Yoshimitsu Shimomura

The Asymmetric Trimeric Architecture of [2Fe-2S] IscU : Implications for Its Scaffolding during Iron-Sulfur Cluster Biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 A, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S(gamma) atom of Cys36 and the N(epsilon) atom of His106, and the other was coordinated by the S(gamma) atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal alpha1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.

Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro

Nepmucin/CLM‐9 is an Ig domain‐containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle‐like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin‐expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti‐nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.

Crystal structure of Escherichia coli YfhJ protein, a member of the ISC machinery involved in assembly of iron–sulfur clusters†‡

Introduction. Iron–sulfur (Fe–S) proteins are ubiquitously distributed in organisms, playing essential roles in various biological processes as electron carriers, enzymes, and sensors of oxygen and iron. These functions depend on the chemical versatility of their cofactors, Fe–S clusters, which consist of several iron and sulfur atoms. Recent genetic and biochemical analyses have revealed that several enzymatic systems are involved in vivo in the synthesis of Fe–S clusters and in their incorporation into apo Fe–S proteins; three Fe–S cluster assembly machineries (NIF, ISC, and SUF) have so far been identified. In the NIF system, NifS and NifU are required for the formation of metalloclusters of nitrogenase in Azotobacter vinelandii. Furthermore, the NIF-like machinery has been shown to work in the maturation of a wide variety of Fe–S proteins. The ISC machinery, discovered from the homology between NifS and IscS, has broad specificity and targets general Fe–S proteins. The third SUF machinery was identified as an alternative pathway of Fe–S cluster assembly in Escherichia coli. The ISC machinery is present in -, -, and -proteobacteria and the mitchondria of eukaryotes, whereas the SUF machinery is widely distributed among archaebacteria, eubacteria, and the plastids of eukaryotes. The components of the ISC machinery are encoded in the so-called isc operon (iscRSUA-hscBA-fdx-yfhJ). This machinery was unambiguously demonstrated to be involved in the Fe–S cluster assembly; the overexpression of the isc operon increases the production of recombinant Fe–S proteins, whereas mutation of the operon decreases the activity of Fe–S proteins. IscS is a cysteine desulfurase, which releases a sulfur atom from L-cystein and serves as a sulfur donor for the Fe–S cluster assembly reaction. IscU and IscA bind unstable Fe–S clusters during the in vitro reconstitution process and appear to function as scaffolds in the Fe–S cluster assembly process. HscA is a molecular chaperone and HscB is a co-chaperone. Fdx is a [2Fe-2S]-type ferredoxin. IscR is a transcription factor that regulates expression of the isc genes. YfhJ (also called ORF3 or IscX) is a small, acidic protein of 66 residues, and pI 3.7. The homologs of YfhJ are distributed in proteobacteria categorized in the and -subdivisions, in which the gene order (iscRSUA-hscBAfdx-yfhJ) is highly conserved in the respective chromosomes as well as the acidic character of the protein. Two-hybrid and pull-down experiments have shown a specific interaction between YfhJ protein and IscS, indicating that YfhJ protein is involved in the ISC function. However, there is limited information concerning how YfhJ protein functions, since the mutation of the yfhJ gene does not show any conspicuous phenotype. Here we report a crystal structure of YfhJ from E. coli at 1.75 Å resolution and the characteristic charge distribution on its protein surface.

Overproduction, crystallization and preliminary X-­ray diffraction analysis of a quinone oxidoreductase from Thermus thermophilus HB8

A probable quinone oxidoreductase (MW = 32.1 kDa) from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography suggested the protein to be in a dimeric state. This protein enhanced the reduction activity of quinones by NADPH. It was crystallized in the absence and the presence of NADPH by the hanging-drop vapour-diffusion method. Both crystals were hexagonal, space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.6, c = 236.7 A for the apo form and a = b = 77.6, c = 235.9 A for the complex with NADPH. They diffract to better than 2.3 A resolution with synchrotron radiation. The asymmetric unit has one protein subunit (V(M) = 3.2 A(3) Da(-1) and V(sol) = 0.62 for the apo form), indicating that the twofold axis of the dimeric protein and the crystallographic twofold axis coincide.

Sildenafil and steroid therapy effectively improved POEMS syndrome-associated pulmonary arterial hypertension

POEMS syndrome is a rare multi-systemic plasma cell disorder characterized by polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes [1]. Pulmonary arterial hypertension (PAH) is one of rare manifestations of POEMS syndrome [1, 2], and steroid therapy is effective in treating POEMS syndrome-associated PAH [3–5]. However, because there is a risk of dosedependent steroid intolerance or insufficient effectiveness in treating POEMS syndrome-associated PAH [5, 6], alternative or adjunctive therapy should be evaluated. In this report, we showed a rare case of POMES syndromeassociated PAH which was successfully treated with sildenafil and relatively low dose of prednisolone. The severity of PAH and effectiveness of therapy were definitively evaluated with right heart catheterization. A 55-year-old man started to suffer from difficulty in walking, and edema in lower extremities from October 2005. He was diagnosed as POEMS syndrome in March 2007 because he met both of 2 major criteria (polyneuropathy and IgG-lambda type monoclonal protein) and 6 of 7 minor criteria (Castleman’s disease, splenomegaly, edema in lower extremities, hypothyroidism, hyperpigmentation, and papilledema) of POEMS syndrome [1]. The vascular endothelial growth factor (VEGF) level was 6630 pg/mL at the time of diagnosis. After improvements of his symptoms with steroid pulse therapy, he was followed up regularly as an outpatient without steroid. Despite relatively low VEGF level of 275 pg/mL, dyspnea and edema in lower extremities exacerbated and transthoracic echocardiography newly revealed possible pulmonary hypertension. He was referred to our institution. This case has also been reported elsewhere from another point of view [7]. PAH was confirmed by right heart catheterization as shown in Table 1. There were no other apparent secondary causes of pulmonary hypertension based on the World Health Organization updated clinical classification of pulmonary hypertension. After recovery from previously reported pulmonary edema [7], sildenafil was started at 20 mg/day and the dose was increased gradually to 60 mg/ day which resulted in improvement of plasma brain natriuretic peptide (BNP) level from 276 to 112 pg/mL [8]. At this point, oral prednisolone (0.4 mg/kg/day) and 2 cycles of intravenous dexamethasone (0.8 mg/kg 9 4 days) administration were added because exacerbation of POEMS syndrome was suspected with accumulation of pericardial effusion. Eventually, 100 mg/day of sildenafil and 0.4 mg/ kg/day of prednisolone administration resulted in dramatic improvement of PAH confirmed by many indexes including right heart catheterization about 3 months after the initiation of sildenafil (Table 1). Until now, steroid therapy has been believed as firstline therapy in treating POEMS syndrome-associated PAH and 1 mg/kg/day prednisolone administration may be We have to inform you that this case has also been reported in Annals of Hematology from another point of view [7]. Especially, laboratory data and part of right heart catheterization findings before the treatment in Table 1 are reproductions from it. However, the concept of this case report is totally different from the other one, and we got reproduction permission from Springer for this submission.

Expression of multiple leukemic stem cell markers is associated with poor prognosis in de novo acute myeloid leukemia

Abstract Leukemic stem cells (LSCs) play a crucial role in chemotherapy resistance in acute myeloid leukemia (AML). Although the association between the expression of individual LSC markers and poor prognosis has been reported, few studies have evaluated the prognostic effect of multiple LSC markers in patients with AML. Herein, we examined three LSC markers (CD25, CD96, and CD123) and the combined effect of their expression on clinical outcome. We retrospectively analyzed 80 adult patients with de novo AML who received intensive chemotherapy. Multiple LSC marker expression was significantly associated with shorter three-year overall survival (OS), compared with single or no LSC marker expression (18.2 vs. 65.0%, p < .001). Multivariate analysis showed that the expression of multiple LSC markers remained significant in terms of three-year OS (hazard ratio: 3.80, p = .001). Therefore, the combined evaluation of several LSC markers can predict the clinical outcome in patients with AML.

Small Cell Carcinoma Mimicking Acute Leukemia

A 72-year-old man presented with a 1-month history of lymphadenopathy. He was admitted to our hospital in May 2016. A complete blood count upon admission showed the following findings: hemoglobin, 13.0 g/dL; platelet count, 84×10/L; and white blood cell count, 6.3×10/L. His laboratory test showed increased lactate dehydrogenase (2,382 U/ L). Computed tomography showed a small pulmonary nodule (1 cm) and lymphadenopathy at multiple sites such as the right neck (6 cm), mediastinum (6 cm), and right axillary lymph nodes (3 cm). A bone marrow sample showed marked hypercellularity, containing 72% blast-like cells, which were not aggregated (Picture 1, 2). We suspected acute leukemia. However, flow cytometry revealed that the blasts were negative for CD19, cytoplasmic CD3, and cytoplasmic myeloperoxidase. Immunohistochemical staining of the bone marrow specimen was positive for cytokeratin (Picture 3) and synaptophysin and negative for chromogranin and thyroid transcription factor 1, which confirmed the diagnosis of small cell carcinoma. Based on the lung involvement and the frequency of occurrence, we considered the lung to be the site of the primary tumor. Small cell carcinoma mimicking acute leukemia is rare (1, 2); however, immunohistochemical staining should be performed as it may assist in making a quick and accurate diagnosis if blasts show the abnormal expression of surface antigen. Therefore, appropriate treatment can be initi-

The asymmetric architecture of 2Fe-2S IscU, a scaffold protein for iron-sulfur cluster biosynthesis

Iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups that are required to maintain fundamental life processes. The assembly of Fe-S clusters in several bacteria as well as eukaryotic mitochondria is achieved by a multicomponet system, called ISC machinery. In this machinery, IscU serves as a scaffold for assembly of a nascent Fe-S cluster, prior to its delivery to an apo-protein. The Hsp70-type molecular chaperon HscA and the J-type cochaperone HscB comprise a specialized chaperone system that selectively binds IscU and facilitate the cluster transfer process. We have determined the crystal structure of holo-IscU from the hyperthermophilic bacterium Aquifex aeolicus at 2.3 A resolution, using multi-wavelength anomalous diffraction of the [2Fe-2S] cluster. IscU formed an asymmetric homotrimeric structure harboring only one [2Fe-2S] cluster that was coordinated by three cysteines and one histidine (Cys36, Cys63, His106, and Cys107) on the surface of just one of the protomers. The cluster was buried inside the homotrimer by the neighboring protomers. The three protomers were conformationally distinct from each other, and associated around a non-crystallographic pseudo-three-fold axis. This unique trimeric holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization would be involved in the scaffolding function of IscU. This oligomeric holoIscU structure provides mechanistic implications concerning the initial event in the assembly of Fe-S cluster, as well as the event in the transfer to target apo-proteins.