Structural basis for substrate recognition in the Phytolacca americana glycosyltransferase PaGT3.

Capsaicinoids are phenolic compounds that have health benefits. However, the pungency and poor water solubility of these compounds limit their exploitation. Glycosylation is a powerful method to improve water solubility and reduce pungency while preserving bioactivity. PaGT3, a uridine diphosphate glycosyltransferase (UGT) from Phytolacca americana, is known for its ability to glycosylate capsaicinoids and other phenolic compounds. While structural information on several UGTs is available, structures of UGTs that can glycosylate a range of phenolic compounds are rare. To fill this gap, crystal structures of PaGT3 with a sugar-donor analogue (UDP-2-fluoroglucose) and the acceptors capsaicin and kaempferol were determined. PaGT3 adopts a GT-B-fold structure that is highly conserved among UGTs. However, the acceptor-binding pocket in PaGT3 is hydrophobic and large, and is surrounded by longer loops. The larger acceptor-binding pocket in PaGT3 allows the enzyme to bind a range of compounds, while the flexibility of the longer loops possibly plays a role in accommodating the acceptors in the binding pocket according to their shape and size. This structural information provides insights into the acceptor-binding mechanism in UGTs that bind multiple substrates.

Crown-ether-mediated crystal structures of the glycosyltransferase PaGT3 from Phytolacca americana.

Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structure-function relationship of PaGT3, its crystal structure was determined. The sugar-donor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptor-binding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein. The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.

An Ambidextrous Polyphenol Glycosyltransferase PaGT2 from Phytolacca americana.

The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.

Progress in the treatment of pulmonary metastases after liver transplantation for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and is the third leading cause of cancer-related death. Liver transplantation (LT) has become a curative treatment for patients with HCC. However, recurrence and metastasis after LT are the main factors reducing long-term survival in patients, and the lung is the most common site of metastasis after LT for HCC, although metastasis to liver, para-aortic lymph nodes and renal periphery are observed. Thus, the treatment of pulmonary metastases after LT for HCC has become a hot research topic, the successful treatment of pulmonary metastases can significantly prolong the survival of LT patients. Although single conventional treatment (chemotherapy, surgery and external beam radiation therapy), immunosuppression, image-guided minimally invasive therapy (radiofrequency ablation, microwave ablation, cryoablation, and brachytherapy) and molecular targeted drugs have had a significant effect, patients do not have durable remission and the long-term survival rate is disappointing. Therefore, improving existing treatments and identifying a more effective combination therapy are important research issues in the prevention and treatment of pulmonary metastases after LT for HCC. The paper reviewed single conventional treatments, new treatments, and combination therapy, to provide a basis for the best treatment of these patients.

125I Seed Implant Brachytherapy for Painful Bone Metastases After Failure of External Beam Radiation Therapy.

The purpose of this study was to evaluate the safety and therapeutic efficacy of computed tomography (CT)-guided I seed implant brachytherapy in patients with painful metastatic bone lesions after failure of external beam radiation therapy (EBRT).From August 2012 to July 2014, 26 patients with painful bone metastases after failure of EBRT were treated with CT-guided I seed implant brachytherapy. Patient pain and analgesic use were measured using the Brief Pain Inventory before treatment, weekly for 4 weeks, and every 4 weeks thereafter for a total of 24 weeks. Opioid analgesic medications and complications were monitored at the same follow-up intervals.Before I seed implantation, the mean score for worst pain in a 24-hour period was 7.3 out of 10. Following treatment, at weeks 1, 4, 8, 12, and 24, worst pain decreased to 5.0 (P < 0.0001), 3.0 (P < 0.0001), 2.8 (P < 0.0001), 2.6 (P < 0.0001), and 2.0 (P = 0.0001), respectively. Opioid usage significantly decreased at weeks 4, 8, and 12. Overall response rates of osseous metastases after I seed implantation at 1, 4, 8, 12, and 24 weeks were 58%, 79%, 81%, 82%, and 80%, respectively. Adverse events were seen in 4 patients, including Grade 1 myelosuppression and Grade 1 late skin toxicity.I seed brachytherapy is a safe and effective treatment for patients with painful bone metastases after failure of EBRT.

Refractory malignant fibrous histiocytoma: CT-guided treatment with a multidisciplinary, minimally invasive approach.

The purpose of this study is to evaluate the effectiveness and safety of a computed tomography (CT)-guided, multidisciplinary, minimally invasive approach to the treatment of patients with large, refractory malignant fibrous histiocytoma. This approach includes microwave ablation and absolute alcohol therapy combined with 125I seed implantation. Seven patients (5 males and 2 females, 26-78 years old, mean 49.7 years old) with large, refractory malignant fibrous histiocytoma participated in this study. The tumors had an average maximum diameter of 14.1 cm (10.0-19.0 cm). Follow-up was conducted for an average of 35.7 months to determine the local control rate, overall survival rate, and clinical complications. Follow-up times ranged from 2 to 45 months. Pain was significantly relieved in patients treated with multidisciplinary, minimally invasive approach. Complete response was achieved in 5 patients (71.4%), partial response in the other 2 patients (28.6%). The response rate of this treatment was 100%. The median survival time was 35.7 months. All patients were alive in 2 years after the treatment. Five patients were still alive after 3 years. The 3 year survival rate was 71.4%. The long-term complications included hyperpigmentation at the operative sites (n = 5) and insensible feeling at the ablation sites (n = 3).This CT-guided multidisciplinary, minimally invasive approach is an effective, safe, and feasible means of treating large, refractory malignant fibrous histiocytoma with minimal damage and few complications, but large-scale randomized clinical trials are necessary to confirm this assessment.

Metastatic malignant melanoma: computed tomography-guided 125I seed implantation treatment.

The aim of this study is to evaluate the therapeutic efficacy of computed tomography (CT)-guided interstitial iodine-125 (I) seed implantation for metastatic malignant melanoma treatment. From November 2008 to May 2011, 24 patients with metastatic malignant melanoma who had undergone surgery for excision of primary lesions and repeated chemotherapy underwent CT-guided I seed implantation. Their clinical situations, biochemical indicators, MRIs, and CTs were observed. The follow-up time ranged from 5 to 24 months (mean 19.6 months). The local control rates of metastatic malignant melanoma after surgery excision for primary lesion after 2, 6, 12, and 24 months were 86.8, 78.6, 62.1, and 55.0%, respectively. One patient died of liver failure 5 months after brachytherapy and another died of a metastatic brain tumor 8 months after brachytherapy. Two patients died of lung dysfunction from pulmonary metastases 15 months after brachytherapy. All other patients survived throughout the follow-up period. The 2-year survival rate was 83.3%. During the procedure, one patient presented with minimal bleeding from the applicator route and another presented with pneumothorax with 20% pulmonary compression, which improved after intraprocedure suctioning. Four patients had low-grade fever on day 3. Three showed mild decreases in their white blood cell counts. CT-guided I seed implantation is a safe, feasible, and promising approach to the treatment of patients with metastatic malignant melanoma after surgery excision for primary lesions and repeated chemotherapy, but large-scale randomized clinical trials should be conducted before the technique can be used routinely.