A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing ß cells. The response of autoreactive T cells to ß cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as ß cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Structural basis of an ERAD pathway mediated by the ER-resident protein disulfide reductase ERdj5.

ER-associated degradation (ERAD) is an ER quality-control process that eliminates terminally misfolded proteins. ERdj5 was recently discovered to be a key ER-resident PDI family member protein that accelerates ERAD by reducing incorrect disulfide bonds in misfolded glycoproteins recognized by EDEM1. We here solved the crystal structure of full-length ERdj5, thereby revealing that ERdj5 contains the N-terminal J domain and six tandem thioredoxin domains that can be divided into the N- and C-terminal clusters. Our systematic biochemical analyses indicated that two thioredoxin domains that constitute the C-terminal cluster form the highly reducing platform that interacts with EDEM1 and reduces EDEM1-recruited substrates, leading to their facilitated degradation. The pulse-chase experiment further provided direct evidence for the sequential movement of an ERAD substrate from calnexin to the downstream EDEM1-ERdj5 complex, and then to the retrotranslocation channel, probably through BiP. We present a detailed molecular view of how ERdj5 mediates ERAD in concert with EDEM1.

[A case of hereditary sensory and autonomic neuropathy type 1E with frontal lobe dysfunction as an initial symptom].

A 49-year-old man had developed gradually personality change, gait disturbance, and hearing loss for five years. On admission, he presented with frontal release signs, stuttering, vertical gaze palsy, sensorineural deafness, muscle rigidity, ataxia, and sensory disturbance with areflexia in the lower extremities. Brain MRI demonstrated atrophy in the cerebellum and midbrain tegmentum as well as cerebral atrophy, predominantly in the frontal lobe. He was tentatively diagnosed as progressive supranuclear palsy on the basis of clinical features and imagings. On nerve conduction study, no sensory nerve action potentials were elicited in the upper and lower extremities. Details of family history revealed a hereditary sensory neuropathy with autosomal dominant inheritance in his relatives. Because genetic analysis showed a rare missense mutation (c.1483T>C, p.Y495H) in DNA methyltransferase 1 gene, we diagnosed him as having hereditary sensory and autonomic neuropathy type 1E (HSAN1E). In addition, p.M232R mutation in prion protein gene was detected. It should be kept in mind that there are some patients with HSAN1E presenting with frontal lobe dysfunction as an initial symptom and with clinical features mimicking progressive supranuclear palsy.

Kinetic mechanism of quinol oxidation by cytochrome bd studied with ubiquinone-2 analogs.

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S(n) on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.

[A case of Creutzfeldt-Jakob disease presenting with arm levitation as an initial symptom].

A 74-year-old, right handed man, developed insidiously with levitation and clumsiness of the right upper limb. His right arm tended to levitate spontaneously, when he was examined. He could put the elevated arm down on command, while the arm resumed to antigravity posture when his attention was diverted. His right arm also exhibited unwilled elevation when performing complex finger movements on the right side. He had a feeling of strangeness of the elevated limb, especially with the eyes closed. In addition to asymmetric limb-kinetic apraxia, combined sensations such as stereognosis were disturbed on the right side. Brain MRI showed high signal lesions predominantly in the left cerebral cortices and basal ganglia. SPECT with (123)I-IMP revealed asymmetric hypoperfusion, predominantly in the left medial frontal and parietal regions. Two months after the onset, levitation of the arm gradually disappeared, with the development of rapidly progressive dementia, frontal signs, dystonia and generalized myoclonus. The diagnosis of Creutzfeldt-Jakob disease (CJD) was made based on the clinical features and cerebrospinal fluid biomarkers. The early manifestation of the patient mimicked corticobasal degeneration which presents with arm levitation or alien hand syndrome. It is suggested that CJD can represent involuntary movements with higher brain dysfunction resembling corticobasal degeneration at the early stage of the illness. Although the underlying mechanism of arm levitation is still unknown, frontal disinhibition and parietal cortical sensory disturbance may contribute to the development of involuntary arm levitation in our patient.

Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus.

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c-dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

Structural basis of biological N2O generation by bacterial nitric oxide reductase.

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (Fe(B)) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

Double stent technique for the treatment of an internal carotid artery pseudoaneurysm caused by zone III stab injury.

A 77-year-old man was transferred to the hospital with swelling of his neck and oropharynx after a stab injury to his oral cavity with pruning shears. Findings at complete neurologic examination were normal. Contrast-enhanced computed tomography (CT) and angiography revealed a pseudoaneurysm at the pharyngeal portion of the right internal carotid artery. Endovascular treatment was undertaken by using the double bare stent technique. The pseudoaneurysm was completely occluded immediately after the procedure. There were no complications. There were no further symptoms or evidence of recurrence of the aneurysm during the 18-month follow-up period. The double bare stent technique is safe and effective for the treatment of zone III carotid artery stab injuries.

Mass spectrometric analysis of the ubiquinol-binding site in cytochrome bd from Escherichia coli.

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).