A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.Posttranslational modification by ubiquitin regulates diverse processes in cells (1, 2). Ubiquitination is catalyzed by three types of enzymes—E1, E2, and E3, with the selectivity for the target protein provided by E3 ubiquitin ligases. Although the human genome encodes more than 600 ubiquitin ligases, many of them remain to be studied (3). The Skp1–Cul1–F-box protein (SCF) complex, one of the best-characterized ubiquitin ligases, is composed of three invariable components (Skp1, Cul1, and Rbx1) and a variable component F-box protein that serves as the substrate recognition module. Among the over 70 F-box proteins found in humans, less than half have been characterized (4).The identification of substrates for a specific ubiquitin ligase has been challenging despite considerable efforts. To date, the physical interaction between an ubiquitin ligase and its substrates has been exploited as the major approach for substrate identification (5–7). In these studies, immunoprecipitation followed by MS has been used to isolate ligase–substrate complexes. However, there are several difficulties associated with this approach: Most ligase–substrate interactions are generally too weak and transient to isolate the substrates by immunoprecipitation, and the abundances of relevant in vivo substrates are often low due to proteasomal degradation.Recently, an antibody that recognizes the ubiquitin remnant motif Lys-ε-Gly-Gly (diGly), which is exposed upon tryptic digestion of ubiquitinated proteins, has been developed for global proteomic applications aimed at identifying ubiquitinated substrates (8, 9). Although a few quantitative proteomics studies have identified a particular ubiquitin ligase substrate using stable isotope labeling utilizing amino acids in cell culture and the anti-diGly antibody (10), these examples required large quantities of samples and advanced techniques.Tandem ubiquitin-binding entity(ies) (TUBE) based on ubiquitin-associated domains have been developed for isolation of polyubiquitinated proteins from cell extracts (11). Notably, TUBE reagents protect polyubiquitin-conjugated proteins in cell lysates from both proteasomal degradation and deubiquitinating enzymes (DUBs) as efficiently as specific inhibitors of these enzymes (11). In this paper, we applied the TUBE technology to in vivo capture of ubiquitinated proteins. To develop a versatile method for identifying substrates of a specific ubiquitin ligase, we designed a mammalian expression vector encoding a FLAG-tagged trypsin-resistant (TR) TUBE, which protects ubiquitin chains from trypsin digestion under native conditions. Using two enrichment methods, TR-TUBE and the anti-diGly antibody, we succeeded in identifying the target substrates of the uncharacterized F-box protein FBXO21.     

Renal Damage in Recurrent Atypical Hemolytic Uremic Syndrome Associated with C3 p.Ile1157Thr Gene Mutation

Patients with atypical hemolytic uremic syndrome (aHUS) associated with a C3 p.Ile1157Thr mutation show a relatively high renal survival and low mortality rates, but renal histopathological findings after recurrence have been rarely reported. A 30-year-old man with a C3 p.Ile1157Thr mutation experienced a third recurrence of thrombotic microangiopathies with neurological and gastrointestinal disorders. A renal biopsy performed during the recovery phase of acute kidney injury revealed collapsed glomeruli and arteriolar vacuolization. Approximately 10% of glomeruli were globally sclerotic, despite the absence of arterio-/arteriolo-sclerosis. These findings suggest substantial progression of irreversible injuries in multiple organs, including kidneys, which occurs in aHUS patients with repeated thrombotic microangiopathies.     

Influence of different smear layers on bond durability of self-etch adhesives

Objective

The purpose of this study was to determine the influence of different smear layers on enamel and dentin bond durability of various types of self-etch adhesives.

A protective role for CD154 in hepatic steatosis in mice

Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild-type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte-derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low-density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X-Box binding protein-1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte-derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER-to-nucleus signaling protein-1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis. Conclusion: Our study identifies CD154 as a new mediator of hepatic steatosis.     

Weakness in intercellular association of keratinocytes in severely brittle nails

The brittle fingernail is a common complaint, but the features of the cellular structure of the nail plate remain unclear. In this study, clipped nailplates from two persons with severely brittle nails, one female aged 26 years and one male aged 82 years, were observed by light and electron microscopy and compared with normal nail plates. Numerous cracks were observed in clipped brittle nails, but not in normal nails, on light microscopy. When the deep areas of nail plates of the clipped normal nails were observed by electron microscopy, intercellular boundaries appeared intermingled, and two thin, electron-dense layers were observed in a narrow intercellular gap. In contrast, in brittle nails, marked dilatation of intercellular spaces was frequently observed and electron-dense layers were either not seen or were disrupted. When clipped normal nails were dehydrated in a desiccation chamber, similar dilatations - though not so severe -were observed, without evident cracks. These results suggest that dilatation of the intercellular space between nail keratinocytes is correlated with brittle nails and that dehydration may result in such intercellular dilatation.     

Subjective ratings and autonomic responses to dental video stimulation in children and their mothers

Children's fear of dental procedures could be related to their parents' attitude toward these procedures. To investigate this relationship, we compared subjective ratings and autonomic responses to dental and neutral video stimulation in children to the ratings and responses of their mothers. We selected 24 healthy children (12 girls and 12 boys; average age, 9.8 years) and their mothers (average age, 42.6 years). During video stimulation using scenes of dental drilling, vacuum suction, and an ordinary landscape, cardiac activity was monitored using electrocardiograms. Autonomic nerve activity was analyzed by power spectral analysis of heart rate variability at low (LF: 0.04–0.15 Hz) and high (HF: 0.15–0.4 Hz) frequencies. Levels of salivary alpha amylase (sAA) were measured after each video stimulation. LF/HF, heart rate (HR), and sAA values were used as indices of autonomic nerve activity. All subjects rated their response (valence, arousal, disgust, fear, and pain) to the video stimulation on a visual analog scale. Statistical analysis was performed using repeated-measures analysis of variance. No significant main effect of group (children vs. mothers) was observed in ratings of arousal, disgust, fear, and pain or in LF/HF and sAA changes. The main effect of video stimulation was significant for ratings of valence, disgust, fear, and pain (P < 0.001) and changes in LF/HF (P = 0.010). Subjective ratings and autonomic responses to virtual dental procedures could be similar between children and their mothers. Even in children, stimulation using dental videos is useful in assessing subjective perspectives and emotional stress.     

Malfunction of vascular control in lifestyle-related diseases: distribution of adrenomedullin-containing perivascular nerves and its alteration in hypertension

The distribution and characteristics of adrenomedullin (AM)-containing perivascular nerves in the rat mesenteric artery were investigated using immunohistochemical techniques. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia of mesenteric arteries, which were densely innervated by calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-LI fibers. AM-LI, CGRP-LI, and NPY-LI fibers were abolished by cold storage denervation. Capsaicin pretreatment abolished AM-LI and NPY-LI fibers but not NPY-LI fibers. NPY-LI fibers but not AM-LI and CGRP-LI fibers disappeared after treatment with 6-hydroxydopamine. There were many AM-LI positive cells in the dorsal root ganglia, where AM mRNA was detected. In a double immunofluorescence study, AM-LI was found in CGRP-LI fibers, although some fibers contained AM-LI alone. The density of AM-LI fibers was lower in SHR than in WKY mesenteric arteries. These results suggest that the mesenteric artery is innervated by AM-containing perivascular nerves and AM may have a neurotransmitter role in the regulation of vascular tone.     

Association of trypsin expression with tumour progression and matrilysin expression in human colorectal cancer

Overexpression of the matrix serine protease (MSP) trypsin has been implicated in tumour growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathological and prognostic significance of trypsin expression in colorectal cancer. This study analysed the association between immunohistochemically detected trypsin expression in colorectal cancer and clinicopathological characteristics, and investigated whether trypsin is a predictor of recurrence and/or survival. Trypsin immunoreactivity was more intense at the invasive front than in the superficial part of the tumour. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 48 cases (48%), were judged to be positive for trypsin. Trypsin positivity was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advanced pathological tumour-node-metastasis (TNM) stage, and recurrence. Patients with trypsin-positive carcinoma had significantly shorter overall and disease-free survival periods than did those with trypsin-negative carcinoma. Trypsin retained its significant predictive value for overall and disease-free survival in multivariate analysis that included conventional clinicopathological factors. It is well known that trypsin activates matrilysin (matrix metalloproteinase-7), which plays an important role in colorectal cancer progression. Patients with concordant overexpression of trypsin and matrilysin at the invasive front, in which they were often co-localized, had the worst prognosis. Trypsinogen-1-transfected HCT116 colon cancer cells showed not only trypsin activity, but also active matrilysin activity and were more invasive in vitro than mock-transfected HCT116 cells. These results suggest that trypsin plays a key role in the progression of colorectal cancer. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence in and poor prognosis of colorectal cancer patients.