The HBx protein from hepatitis B virus coordinates a redox-active Fe-S cluster.

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.

Characterization of Fe-S Clusters in Proteins by MÓ§ssbauer Spectroscopy.

57Fe MÓ§ssbauer spectroscopy is unparalleled in the study of Fe-S cluster-containing proteins because of its unique ability to detect all forms of iron. Enrichment of biological samples with the 57Fe isotope and manipulation of experimental parameters such as temperature and magnetic field allow for elucidation of the number of Fe-S clusters present in a given protein, their nuclearity, oxidation state, geometry, and ligation environment, as well as any transient states relevant to enzyme chemistry. This chapter is arranged in five sections to help navigate an experimentalist to utilize 57Fe MÓ§ssbauer spectroscopy for delineating the role and structure of biological Fe-S clusters. The first section lays out the tools and technical considerations for the preparation of 57Fe-labeled samples. The choice of experimental parameters and their effects on the MÓ§ssbauer spectra are presented in the following two sections. The last two sections provide a theoretical and practical guide on spectral acquisition and analysis relevant to Fe-S centers.

The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries.

The histidine-aspartate (HD)-domain protein superfamily contains metalloproteins that share common structural features but catalyze vastly different reactions ranging from oxygenation to hydrolysis. This chemical diversion is afforded by (i) their ability to coordinate most biologically relevant transition metals in mono-, di-, and trinuclear configurations, (ii) sequence insertions or the addition of supernumerary ligands to their active sites, (iii) auxiliary substrate specificity residues vicinal to the catalytic site, (iv) additional protein domains that allosterically regulate their activities or have catalytic and sensory roles, and (v) their ability to work with protein partners. More than 500 structures of HD-domain proteins are available to date that lay out unique structural features which may be indicative of function. In this respect, we describe the three known classes of HD-domain proteins (hydrolases, oxygenases, and lyases) and identify their apparent traits with the aim to portray differences in the molecular details responsible for their functional divergence and reconcile existing notions that will help assign functions to yet-to-be characterized proteins. The present review collects data that exemplify how nature tinkers with the HD-domain scaffold to afford different chemistries and provides insight into the factors that can selectively modulate catalysis.

Differential S-palmitoylation of the human and rodent ß3-adrenergic receptors.

With few reported exceptions, G protein-coupled receptors (GPCRs) are modified by Cys palmitoylation (S-palmitoylation). In multiple GPCRs, S-palmitoylation targets a canonical site within the C-terminal cytoplasmic tail adjacent to the C terminus of the seventh transmembrane domain, but modification of additional sites is exemplified by the ß-adrenergic receptors (ßARs). The ß1AR is S-palmitoylated at a second, more distal site within the C-terminal tail, and the ß2AR is modified at a second site within the third intracellular loop, neither of which is conserved in other ßAR isoforms. The functional roles of S-palmitoylation of disparate sites are incompletely characterized for any GPCR family. Here, we describe S-palmitoylation of the ß3AR. We compared mouse and human ß3ARs and found that both were S-palmitoylated at the canonical site within the C-terminal tail, Cys-358 and Cys-361/363 in mouse and human ß3ARs, respectively. Surprisingly, the human ß3AR was S-palmitoylated at two additional sites, Cys-153 and Cys-292 within the second and third intracellular loops, respectively. Cys-153 is apparently unique to the human ß3AR, and Cys-292 is conserved primarily in primates. Mutational substitution of C-tail Cys in human but not mouse ß3ARs resulted in diminished ligand-induced cAMP production. Substitution of Cys-153, Cys-292, or Cys-361/363 within the human ß3AR diminished membrane-receptor abundance, but only Cys-361/363 substitution diminished membrane-receptor half-life. Thus, S-palmitoylation of different sites differentially regulates the human ß3AR, and differential S-palmitoylation distinguishes human and rodent ß3ARs, potentially contributing to species-specific differences in the clinical efficacy of ß3AR-directed pharmacological approaches to disease.

Redox-dependent rearrangements of the NiFeS cluster of carbon monoxide dehydrogenase.

The C-cluster of the enzyme carbon monoxide dehydrogenase (CODH) is a structurally distinctive Ni-Fe-S cluster employed to catalyze the reduction of CO2 to CO as part of the Wood-Ljungdahl carbon fixation pathway. Using X-ray crystallography, we have observed unprecedented conformational dynamics in the C-cluster of the CODH from Desulfovibrio vulgaris, providing the first view of an oxidized state of the cluster. Combined with supporting spectroscopic data, our structures reveal that this novel, oxidized cluster arrangement plays a role in avoiding irreversible oxidative degradation at the C-cluster. Furthermore, mutagenesis of a conserved cysteine residue that binds the C-cluster in the oxidized state but not in the reduced state suggests that the oxidized conformation could be important for proper cluster assembly, in particular Ni incorporation. Together, these results lay a foundation for future investigations of C-cluster activation and assembly, and contribute to an emerging paradigm of metallocluster plasticity.

Medical Treatment of Postendoscopic Submucosal Dissection Phlegmonous Gastritis in an Elderly Diabetic Woman with Myelodysplastic Syndrome.

Phlegmonous gastritis is a rare, suppurative disease characterized by full-thickness exudative changes, infiltration of inflammatory cells, and edema primarily in the submucosal layer. A 76-year-old woman with type 2 diabetes and myelodysplastic syndrome underwent endoscopic submucosal dissection (ESD) for early gastric cancer. Postoperatively, she developed persistent fever and computed tomography displayed full-circumference thickening of the gastric wall and increased levels of fat stranding. Endoscopy showed post-ESD ulcer floor expansion, formation of a false lumen between the ulcer floor and surrounding folds, and adhesion of purulent matter. Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans were detected from pus culture and Klebsiella pneumoniae from blood culture, leading to a diagnosis of phlegmonous gastritis. Contrast examination showed no leakage outside the gastric wall; therefore, the patient fasted and was given antibiotics. She was successfully treated with medical therapy, as demonstrated by repeat endoscopy. Based on our experience, we recommend antibiotics before and after ESD in patients thought to be at high risk of infection, as well as careful postoperative management including postoperative endoscopy.

1,635 Endoscopic submucosal dissection cases in the esophagus, stomach, and colorectum: complication rates and long-term outcomes.

BACKGROUND: Endoscopic submucosal dissection (ESD) enables en bloc resection of early gastrointestinal neoplasms; however, most ESD articles report small series, with short-term outcomes performed by multiple operators on single organ. We assessed short- and long-term treatment outcomes following ESD for early neoplasms throughout the gastrointestinal tract. METHODS: We performed a longitudinal cohort study in single tertiary care referral center. A total of 1,635 early gastrointestinal neoplasms (stomach 1,136; esophagus 138; colorectum 361) were treated by ESD by single operator. Outcomes were complication rates, en bloc R0 resection rates, and long-term overall and disease-specific survival rates at 3 and 5 years for both guideline and expanded criteria for ESD. RESULTS: En bloc R0 resection rates were: stomach: 97.1 %; esophagus: 95.7 %; colorectum: 98.3 %. Postoperative bleeding and perforation rates respectively were: stomach: 3.6 and 1.8 %; esophagus: 0 and 0 %; colorectum: 1.7 and 1.9 %. Intra criteria resection rates were: stomach: 84.9 %; esophagus: 81.2 %; colorectum: 88.6 %. Three-year survival rates for lesions meeting Japanese ESD guideline/expanded criteria were for all organ-combined: 93.4/92.7 %. Five-year rates were: stomach: 88.1/84.6 %; esophagus: 81.6/57.3 %; colorectum: 94.3/100 %. Median follow-up period was 53.4 (range, 0.07-98.6) months. Follow-up rate was 94 % (1,020/1,085). There was no recurrence or disease-related death. CONCLUSIONS: In this large series by single operator, ESD was associated with high curative resection rates and low complication rates across the gastrointestinal tract. Disease-specific and overall long-term prognosis for patients with lesions within intra criteria after curative resection appeared to be excellent.

A case of very large intrahepatic bile duct adenoma followed for 7 years.

A 70-year-old man was referred to our hospital due to abnormal liver function. A tumor of 92 mm × 61 mm was detected on ultrasound screening of the left liver lobe. Although the tumor was suspected to be intrahepatic bile duct carcinoma, he had chronic heart disease and was unable to undergo surgery. Therefore, he was followed without further testing. No increase in tumor serum markers or tumor size was observed for the subsequent 7 years. We continued to suspect intrahepatic bile duct carcinoma, and we decided to perform a tumor biopsy. Tumor biopsy findings indicated intrahepatic bile duct adenoma (BDA), which is a rare benign epithelial liver tumor typically ranging from 1 mm to 20 mm. We herein report a case of very large BDA followed for 7 years.

Anti-androgenic activities of Ganoderma lucidum.

The inhibitory effects of methanol extracts of 19 edible and medicinal mushrooms on 5alpha-reductase activity were examined. The extract of Ganoderma lucidum Fr. Krast (Ganodermataceae) showed the strongest 5alpha-reductase inhibitory activity. The treatment of the fruit body of Ganoderma lucidum or the extract prepared from it significantly inhibited the testosterone-induced growth of the ventral prostate in castrated rats. These results showed that Ganoderma lucidum might be a useful ingredient for the treatment of benign prostatic hyperplasia (BPH).