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author:("Tanaka, kenji")
1.  The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates 
Nature Communications  2015;6:6116.
Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.
Ubiquitin is a stable and soluble protein, but it is commonly found in inclusion bodies in neurodegenerative disorders and cancer. Here, Morimoto et al. report that increasing ubiquitin chain length leads to the formation of amyloid-like fibrils, which are degraded by an autophagy mechanism.
doi:10.1038/ncomms7116
PMCID: PMC4309437  PMID: 25600778
2.  Neural Substrates of View-Invariant Object Recognition Developed without Experiencing Rotations of the Objects 
The Journal of Neuroscience  2014;34(45):15047-15059.
One fails to recognize an unfamiliar object across changes in viewing angle when it must be discriminated from similar distractor objects. View-invariant recognition gradually develops as the viewer repeatedly sees the objects in rotation. It is assumed that different views of each object are associated with one another while their successive appearance is experienced in rotation. However, natural experience of objects also contains ample opportunities to discriminate among objects at each of the multiple viewing angles. Our previous behavioral experiments showed that after experiencing a new set of object stimuli during a task that required only discrimination at each of four viewing angles at 30° intervals, monkeys could recognize the objects across changes in viewing angle up to 60°. By recording activities of neurons from the inferotemporal cortex after various types of preparatory experience, we here found a possible neural substrate for the monkeys' performance. For object sets that the monkeys had experienced during the task that required only discrimination at each of four viewing angles, many inferotemporal neurons showed object selectivity covering multiple views. The degree of view generalization found for these object sets was similar to that found for stimulus sets with which the monkeys had been trained to conduct view-invariant recognition. These results suggest that the experience of discriminating new objects in each of several viewing angles develops the partially view-generalized object selectivity distributed over many neurons in the inferotemporal cortex, which in turn bases the monkeys' emergent capability to discriminate the objects across changes in viewing angle.
doi:10.1523/JNEUROSCI.1898-14.2014
PMCID: PMC4220033  PMID: 25378169
monkey inferotemporal cortex; object; view invariance
3.  The Essential Role of Primate Orbitofrontal Cortex in Conflict-Induced Executive Control Adjustment 
The Journal of Neuroscience  2014;34(33):11016-11031.
Conflict in information processing evokes trial-by-trial behavioral modulations. Influential models suggest that adaptive tuning of executive control, mediated by mid-dorsal lateral prefrontal cortex (mdlPFC) and anterior cingulate cortex (ACC), underlies these modulations. However, mdlPFC and ACC are parts of distributed brain networks including orbitofrontal cortex (OFC), posterior cingulate cortex (PCC), and superior-dorsal lateral prefrontal cortex (sdlPFC). Contributions of these latter areas in adaptive tuning of executive control are unknown. We trained monkeys to perform a matching task in which they had to resolve the conflict between two behavior-guiding rules. Here, we report that bilateral lesions in OFC, but not in PCC or sdlPFC, impaired selection between these competing rules. In addition, the behavioral adaptation that is normally induced by experiencing conflict disappeared in OFC-lesioned, but remained normal in PCC-lesioned or sdlPFC-lesioned monkeys. Exploring underlying neuronal processes, we found that the activity of neurons in OFC represented the conflict between behavioral options independent from the other aspects of the task. Responses of OFC neurons to rewards also conveyed information of the conflict level that the monkey had experienced along the course to obtain the reward. Our findings indicate dissociable functions for five closely interconnected cortical areas suggesting that OFC and mdlPFC, but not PCC or sdlPFC or ACC, play indispensable roles in conflict-dependent executive control of on-going behavior. Both mdlPFC and OFC support detection of conflict and its integration with the task goal, but in contrast to mdlPFC, OFC does not retain the necessary information for conflict-induced modulation of future decisions.
doi:10.1523/JNEUROSCI.1637-14.2014
PMCID: PMC4131015  PMID: 25122901
conflict; executive control; lesion study; orbitofrontal cortex
4.  Cognitive Control Functions of Anterior Cingulate Cortex in Macaque Monkeys Performing a Wisconsin Card Sorting Test Analog 
The Journal of Neuroscience  2014;34(22):7531-7547.
Monkeys were trained to select one of three targets by matching in color or matching in shape to a sample. Because the matching rule frequently changed and there were no cues for the currently relevant rule, monkeys had to maintain the relevant rule in working memory to select the correct target. We found that monkeys' error commission was not limited to the period after the rule change and occasionally occurred even after several consecutive correct trials, indicating that the task was cognitively demanding. In trials immediately after such error trials, monkeys' speed of selecting targets was slower. Additionally, in trials following consecutive correct trials, the monkeys' target selections for erroneous responses were slower than those for correct responses. We further found evidence for the involvement of the cortex in the anterior cingulate sulcus (ACCs) in these error-related behavioral modulations. First, ACCs cell activity differed between after-error and after-correct trials. In another group of ACCs cells, the activity differed depending on whether the monkeys were making a correct or erroneous decision in target selection. Second, bilateral ACCs lesions significantly abolished the response slowing both in after-error trials and in error trials. The error likelihood in after-error trials could be inferred by the error feedback in the previous trial, whereas the likelihood of erroneous responses after consecutive correct trials could be monitored only internally. These results suggest that ACCs represent both context-dependent and internally detected error likelihoods and promote modes of response selections in situations that involve these two types of error likelihood.
doi:10.1523/JNEUROSCI.3405-13.2014
PMCID: PMC4035517  PMID: 24872558
cognitive control; error; lesion; prefrontal cortex; response selection; response time
5.  New crystal structure of the proteasome-dedicated chaperone Rpn14 at 1.6 Å resolution 
A new crystal structure of yeast Rpn14 with an E384A mutation was determined at 1.6 Å resolution. The improved high-resolution structure provides a framework for understanding proteasome assembly.
The 26S proteasome is an ATP-dependent protease responsible for selective degradation of polyubiquitylated proteins. Recent studies have suggested that proteasome assembly is a highly ordered multi-step process assisted by specific chaperones. Rpn14, an assembly chaperone for ATPase-ring formation, specifically recognizes the ATPase subunit Rpt6. The structure of Rpn14 at 2.0 Å resolution in space group P64 has previously been reported, but the detailed mechanism of Rpn14 function remains unclear. Here, a new crystal structure of Rpn14 with an E384A mutation is presented in space group P21 at 1.6 Å resolution. This high-resolution structure provides a framework for understanding proteasome assembly.
doi:10.1107/S1744309112011359
PMCID: PMC3374504  PMID: 22691779
Rpn14; chaperones; 26S proteasome
6.  Proteasome Dysfunction Mediates Obesity-Induced Endoplasmic Reticulum Stress and Insulin Resistance in the Liver 
Diabetes  2013;62(3):811-824.
Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30–40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH2-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro—responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.
doi:10.2337/db11-1652
PMCID: PMC3581221  PMID: 23209186
7.  The rare sugar d-allose acts as a triggering molecule of rice defence via ROS generation 
Journal of Experimental Botany  2013;64(16):4939-4951.
Only d-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to d-allose. d-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-d-allose, a structural derivative of d-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding d-allose kinase to increase d-allose 6-phosphate synthesis were more sensitive to d-allose, but E. coli AlsI encoding d-allose 6-phosphate isomerase expression to decrease d-allose 6-phosphate reduced sensitivity. A d-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, d-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of d-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of d-allose to d-allose 6-phosphate, and treatment with d-allose might prove to be useful for reducing disease development in rice.
doi:10.1093/jxb/ert282
PMCID: PMC3830479  PMID: 24014866
d-Allose; d-glucose 6-phosphate dehydrogenase; hexokinase; NADPH oxidase; Oryza sativa L; rare sugar.
8.  Haemolytic anaemia due to stenosed double-reinforced grafts after surgical repaired aortic dissection 
Haemolytic anaemia due to a stenosed graft is a rare complication after surgery for aortic dissection. We present the case of a patient with haemolytic anaemia and heart failure, who had undergone emergent ascending aorta replacement for type A acute aortic dissection 5 years earlier. Chest computed tomography revealed severe graft stenosis of the proximal anastomosis and transthoracic echocardiography showed severe aortic regurgitation. Surgical treatment was necessary because of heart failure and myocardial ischaemia due to haemolytic anaemia and aortic regurgitation. During the operation, we found an inner graft surrounded by an outer graft and a dilated lumen between the double-reinforced grafts compressing the inner graft. We successfully reconstructed the aortic root with a total arch replacement. To the best of our knowledge, there are no cases in which haemolytic anaemia and AR developed in a patient with acute aortic dissection surgically treated by such a mechanism.
doi:10.1093/icvts/ivs272
PMCID: PMC3422964  PMID: 22695513
Haemolytic anaemia; Surgical repaired aortic dissection and aortic regurgitation
9.  Safety and efficacy of dexmedetomidine for long-term sedation in critically ill patients 
Journal of Anesthesia  2013;28(1):38-50.
Purpose
We evaluated the safety and efficacy of long-term administration of dexmedetomidine in patients in the intensive care unit (ICU). Primary endpoint was the incidence of hypotension, hypertension, and bradycardia. Secondary endpoints were withdrawal symptoms, rebound effects, the duration of sedation with Richmond Agitation-Sedation Scale (RASS) ≤ 0 relative to the total infusion time of dexmedetomidine, and the dose of additional sedatives or analgesics.
Methods
Dexmedetomidine 0.2–0.7 μg/kg/h was continuously infused for maintaining RASS ≤ 0 in patients requiring sedation in the ICU. Safety and efficacy of short-term (≤24 h) and long-term (>24 h) dexmedetomidine administration were compared.
Results
Seventy-five surgical and medical ICU patients were administered dexmedetomidine. The incidence of hypotension, hypertension, and bradycardia that occurred after 24 h (long-term) was not significantly different from that occurring within 24 h (short-term) (P = 0.546, 0.513, and 0.486, respectively). Regarding withdrawal symptoms, one event each of hypertension and headache occurred after the end of infusion, but both were mild in severity. Increases of mean arterial blood pressure and heart rate after terminating the infusion of dexmedetomidine were not associated with the increasing duration of its infusion. The ratio of duration with RASS ≤ 0 was ≥ 85 % until day 20, except day 9 (70 %) and day 10 (75 %). There was no increase in the dose of additional sedatives or analgesics after the first 24-h treatment period.
Conclusions
Long-term safety of dexmedetomidine compared to its use for 24 h was confirmed. Dexmedetomidine was useful to maintain an adequate sedation level (RASS ≤ 0) during long-term infusion.
doi:10.1007/s00540-013-1678-5
PMCID: PMC3921449  PMID: 23912755
Dexmedetomidine; Long term; Sedation; Intensive care unit; Withdrawal
10.  Acute renal failure as the presenting sign of disseminated intravascular coagulation in a patient with metastatic prostate cancer 
Disseminated intravascular coagulation (DIC) is the most frequent coagulation disorder in patients with prostate cancer. However, renal involvement in DIC associated with prostate cancer has rarely been documented. Herein, we present a case of metastatic prostate cancer presenting with acute renal failure (RF) triggered by DIC. An 80 year old man with metastatic prostate cancer was treated with antihormone therapy at an outpatient clinic. He was admitted to our hospital because of severe dyspnea and progressive RF. A hemorrhagic tendency was not clinically evident. Laboratory tests exhibited a significant coagulation disorder, suggestive of DIC. Despite treatment, his RF and dyspnea worsened, and he eventually passed away. An autopsy study revealed hypertensive nephrosclerosis superimposed by fibrin rich thrombi formation involving glomerular capillaries and arterioles characteristic of DIC. Additionally, focal segmental glomerulosclerosis was identified, which was presumably secondary to the glomerular endothelial and/or podocyte injury augmented by DIC. Those findings showed that glomerular injury, which was induced and subsequently exacerbated by DIC associated with prostate cancer, highly contributed to the progression of RF in our case. A differential diagnosis of DIC should be considered when a patient with prostate cancer presents with renal dysfunction.
doi:10.2147/IJNRD.S41813
PMCID: PMC3595975  PMID: 23525355
disseminated intravascular coagulation; prostate cancer; renal pathology
11.  A non-HIV case with disseminated Mycobacterium kansasii disease associated with strong neutralizing autoantibody to interferon-γ 
Disseminated non-tuberculous mycobacterium (dNTM) infection is rare in humans without human immunodeficiency virus (HIV) infection. Previous reports have shown autoantibodies to human interferon-gamma (IFN-γ), which play important roles in mycobacterium infection, in the sera of patients with non-HIV dNTM disease. Herein, we describe a 53-year-old male who was strongly suspected to have multicentric Castleman disease (MCD) based on bone marrow study and chest radiological findings. However, Mycobacterium kansasii was detected in respiratory samples including pleural effusion. We initiated anti-mycobacterial therapy under intensive care; he died on the 48th hospital day. We detected no hematological disorders, ruling out MCD postmortem. However, we detected M. kansasii in pulmonary, liver, spleen and bone marrow tissues. Moreover, anti-IFN-γ autoantibody was detected with strong neutralizing capacity for IFN-γ. We consider our present report to contribute to understanding of the relationship between anti-IFN-γ autoantibody and disease development.
doi:10.1016/j.rmcr.2012.11.003
PMCID: PMC3920570
Disseminated non-tuberculous mycobacterium disease; Anti-IFN-γ autoantibody; Non-HIV patient
13.  Clinical evaluation of sivelestat for acute lung injury/acute respiratory distress syndrome following surgery for abdominal sepsis 
Background
The efficacy of sivelestat in the treatment of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) has not been established. In part, this is due to the wide variety of factors involved in the etiology of ALI/ARDS. In this study, we examined the efficacy of sivelestat in patients with ALI/ARDS associated with abdominal sepsis.
Methods
The subjects were 49 patients with ALI/ARDS after surgery for abdominal sepsis. The efficacy of sivelestat was retrospectively assessed in two treatment groups, ie, a sivelestat group (n = 34) and a non-sivelestat group (n = 15).
Results
The sivelestat group showed significant improvements in oxygenation, thrombocytopenia, and multiple organ dysfunction score. The number of ventilator days (6.6 ± 6.1 versus 11.1 ± 8.4 days; P = 0.034) and length of stay in the intensive care unit (8.5 ± 6.2 versus 13.3 ± 9.5 days; P = 0.036) were significantly lower in the sivelestat group. The hospital mortality rate decreased by half in the sivelestat group, but was not significantly different between the two groups.
Conclusion
Administration of sivelestat to patients with ALI/ARDS following surgery for abdominal sepsis resulted in early improvements of oxygenation and multiple organ dysfunction score, early ventilator weaning, and early discharge from the intensive care unit.
doi:10.2147/DDDT.S36436
PMCID: PMC3471461  PMID: 23091371
sivelestat; acute lung injury; acute respiratory distress syndrome; abdominal sepsis
14.  Demonstration of Tuning to Stimulus Orientation in the Human Visual Cortex: A High-Resolution fMRI Study with a Novel Continuous and Periodic Stimulation Paradigm 
Cerebral Cortex (New York, NY)  2012;23(7):1618-1629.
Cells in the animal early visual cortex are sensitive to contour orientations and form repeated structures known as orientation columns. At the behavioral level, there exist 2 well-known global biases in orientation perception (oblique effect and radial bias) in both animals and humans. However, their neural bases are still under debate. To unveil how these behavioral biases are achieved in the early visual cortex, we conducted high-resolution functional magnetic resonance imaging experiments with a novel continuous and periodic stimulation paradigm. By inserting resting recovery periods between successive stimulation periods and introducing a pair of orthogonal stimulation conditions that differed by 90° continuously, we focused on analyzing a blood oxygenation level-dependent response modulated by the change in stimulus orientation and reliably extracted orientation preferences of single voxels. We found that there are more voxels preferring horizontal and vertical orientations, a physiological substrate underlying the oblique effect, and that these over-representations of horizontal and vertical orientations are prevalent in the cortical regions near the horizontal- and vertical-meridian representations, a phenomenon related to the radial bias. Behaviorally, we also confirmed that there exists perceptual superiority for horizontal and vertical orientations around horizontal and vertical meridians, respectively. Our results, thus, refined the neural mechanisms of these 2 global biases in orientation perception.
doi:10.1093/cercor/bhs149
PMCID: PMC3673175  PMID: 22661413
fMRI; oblique effect; orientation; primary visual cortex; radial bias
15.  Aggressive treatment with noninvasive ventilation for mild acute hypoxemic respiratory failure after cardiovascular surgery: Retrospective observational study 
Background
Acute hypoxemic respiratory failure (AHRF) is one of the most serious complications after cardiovascular surgery. It remains unclear whether noninvasive ventilation (NIV) has potential as an effective therapy for AHRF after cardiovascular surgery, although many reports have described the use of NIV for AHRF after extubation. The aim of this study was to investigate the effectiveness of NIV in the early stage of mild AHRF after cardiovascular surgery.
Methods
We retrospectively analyzed all patients admitted to the intensive care unit after cardiovascular surgery, whose oxygenation transfer (PaO2/FIO2) deteriorated mildly after extubation, and in whom NIV was initiated. A two-way analysis of variance and the Bonferroni multiple comparisons procedure, the Mann–Whitney test, Fisher’s exact test or the χ2test was performed.
Results
A total of 94 patients with AHRF received NIV, of whom 89 patients (94%) successfully avoided endotracheal intubation (successful group) and five patients required reintubation (reintubation group). All patients, including the reintubated patients, were successfully weaned from mechanical ventilation and discharged from the intensive care unit. In the successful group, PaO2/FIO2 improved and the respiratory rate decreased significantly within 1 h after the start of NIV, and the improvement in PaO2/FIO2 remained during the whole NIV period.
Conclusion
We conclude that NIV is beneficial for mild AHRF after cardiovascular surgery when it is started within 3 h after mild deterioration of PaO2/FIO2. We also think that it is important not to hesitate before performing reintubation when NIV is judged to be ineffective.
doi:10.1186/1749-8090-7-41
PMCID: PMC3434065  PMID: 22554005
Acute hypoxemic respiratory failure; Cardiovascular surgery; Post-extubation; Noninvasive ventilation; PaO2/FIO2
16.  Association of body temperature and antipyretic treatments with mortality of critically ill patients with and without sepsis: multi-centered prospective observational study 
Critical Care  2012;16(1):R33.
Introduction
Fever is frequently observed in critically ill patients. An independent association of fever with increased mortality has been observed in non-neurological critically ill patients with mixed febrile etiology. The association of fever and antipyretics with mortality, however, may be different between infective and non-infective illness.
Methods
We designed a prospective observational study to investigate the independent association of fever and the use of antipyretic treatments with mortality in critically ill patients with and without sepsis. We included 1,425 consecutive adult critically ill patients (without neurological injury) requiring > 48 hours intensive care admitted in 25 ICUs. We recorded four-hourly body temperature and all antipyretic treatments until ICU discharge or 28 days after ICU admission, whichever occurred first. For septic and non-septic patients, we separately assessed the association of maximum body temperature during ICU stay (MAXICU) and the use of antipyretic treatments with 28-day mortality.
Results
We recorded body temperature 63,441 times. Antipyretic treatment was given 4,863 times to 737 patients (51.7%). We found that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) or acetaminophen independently increased 28-day mortality for septic patients (adjusted odds ratio: NSAIDs: 2.61, P = 0.028, acetaminophen: 2.05, P = 0.01), but not for non-septic patients (adjusted odds ratio: NSAIDs: 0.22, P = 0.15, acetaminophen: 0.58, P = 0.63). Application of physical cooling did not associate with mortality in either group. Relative to the reference range (MAXICU 36.5°C to 37.4°C), MAXICU ≥ 39.5°C increased risk of 28-day mortality in septic patients (adjusted odds ratio 8.14, P = 0.01), but not in non-septic patients (adjusted odds ratio 0.47, P = 0.11).
Conclusions
In non-septic patients, high fever (≥ 39.5°C) independently associated with mortality, without association of administration of NSAIDs or acetaminophen with mortality. In contrast, in septic patients, administration of NSAIDs or acetaminophen independently associated with 28-day mortality, without association of fever with mortality. These findings suggest that fever and antipyretics may have different biological or clinical or both implications for patients with and without sepsis.
Trial registration
ClinicalTrials.gov: NCT00940654
doi:10.1186/cc11211
PMCID: PMC3396278  PMID: 22373120
body temperature; antipyretic; fever; critical illness; mortality
17.  Crystallization and preliminary X-ray characterization of the Skp1–Fbg3 complex 
The crystallization and preliminary X-ray diffraction studies of the Skp1–Fbg3 complex are reported. Crystallization by repeated microseeding using selected crystals as a source of microseeds is described.
F-box proteins are the substrate-recognition components of Skp1–Cullin1–F-­box protein–Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P212121, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9 Å and one molecule per asymmetric unit.
doi:10.1107/S1744309109050581
PMCID: PMC2805547  PMID: 20057081
F-box proteins; Fbg3; Skp1–Fbg3 complex; ubiquitin ligases
18.  Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells 
The Journal of Cell Biology  2011;193(2):275-284.
Impaired autophagy stabilizes p62 and promotes tumorigenesis through activation of the Nrf2 transcription factor.
Suppression of autophagy is always accompanied by marked accumulation of p62, a selective autophagy substrate. Because p62 interacts with the Nrf2-binding site on Keap1, which is a Cullin 3–based ubiquitin ligase adapter protein, autophagy deficiency causes competitive inhibition of the Nrf2–Keap1 interaction, resulting in stabilization of Nrf2 followed by transcriptional activation of Nrf2 target genes. Herein, we show that liver-specific autophagy-deficient mice harbor adenomas linked to both the formation of p62- and Keap1-positive cellular aggregates and induction of Nrf2 targets. Importantly, similar aggregates were identified in more than 25% of human hepatocellular carcinomas (HCC), and induction of Nrf2 target genes was recognized in most of these tumors. Gene targeting of p62 in an HCC cell line markedly abrogates the anchorage-independent growth, whereas forced expression of p62, but not a Keap1 interaction-defective mutant, resulted in recovery of the growth defect. These results indicate the involvement of persistent activation of Nrf2 through the accumulation of p62 in hepatoma development.
doi:10.1083/jcb.201102031
PMCID: PMC3080263  PMID: 21482715
19.  A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans  
The Journal of Clinical Investigation  2011;121(10):4150-4160.
Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional β subunits, expression of which is induced by IFN-γ, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, β type 8 (PSMB8), which encodes one of the β subunits induced by IFN-γ in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient’s tissues. In the patient’s skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.
doi:10.1172/JCI58414
PMCID: PMC3195477  PMID: 21881205
20.  Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t 
Chemistry & biology  2010;17(8):795-801.
SUMMARY
Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered β5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the β5t subunit is catalytically active in the murine thymus. A panel of established protea-some inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the β5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit β5t. We show that β5t has a substrate preference distinct from β5/β5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection.
doi:10.1016/j.chembiol.2010.05.027
PMCID: PMC3039300  PMID: 20797608
21.  AKT suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy 
Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Such degeneration is not a passive event, but rather is an active process, mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigro-striatal projection that a constitutively active form of the kinase Akt (Myr-Akt) demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve their axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mTor activity, and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.
doi:10.1523/JNEUROSCI.5519-10.2011
PMCID: PMC3075005  PMID: 21307249
degeneration; Parkinson’s disease; apoptosis; axon; autophagy; substantia nigra
22.  Matching Categorical Object Representations in Inferior Temporal Cortex of Man and Monkey 
Neuron  2008;60(6):1126-1141.
SUMMARY
Inferior temporal (IT) object representations have been intensively studied in monkeys and humans, but representations of the same particular objects have never been compared between the species. Moreover, IT’s role in categorization is not well understood. Here, we presented monkeys and humans with the same images of real-world objects and measured the IT response pattern elicited by each image. In order to relate the representations between the species and to computational models, we compare response-pattern dissimilarity matrices. IT response patterns form category clusters, which match between man and monkey. The clusters correspond to animate and inanimate objects; within the animate objects, faces and bodies form subclusters. Within each category, IT distinguishes individual exemplars, and the within-category exemplar similarities also match between the species. Our findings suggest that primate IT across species may host a common code, which combines a categorical and a continuous representation of objects.
doi:10.1016/j.neuron.2008.10.043
PMCID: PMC3143574  PMID: 19109916
23.  Liver autophagy contributes to the maintenance of blood glucose and amino acid levels 
Autophagy  2011;7(7):727-736.
Both anabolism and catabolism of the amino acids released by starvation-induced autophagy are essential for cell survival, but their actual metabolic contributions in adult animals are poorly understood. Herein, we report that, in mice, liver autophagy makes a significant contribution to the maintenance of blood glucose by converting amino acids to glucose via gluconeogenesis. Under a synchronous fasting-initiation regimen, autophagy was induced concomitantly with a fall in plasma insulin in the presence of stable glucagon levels, resulting in a robust amino acid release. In liver-specific autophagy (Atg7)-deficient mice, no amino acid release occurred and blood glucose levels continued to decrease in contrast to those of wild-type mice. Administration of serine (30 mg/animal) exerted a comparable effect, raising the blood glucose levels in both control wild-type and mutant mice under starvation. Thus, the absence of the amino acids that were released by autophagic proteolysis is a major reason for a decrease in blood glucose. Autophagic amino acid release in control wild-type livers was significantly suppressed by the prior administration of glucose, which elicited a prompt increase in plasma insulin levels. This indicates that insulin plays a dominant role over glucagon in controlling liver autophagy. These results are the first to show that liver-specific autophagy plays a role in blood glucose regulation.
doi:10.4161/auto.7.7.15371
PMCID: PMC3149698  PMID: 21471734
amino acid; autophagy; liver; gluconeogenesis; insulin; phosphoenolpyruvate carboxykinase
24.  Parkin Mediates Apparent E2-Independent Monoubiquitination In Vitro and Contains an Intrinsic Activity That Catalyzes Polyubiquitination 
PLoS ONE  2011;6(5):e19720.
Background
Mutations in the parkin gene, which encodes a ubiquitin ligase (E3), are a major cause of autosomal recessive parkinsonism. Although parkin-mediated ubiquitination was initially linked to protein degradation, accumulating evidence suggests that the enzyme is capable of catalyzing multiple forms of ubiquitin modifications including monoubiquitination, K48- and K63-linked polyubiquitination. In this study, we sought to understand how a single enzyme could exhibit such multifunctional catalytic properties.
Methods and Findings
By means of in vitro ubiquitination assays coupled with mass spectrometry analysis, we were surprised to find that parkin is apparently capable of mediating E2-independent protein ubiquitination in vitro, an unprecedented activity exhibited by an E3 member. Interestingly, whereas full length parkin catalyzes solely monoubiquitination regardless of the presence or absence of E2, a truncated parkin mutant containing only the catalytic moiety supports both E2-independent and E2-dependent assembly of ubiquitin chains.
Conclusions
Our results here suggest a complex regulation of parkin's activity and may help to explain how a single enzyme like parkin could mediate diverse forms of ubiquitination.
doi:10.1371/journal.pone.0019720
PMCID: PMC3100294  PMID: 21625422
25.  Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection 
The Journal of Cell Biology  2010;191(3):537-552.
Inactivation of the essential autophagy gene Atg5 results in selective accumulation of aggregation-prone proteins independently of substrate ubiquitination.
Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub–Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7−/− liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.
doi:10.1083/jcb.201005012
PMCID: PMC3003313  PMID: 21041446

Results 1-25 (63)