only account for the importance of TRAF RING dimers, but also provide a mechanism by which clustering of cytokine receptors could elicit differential ubiquitin ligase activity—and the assembly of distinct ubiquitin chains from homo- and heterodimeric TRAF complexes. The nature and extent to which such signalling occurs in a cellular setting will depend on identification of the E2s bound by other TRAFs, and thus the ubiquitin chains assembled, as well as systematic analysis of TRAF RING heterodimer formation. Despite these uncertainties, heterodimeric TRAF RING domains bridging a network of trimeric TRAF proteins is an attractive mechanism to account for the complexity of signals elicited downstream of many cytokine receptors.Escherichia coli BL21 (DE3) (Novagen). Ubiquitin and ubiquitin variants were expressed as untagged proteins40. After overnight expression at 18 °C, the E. coli cells were harvested and re-suspended in 50 mM ammonium acetate pH 4.5 containing 1 mM EDTA. After sonication and clarification, the supernatant was injected on a 5 mL HiTrap SP column (GE) and eluted with a 50 mL linear gradient from 0 to 1 M NaCl, in 50 mM ammonium acetate pH 4.5, 1 mM EDTA. The peak fractions were pooled and injected on a HiLoad Superdex 75 16/600 column (GE) equilibrated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl.All proteins were expressed in 6 tag41. After lysis in 20 mM Tris-HCl pH 8.5, 100 mM NaCl, 5 mM imidazole, the supernatant was loaded on a 5 mL HisTrap column. A linear 50 mL gradient from 5 to 250 mM imidazole was applied. The fractions containing E1 were injected on a HiLoad Superdex 200 16/600 column (GE) equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl.The human E1 protein was expressed with an N-terminal HisHomo sapiens TRAF6 constructs  were cloned into pET21d encoding a C-terminal His tag, while Danio rerio TRAF6 constructs  were in pET24b. All TRAF6 constructs were induced at an O.D.600nm of 0.6 with 0.2 mM IPTG and 0.1 mM ZnCl2 and incubated at 28 °C for 4 h. Cells were sonicated in 50 mM Tris-HCl pH 7.5 and 350 mM NaCl, before being clarified and loaded into a 5 mL HisTrap FF column (GE). After elution, the peak fractions were loaded into a 10/300 Superdex 200 column (GE). TRAF5 was cloned into the pGEX6P3 vector containing an N-terminal GST tag. Protein expression was induced as for TRAF6, but was incubated at 18 °C overnight. Cells were sonicated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and the lysate was clarified and bound to glutathione sepharose resin for 1 h. The GST tag was cleaved by overnight incubation with 3C protease. The soluble fraction was loaded on a 5 mL HiTrap Q column, eluted with a linear 1 M NaCl gradient, and cleaned up by size-exclusion chromatography as for TRAF6. Ubc13 and Uev1A were expressed as N-terminal GST-fusion proteins and purified using glutathione affinity, proteolysis of the GST tag, and size-exclusion chromatography.2, 10 mM phosphocreatine (Sigma) and 0.5 U mL−1 creatine phosphokinase (Sigma)42. The reaction was incubated at 37 °C for 16 h. The resulting Ubc13~Ub conjugate was purified using a 26/600 Superdex 75 column (GE) equilibrated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl.For stable Ubc13~Ub conjugate formation, a mutated form of Ubc13 was used. This variant included C87K, K92T and K94Q mutations to allow formation of a stable isopeptide-linked conjugate (Ubc13~Ub) while disrupting formation of other species as a result of non-specific transfer from the E1. For production of isopeptide-linked conjugates, 40 µM of pure Ubc13  was mixed with 80 µM wild-type ubiquitin, 1 μM E1, 1 mM ATP, and ATP cycling buffer containing 25 mM Tris-HCl pH 9.0, 5 mM MgCl3 to Ubc13 and Ubc13~Ub conjugates, GST-fused Uev1A was mixed with TRAF6, Ubc13 and Ubc13~Ub conjugates. The incubations were performed in PBS containing 0.2% TWEEN 20 and 1 mM DTT, at 4 °C for 1 h. Subsequently, the resin was washed three times with PBS with 0.2% TWEEN 20 before being mixed with SDS loading dye and separated by SDS PAGE. The gels were stained with Coomassie Blue.To assess binding of TRAF6 RZDrRZ3 and Ubc13~Ub at 250 µM; HsRZ3 and Ubc13/Ubc13~Ub at 50 µM), over a 10/300 Superdex 200 (GE) equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl at 0.5 mL min−1. The recovered fractions were analysed by SDS PAGE. For the analysis of TRAF6 proteins, each protein was injected over a 10/300 Superdex 200 Increase at 40 µM. For determination of the oligomeric status of TRAF6, multiangle light scattering  coupled to a Superdex 200 10/300 GL (GE) was used. Each TRAF5 protein was analysed at 40 µM using a 10/300 Superdex 75 Increase column.Analytical size-exclusion chromatography (SEC) was performed by injecting 200 µL of purified protein, or mixtures of proteins (D. rerio TRAF6 (RZ1 and RZ3) were mixed with isopeptide-linked Ubc13~Ub conjugate for 30 min on ice before being separated using size-exclusion chromatography over a 10/300 Superdex 200 column (GE). The fractions corresponding to the TRAF6-Ubc13~Ub complex were pooled, concentrated to ~2 mg mL−1, and the PACT and JCSG+ crystal screens (Molecular Dimensions) were set up with a 1:1 ratio of protein to precipitant in sitting well plates (Swissci). Trays containing DrRZ3 and DrRZ1 resulted in crystals within 12 h in many conditions, which diffracted to ~8 Å. Crystals of DrRZ3 were grown in 100–200 mM Na/K tartrate, 11–15% PEG 3350 and 100 mM Bis-Tris propane pH 7.5; DrRZ1 crystals were produced in 0.05–0.3 mM sodium citrate, 100 mM Bis-Tris propane, and 17–23% PEG 3350. Fine tuning of the crystal conditions, changing drop ratios to 2:1 (protein: well solution), and slowing down the growth of crystals using microbatch resulted in DrRZ1 and DrRZ3 crystals that diffracted to 3.4 and 3.9 Å, respectively, on MX2 at the Australian Synchrotron43.Purified 44. Data were merged with Aimless45, and the structures were solved with Phaser-MR46 using the complex of Ubc13-TRAF6 RING domain (PDB: 3HCT12) followed by ubiquitin (PDB: 1UBQ47). For one of the DrRZ3 molecules, two of the three ZF could be automatically placed, while the third ZF had to be manually built using Coot48. Structures were refined using Refmac49, while Coot was used to iteratively build missing residues, including the C-terminal tail of ubiquitin. For DrRZ3, 92.1% of residues are in favoured regions, 6.6% in allowed regions and 1.3% are outliers. For DrRZ1, 96.3% of residues are in favoured regions, 3.5% in allowed regions and 0.2% are outliers.Data sets were collected for each crystal form, and processed and scaled using XDS−1. Thioester-linked conjugate containing fluorescent ubiquitin was produced and purified using similar conditions as before,50 except Ubc13 with a K97R mutation was mixed with ubiquitin, E1 and ATP cycling buffer at pH 7.5. The thioester charging reaction was incubated at 37 °C for 20 min before the conjugate was purified using a 10/300 Superdex 75 column (GE) equilibrated with 20 mM MES pH 6.5, 150 mM NaCl. The fractions containing conjugate were immediately flash frozen in liquid nitrogen.Fluorescently tagged ubiquitin was produced by introducing a Cys residue before the beginning of the ubiquitin sequence, resulting in Met-Cys-ubiquitin. To label the free Cys purified ubiquitin was reduced with 1 mM TCEP and mixed with 5 µL of Cy3 for 1 h. The sample was desalted  to remove excess dye and incubated in the dark at room temperature for ~16 h to allow unconjugated ubiquitin to form disulphide-linked dimers. Subsequently, the mixture was separated on a 16/600 HiLoad Superdex 75 column (GE) and the second peak corresponding to monomeric ubiquitin was pooled and concentrated to ~2 mg mL2 and 0.1 M NaCl, and incubated at 37 ºC. For fluorescently labelled multi-turnover assays, the reaction mixture was spiked with 10% fluorescent ubiquitin. At the indicated times, the reactions were quenched by adding SDS-PAGE loading buffer containing β-mercaptoethanol. Proteins were separated using 16% SDS-PAGE, 10–18% gradient SDS-PAGE (homemade) or commercial  gels. Fluorescence was imaged using an Odyssey FC imaging system (LI-COR) at 600 nm with a 2 min exposure. Gels were stained with Coomassie blue.For multi-turnover ubiquitylation assays, 0.1 µM E1, 6 µM Ubc13 and Uev1A, 50 µM ubiquitin, and 4 µM TRAF proteins (wild-type or mutants) were mixed together in a buffer containing 20 mM Tris-HCl pH 7.5, 2 mM DTT, 2 mM ATP, 5 mM MgClK63R conjugate was mixed with 15 µM Uev1A, 50 µM D77 ubiquitin , and 4 µM TRAF proteins and incubated at 20 or 37 °C for the indicated time. The final pH of the discharge reactions was 7.0. Reactions were quenched with non-reducing SDS-PAGE loading dye containing 10 mM N-ethylmaleimide. For quantification of fluorescently labelled conjugate, gels were scanned as for the multi-turnover assays and the intensity of fluorescent bands was quantified using Image Studio Lite (LI-COR).For single-turnover discharge assays, 10 µM purified thioester linked Ubc13~UbThe coordinates and structure factors for TRAF6 RZ3 and RZ1 complexes with Ubc13~Ub have been deposited to the Protein Data Bank under the accession codes 5VO0 and 5VNZ, respectively. All other data are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review file


Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested , this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis. Amyotrophic lateral sclerosis (ALS) is an age-associated neurodegenerative disease characterized by progressive muscle paralysis and death . ApproxiMore than 150 individual mutations have been identified in human Sod1 (hSod1), affecting in different ways the onset and the prognosis of fALS , 7. NeveIn vitro studies have shown that metal coordination and disulfide bond formation are relevant for Sod1 structural stability –15, suggAltogether, our results clearly define a common mechanism of action of mutant Sod1, resulting in cytotoxicity, impaired function, aggregation, and in alterations in intracellular distribution, opening novel territory for the identification of targets for therapeutic intervention in ALS.SOD1 and the mutants A4V, L38V, G93A, and G93C were subcloned from the yeast plasmid YEp351 [SOD1 cDNA  was cloned to the 3′-end of the VN-fragment (VN-SOD1) and upstream of the VC-fragment (SOD1-VC) by PCR, using specific primers including restriction enzyme sites AflII at the 5′ and XhoI at the 3′-end. The primers used were as follows:Forward: 5′-GGGCTTAAGATGGCGACGAAGGCCGTG -3′Reverse: 5′-CCCCTCGAGTTATTGGGCGATCCCAATTACACC -3′VN-SOD1 Forward: 5′-GGGCTTAAGATGGCGACGAAGGCCGTG -3′Reverse: 5′-CCCCTCGAGTTGGGCGATCCCAATTACACCACAAG -3′SOD1-VC Forward: 5′-GGGCTTAAGATGGCGACGAAGGTCGTGTGCG -3′Reverse: 5′-CCCCTCGAGTTATTGGGCGATCCCAATTACACC -3′VN-SOD1 (A4V)Forward: 5′-GGGCTTAAGATGGCGACGAAGGTCGTGTGCG -3′Reverse: 5′-CCCCTCGAGTTGGGCGATCCCAATTACACCACAAG -3′SOD1-VC (A4V)The cDNA sequence of human WT (wild-type) d YEp351 , 19 intod YEp351 . In partPCR fragments were restriction digested and cloned into alpha-synuclein BiFC constructs by replacing the alpha-synuclein insert . All conv/v) fetal bovine serum (FBS) gold and 1% (v/v) penicillin-streptomycin, at 37 °C, and 5% CO2 humidified atmosphere. Transfections were performed by calcium phosphate using equal amounts of plasmids encoding the wild-type (WT) or mutant  hSod1 fused to Venus BiFC system and the JUNQ substrate (mCherry-VHL). To improve the visualization of VHL-mCherry proteins into JUNQ compartments, 48 h transfected H4 cells were incubated with proteasome inhibitor MG132 (10 μM) for 7 h.Human neuroglioma cells (H4) were cultured in Dulbecco’s Modified Eagle Medium , supplemented with 10%  and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Followed by three washing steps with DPBS, cells were stained with Hoechst 33258  (1∶5000 in DPBS) for 5 min and maintained in DPBS for fluorescence microscopy. Fluorescence images were acquired with a Leica DMI 6000B microscope , with a ×40 objective. Scale bars were calculated by using ImageJ software and were included in the figure legends together with the actual magnification.http://rsbweb.nih.gov/ij/). Using the freehand tool, the nucleus and cytosol were selected and the respective intensities were measured. The results reflect the counting of at least 50 cells per condition.Nuclear and cytoplasmic fluorescence intensities were quantified using ImageJ software , and more than five inclusions (≥ 5 inclusions). Results reflect the counting of at least 50 cells per condition.v/v) PFA, for 15 min at RT. After washing with ×1 PBS, cells were permeabilized with 0.1% (v/v) Triton/PBS, for 15 min at RT. After blocking with 3% (w/v) bovine serum albumin (BSA)/PBS for 1 h at RT, cells were incubated for 2 h with primary antibody anti-G3BP 1:200  diluted in blocking solution. Cells were washed with ×1 PBS before incubation with secondary Alexa Fluor antibody , prepared at 1:1000 in blocking solution, for 1 h at RT. Before mounting the coverslips with Mowiol , nuclei were stained with 4′,6-diamidino-2-phenylindole . Immunofluorescence images were acquired with Leica DMI 6000B microscope , using ×63 magnification objective.Forty-eight hours after transfection, cells were fixed on coverslips with 4% (v/v) FBS and 1% (v/v) penicillin-streptomycin at 5% atmospheric CO2, with the incubation unit set to a temperature of 37 °C. Live cells were imaged every 30 min over a time course of 20 h overnight.Images of H4 cells expressing BiFC-tagged hSod1 were recorded by using the Olympus IX81-ZDC microscope system . Cells were maintained in DMEM, , supplemented with 10% (w/v) SDS, 1% NP40 (v/v), 0.5% (w/v) Na-deoxycholate), 2 mM EDTA, and a protease inhibitor cocktail (one tablet/10 mL) . Protein concentration was determined using the Bradford assay , and the gels were loaded with 30 μg protein after denaturation for 5 min at 95 °C in a protein sample buffer  SDS, 0.5% (w/v) bromophenol blue, 4 mM EDTA 20% (v/v) glycerol 10% (v/v) β-mercaptoethanol). Samples were separated on 15% (w/v) SDS-polyacrylamide gels (SDS-PAGE) with a constant voltage of 120 V using Tris-Glycine SDS 0.5% (w/v) running buffer  SDS, pH 8.3) for 75 min. Protein transference to PVDF membrane was carried out by using Trans-Blot® Turbo™ Transfer System  during 30 min with constant current at 0.3 A. The membranes were blocked with 4% (w/v) BSA  in ×1 TBS-Tween  Tween, pH 7.5) for 60 min at RT. The membranes were further incubated with primary antibody 1:2000 anti-hSod1  and mouse 1:5000 anti-γ-tubulin  in 4% (w/v) BSA/TBS-Tween overnight at 4 °C. After washing three times in TBS-Tween for 5 min, the membranes were incubated for 1 h with anti-mouse IgG and anti-goat IgG horseradish peroxidase labeled secondary antibody  at 1∶6000 in 4% (w/v) BSA/TBS-Tween. Detection was carried out using luminol reagent and peroxide solution  and applied to the membrane 1 min before scanning with in Fusion FX . The band intensity was estimated using the ImageJ software  and normalized against γ-tubulin.Forty-eight hours post-transfection cells were washed with room temperature DPBS and then harvested. H4 cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer  low-gelling temperature agarose (PeqLab) and allowed to air-dry. Approximately 5 × 104 cells/mL were harvested in DPBS, mixed with 1% agarose and placed on a precoated slide. After agarose has gelled, alkaline lysis was performed by submerging the slides in alkaline buffer  sodium lauryl sarcosinate, 0.26 M NaOH, pH > 13) and stored overnight at 4 °C. After overnight lysis, the alkaline rinse solution  was used to wash the slides three times and to conduct the electrophoresis for 25 min at a constant voltage of 0.6 V/cm. Slides were neutralized with distilled water and stained with 2.5 μg/mL of propidium iodide for 20 min at RT. DNA damage quantification was carried out by examining at least 50 comet images from each slide. The images were acquired with an epifluorescence microscope . CometScore software (TriTek Corp) was used to determine the tail moment  of each individual comet image.The comet assay, a single cell gel electrophoresis-based method, was used to the detect DNA single- and double-strand breaks, according to the protocol previously described in . Agarosev/v) H2O2. The fluorescence values were recorded up to 45 min and normalized to those obtained from non-H2O2-induced cells.The generation of intracellular reactive oxygen species (ROS) was assessed in H4 cells expressing BiFC-tagged hSod1 or untagged hSod1 (WT and mutants). Briefly, the cells were seeded in 48-well plates and washed once with DPBS. For ROS production measurement, cells were incubated with 25 μM 2′,7′-dichlorofluorescin diacetate  at 37 °C for 30 min. Following the incubation period, two washing steps with DPBS were performed to remove excess probe and fluorescence intensity  was measured using the microplate reader Infinite M2000 PRO, Tecan. After three basal measurements, cells were challenged with 5%  software and were expressed as the mean ± SD of at least three replicates. Statistical differences from WT Sod1 were calculated using one-way ANOVA and two-way ANOVA with Bonferroni correction. Significance was assessed for, where an asterisk corresponds to In order to assess the effect of hSod1 mutations on oligomerization and subcellular localization, we used the BiFC-based assay which involves the fusion of Sod1 with non-fluorescent fragments of the Venus protein . This asIn addition, we also found that all fALS hSod1 mutations reduced the nuclear localization of Sod1 to half of that observed with WT hSod1 , and costained cells with an antibody against G3BP, an established marker of SGs , 24. We In order to investigate the site of accumulation of hSod1 in H4 cells, we monitored the localization of the inclusions using von Hippel-Lindau (VHL) fused to mCherry as a marker for the JUNQ compartment. VHL is a heterologous protein that misfolds and forms soluble aggregates that are targeted to the JUNQ compartment, where they colocalize with the quality control machinery . To imprTo investigate the dynamics of the formation of Sod1 inclusions in vivo, live-cell imaging was performed during 20 h post-transfection. The time of appearance of the first inclusions was recorded and compared among the variants of hSod1 tested. Upon careful analysis of the data, we verified that A4V and G93A hSod1 inclusions appeared about 3 h earlier than L38V and G93C hSod1 inclusions , we found that the WT, but not the mutant hSod1, reduced the levels of ROS  probe. Using this approach, we also analyzed whether A4V, L38V, G93A, and G93C fALS mutations affected hSod1 function. We found that the levels of ROS observed in cells carrying an empty vector were similar to those observed in cells transfected with hSod1 constructs. Thus, we then asked whether we could detect differences in the handling of ROS in cells transfected with the hSod1 constructs. After 30 min of exposure of the cells expressing the different hSod1 variants to hydrogen peroxide  and the fraction of total DNA in the tail (the number of relaxed/broken pieces) . IntereshSod1 mutations associated with fALS are prone to misfolding and aggregation . VariousThe aggregation of mutant Sod1 in living cells and the alteration in conformation and localization have been monitored by tagging the protein with fluorescent fluorophores , 38. AltGiven that hSod1 spontaneously dimerizes in normal conditions and forms a homodimer , we tookInitially, we observed that BiFC-tagged Sod1 A4V, L38V, G93A, and G93C dimerized and formed intracellular inclusions. This effect was particularly strong for the A4V mutant, which significantly increase in the percentage of cells with inclusions.It is still unclear whether Sod1 inclusions are toxic, and additional studies using different model systems will be instrumental to address this. Recent studies have connected protein inclusions with SGs . Using sSod1 aggregation is directly dependent of dimer dissociation, which is promoted by mutations that weaken the interactions at the dimer interface , 42. FREIn addition to the observation of Sod1 inclusions, we observed that about 40% of dimeric Sod1 WT is located in the nucleus, while only 20% of mutant Sod1 was found in this compartment, showing a significant increase in fluorescence intensity of mutSod1 in the cytoplasm. These results indicate that the presence of Sod1 mutations decreases the ability to this protein to move into the nucleus. It has been shown in yeast cells that exposure to oxidants is sufficient to promote Sod1 nuclear localization through Dun1-Sod1 interaction and regulation of Sod1 by phosphorylation at S60, 99 . In the 2O2 in comparison to WT Sod1. Sod1 mutants do not properly respond against intracellular ROS production. One possible explanation is that A4V, G93A, G93C, and L38V hSod1 as aggregates at cytoplasm are unable to enter the nucleus , leading to an impaired ability to prevent DNA damage when compared to WT Sod1, G93A hSod1 exhibited the worst performance. The levels of damage were twofold higher in G93A hSod1 expressing cells than in cells which contain only the endogenous Sod1, empty vector, meaning that G93A hSod1 is non-active and harmful with respect to DNA protection.Our study reveals an in vivo aggregation timeline between Sod1 mutants, which were probably responsible to induce DNA damage as well as a decreased nuclear localization in comparison to the WT form. Regarding the stages of ALS disease when Sod1 aggregates are formed, some aspects have been reported. Sod1 aggregate was found to accumulate at the highest levels as symptoms appear in mouse models of fALS , 51. Mor


Galleria mellonella) infected with the entomopathogenic nematodes: Heterorhabditis bacteriophora HP88, Heterorhabditis indicus RM1 and Heterorhabditis sp (S1), Steinernema abbasi and Steinernema sp. (S II). Strains were identified as Photorhabdus luminescens HRM1, P. luminescens HS1, P. luminescens HP88, Xenorhabdus indica and X. nematophila ATTC19061 using 16S rDNA sequence analysis. To reveal the genetic diversity among these strains, three molecular markers  were employed. RAPD analysis showed 73.8 and 54.5 polymorphism percentages for the Photorhabdus and Xenorhabdus strains, respectively. ISSR analysis resulted in 70.1 and 75.2 polymorphism percentages among the Photorhabdus and Xenorhabdus strains, respectively. The SRAP analysis indicated that 75.6 and 61.2% genetic polymorphism was detected among Photorhabdus and Xenorhabdus strains, respectively. The cluster analysis grouped the three Photorhabdus strains together in one cluster and the two Xenorhabdus strains together in another cluster indicating the phylogenetic relationships among them. The genotype-specific markers detected from the three molecular markers  were sufficient to distinguish between the different bacterial strains tested and can be used in the future IBM program that could be built on the use of these strains.Five bacterial strains were isolated from the hemocoel of the greater wax moth larvae ( Xenorhabdus spp. and Photorhabdus spp. are motile, Gram-negative bacteria belonging to the family Enterobacteriaceae. These bacteria are symbiotically associated with nematodes of the families, Steinernematidae and Heterorhabditidae, respectively  system was developed by Li and Quiros  to targePhotorhabdus and two Xenorhabdus) were identified at the molecular level based on the 16S rDNA region. Furthermore, the genetic polymorphism among the five bacterial isolates was investigated at the molecular levels using RAPD, ISSR and SRAP analyses. The obtained data were used to construct the phylogenetic tree. The genotype-specific markers were also determined.In the present work, five bacterial isolates  infected with different entomopathogenic nematode strains , Heterorhabditis bacteriophora HP88, Heterorhabditis indicus RM1 and Heterorhabditis sp. (S1)). The bacterial isolates were plated on Nutrient Bromothymol Blue Agar (NBTA) medium . The amplification was carried out in a DNA thermocycler (MWG BIOTECH Primuse) programmed as follows: (94 °C/4 min) 1,  35, (72 °C/7 min) 1  following the manufacturer’s instructions. Isolated symbiotic bacteria were identified by nucleotide sequence analysis of 16S ribosomal DNA (rDNA). The universal primer set used was a forward 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and a reverse 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′)  programmed as follows: (94 °C/4 min) 1,  35, (72 °C/7 min) 1.RAPD-PCR was carried out according to the procedure reported by Williams et al. . Eleven 2, 1× buffer and 2 units of Taq DNA polymerase. The amplification was carried out in a DNA thermocycler (MWG-BIOTECH Primuse) programmed as follows: (94 °C/4 min) 1,  40, and (70 °C/5 min) 1.ISSR amplification was performed according to Kafkas et al.  using 122, 1× buffer and 2 units of Taq DNA polymerase. The amplification was carried out in a DNA thermocycler (MWG-BIOTECH Primuse) programmed as follows: (94 °C/4 min) 1,  5,  35, and (70 °C/5 min) 1.Twenty-five SRAP combinations of five forward and five reverse primers were used Table . The polAll the genotypes were scored for the presence and absence of the RAPD, ISSR and SRAP bands. And the data were entered into a binary matrix as discrete variables, 1 for presence and 0 for absence of the character and this data matrix was subjected to further analysis.The Excel file containing the binary data was imported into NT Edit of NTSYS-pc 2.02J. The 0/1 matrix was used to calculate similarity as DICE coefficient using SIMQUAL subroutine in SIMILARITY routine. The resultant similarity matrix was employed to construct dendrogram using sequential agglomerative hierarchical nesting (SAHN) based on the unweighted pair group method with arithmetic means (UPGMA) to infer genetic relationships and phylogeny  to determine the genetic polymorphism among them and also to determine the genotype-specific markers for each strain.Photorhabdus strains generated 126 bands: 93 bands out of them were polymorphic and can be considered as useful bands for the three bacterial isolates with 73.8 polymorphism percentages. The Xenorhabdus strains showed 123 total bands and 76 bands out of the total bands were polymorphic with 54.5% polymorphism  followed by P. luminescens HS1 (2 markers) and P. luminescens RM1 (2 markers). Xenorhabdus strains showed 76 specific markers: 39 markers of them are specific to X. nematophila S2 and 37 for X. indica AB. The genotype-specific markers obtained for Xenorhabdus strains represent 61.7% of the total band number and 100% of the polymorphic bands  in prokaryotes is well documented  followed by P. luminescens RM1 (9 markers) and P. luminescens HS1 (4 markers). Xenorhabdus strains showed 85 specific markers: 45 markers of them are specific to X. nematophila S2 and 40 for X. indica AB. The genotype-specific markers obtained for Xenorhabdus strains represent 75.2% of the total band numbers and 100% of the polymorphic bands  followed by P. luminescens HS1 (9 markers) and P. luminescens RM1 (4 markers). Xenorhabdus strains showed 117 specific markers: 45 markers of them are specific to X. nematophila S2 and 72 for X. indica AB. The genotype-specific markers obtained for Xenorhabdus strains represent 61.2% of the total band number and 100% of the polymorphic bands  in addition to the 16S rDNA could be efficiently used to identify and characterize the five bacterial strains. The genotype-specific markers were sufficient to distinguish between the different bacterial strains and can be used in the future IBM program that could be built on the use of these strains.