The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.     

Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction

BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif “KRXK” in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.     

Tbx18 regulates development of the epicardium and coronary vessels

The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18^−/− mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α^lacZ/+ activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18^−/− hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.     

Structure-activity relationship of 5'-substituted fluoro-neplanocin a analogues as potent inhibitors of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues la-d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen bonding donor is essential for inhibitory activity.     

Periplaneta Americana extract inhibits osteoclastic differentiation in vitro

ObjectivesPeriplaneta americana extract (PAE) is proven to be promising in treating fever, wound healing, liver fibrosis, and cardiovascular disease. However, the role of PAE in skeletal disorders remains unclear. This study investigated whether PAE regulates osteoclastic differentiation in vitro via the culture using RAW264.7 cells and bone marrow derived macrophages (BMDMs).Materials and MethodsRAW264.7 cells and BMDMs were cultured and induced for osteoclastic differentiation supplementing with different concentrations of PAE (0, 0.1, 1, and 10 mg/mL). Cell counting kit‐8 (CCK‐8) assay was used to detect the cytotoxicity and cell proliferation. TRAP staining, actin ring staining, real‐time quantitative PCR (RT‐qPCR), and bone resorption activity test were performed to detect osteoclastic differentiation. RT‐qPCR and enzyme‐linked immunosorbent assay (ELISA) were conducted to assay the expression and secretion of inflammatory factors. RNA sequencing (RNA‐seq) and western blot analysis were carried out to uncover the underlying mechanism.ResultsCCK‐8 results showed that 10 mg/mL and a lower concentration of PAE did not affect cell proliferation. RT‐qPCR analysis verified that PAE down‐regulated the osteoclastic genes Nfatc1, Ctsk, and Acp5 in macrophages. Moreover, PAE restrained the differentiation, formation, and function of osteoclasts. Besides, RT‐qPCR and ELISA assays showed that PAE decreased inflammatory genes expression and reduced the secretion of inflammatory factors, including IL1β, IL6, and TNFα. Subsequent RNA‐seq analysis identified possible genes and signaling pathways of PAE‐mediated osteoclastogenesis suppression.ConclusionsOur study indicates that PAE has inhibitive effects on osteoclastogenesis and may be a potential therapeutic alternative for bone diseases.

Periplaneta americana extract (PAE), the animal medicine material extracted from the insects Periplaneta americana, is proven to possess a variety of pharmacological functions. However, the role of PAE in skeletal disorders remains unclear. In this study, we found that PAE decreased osteoclast genes expression Nfatc1, Ctsk, and Acp5 in macrophages. Besides, PAE restrained the differentiation, formation, and function of osteoclasts. Moreover, PAE suppressed the LPS‐induced inflammation. Subsequent RNA‐seq analysis identified the signaling pathways of PAE‐mediated osteoclastogenesis suppression. Our study indicated that PAE has inhibitive effects on osteoclastic differentiation and may be a potential therapeutic Chinese medicine for bone diseases.     

传输靶向COX-2 siRNA联合化疗药物对大鼠胃癌细胞生长

目的：通过动物实验探讨传输靶向COX-2 siRNA联合化疗药物对大鼠胃癌细胞生长的抑制作用。方法：24 只健康SD 大鼠 平分为三组,治疗组用COX-2-siRNA转染的胃癌SGC7901 细胞接种,同时进行环磷酰胺、丝裂霉素C 化疗治疗;阴性对照组,用 阴性对照siRNA 转染的胃癌SGC7901 细胞接种,同时进行环磷酰胺、丝裂霉素C 化疗治疗;对照组(n=8),用未经转染的胃癌 SGC7901 细胞接种,不进行化疗治疗;三组转染后都接种了裸鼠。结果：治疗组、阴性对照组及对照组胃癌细胞凋亡率分别为 （22.28± 0.12）%、（1.23± 0.17）%和（1.03± 0.14）%,治疗组与阴性对照组和对照组比较差异都有统计学意义(t=18.152,17.555, P<0.05)。治疗组的抑瘤率为76.7%,阴性对照组和对照组分别为12.8%和6.89%,治疗组的抑瘤率明显高于其他两组(x2=15. 211,13.899,P<0.05)。Western blotting检测结果显示治疗组的COX-2 蛋白表达含量得到了明显抑制。结论：传输靶向COX-2 siRNA和化疗药物的配合应用可有效抑制COX-2 蛋白的表达,从而抑制胃癌细胞的生长,从而起到更好的治疗效果。     

Entropy-driven mechanism of an E3 ligase

Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions.     

Effects of culture conditions on osteogenic differentiation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), beta-glycerophosphate (betaG), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, betaG, and HA had the second highest positive effect on ALP activity.