Insights into the Catalytic Mechanism of Unsaturated Glucuronyl Hydrolase of <i>Bacillus</i> sp. GL1

Together with polysaccharide lyases (PLs), the unsaturated glucuronyl hydrolase of Bacillus sp., GL1, is responsible for the metabolism of glycosaminoglycans (GAGs), which plays an important role in various crucial physiological events. More importantly, the degradation mechanism of GAGs often causes extracellular bacterial infection and is thought to be one of the virulence factors. We have previously studied the first degradation step catalyzed by PLs. In this work, we focused on the degradation of the unsaturated chondroitin disaccharide, which is produced from chondroitin by chondroitin lyase. A combined quantum mechanical and molecular mechanical method was employed in all simulations. First of all, molecular dynamics simulations were performed to obtain a stable initial enzyme–substrate complex structure. Almost all interactions between the substrate and enzyme were found to be related to the d-glucuronic acid unit, whereas no recognition specificity was observed for the N-acetyl-d-galactosamine unit. Experimentally, two different pathways have been proposed on the basis of X-ray structures and kinetic isotopic effects. In our simulation, the pathway involving the formation of an epoxide intermediate has been found to be favorable rather than that involving direct hydration of the vinyl ether group around carbons 4 and 5. A metastable oxocarbenium-ion-like intermediate can be found in our simulation

Data_Sheet_1_Transient Receptor Potential Cation Channel Subfamily V Member 4 Mediates Pyroptosis in Chronic Obstructive Pulmonary Disease.docx

TRPV4, a calcium permeable cation selective channel, was found to be involved in chronic obstructive pulmonary disease (COPD) through releasing ATP and IL-1β. Pyroptosis, a newly discovered pro-inflammatory cell death, was induced by cigarette smoke (CS) in airway epithelial cells (AECs). More recent studies indicated that blocking Ca2+ influx effectively inhibited pyroptosis. Therefore, we asked whether TRPV4 mediated CS-induced pyroptosis of AECs and hence participated in the pathogenesis of COPD. We found that pyroptosis and TRPV4 were upregulated in AECs from patients with COPD and long-term CS-exposed mice. Moreover, pharmacological inhibition or knockdown of TRPV4 function alleviated CS extract (CSE)-induced pyroptosis by inhibiting NACHT, LRP, PYD domains-containing protein 3 (NLRP3) inflammasome/activated caspase-1/gasdermin D pathway, decreasing the number of PI positive cells and lactate dehydrogenase (LDH) release, decreasing the expression of pro- inflammatory interleukin gene (IL)-1β, IL-8, and IL-18 expression, as well as increasing anti-inflammatory gene expression [NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase 2 (mitochondrial) (MNSOD), and catalase, (CAT)]. Moreover, pharmacological inhibition or knockdown of TRPV4 function significantly relieved CSE-induced mitochondrial damage including decreased mitochondrial membrane potential, mitochondrial fusion protein (OPA1, MFN2) expression, and increased mitochondrial fission protein (DRP1, MFF) expression. Taken together, these findings indicate that TRPV4 mediates AEC pyroptosis via NLRP3/caspase-1/GSDMD pathway in COPD.</p

Image1_Honokiol Inhibits HIF-1α-Mediated Glycolysis to Halt Breast Cancer Growth.PDF

Background: Hypoxia-inducible factor-1α (HIF-1α) induces the expression of glycolysis-related genes, which plays a direct and key role in Warburg effect. In a recent study, honokiol (HNK) was identified as one of the potential agents that inhibited the HIF-1α signaling pathway. Because the HIF- 1α pathway is closely associated with glycolysis, we investigated whether HNK inhibited HIF-1α-mediated glycolysis.Methods: The effects of HNK on HIF-1α-mediated glycolysis and other glycolysis-related genes’ expressions, cancer cells apoptosis and tumor growth were studied in various human breast cancer models in vitro and in vivo. We performed the following tests: extracellular acidification and oxygen consumption rate assays, glucose uptake, lactate, and ATP assays for testing glycolysis; WST-1 assay for investigating cell viability; colony formation assay for determining clonogenicity; flow cytometry for assessing cell apoptosis; qPCR and Western blot for determining the expression of HIF-1α, GLUT1, HK2 and PDK1. The mechanisms of which HNK functions as a direct inhibitor of HIF-1α were verified through the ubiquitination assay, the Co-IP assay, and the cycloheximide (CHX) pulse-chase assay.Results: HNK increased the oxygen consumption rate while decreased the extracellular acidification rate in breast cancer cells; it further reduced glucose uptake, lactic acid production and ATP production in cancer cells. The inhibitory effect of HNK on glycolysis is HIF-1α-dependent. HNK also downregulated the expression of HIF-1α and its downstream regulators, including GLUT1, HK2 and PDK1. A mechanistic study demonstrated that HNK enhanced the self-ubiquitination of HIF-1α by recruiting two E3 ubiquitin ligases (UFL1 and BRE1B). In vitro, HNK inhibited cell proliferation and clonogenicity, as well as induced apoptosis of cancer cells. These effects were also HIF1α-dependent. In vivo, HNK inhibited tumor growth and HIF-1α-mediated glycolysis.Conclusion: HNK has an inhibitory effect on HIF-1α-mediated glycolysis in human breast cancer. Our research revealed a new mechanism of HNK as an anti-cancer drug, thus representing a novel strategy to improve the prognosis of cancer.</p

Table_1_The coherence between PSMC6 and α-ring in the 26S proteasome is associated with Alzheimer’s disease.DOCX

Alzheimer’s disease (AD) is a heterogeneous age-dependent neurodegenerative disorder. Its hallmarks involve abnormal proteostasis, which triggers proteotoxicity and induces neuronal dysfunction. The 26S proteasome is an ATP-dependent proteolytic nanomachine of the ubiquitin-proteasome system (UPS) and contributes to eliminating these abnormal proteins. This study focused on the relationship between proteasome and AD, the hub genes of proteasome, PSMC6, and 7 genes of α-ring, are selected as targets to study. The following three characteristics were observed: 1. The total number of proteasomes decreased with AD progression because the proteotoxicity damaged the expression of proteasome proteins, as evidenced by the downregulation of hub genes. 2. The existing proteasomes exhibit increased activity and efficiency to counterbalance the decline in total proteasome numbers, as evidenced by enhanced global coordination and reduced systemic disorder of proteasomal subunits as AD advances. 3. The synergy of PSMC6 and α-ring subunits is associated with AD. Synergistic downregulation of PSMC6 and α-ring subunits reflects a high probability of AD risk. Regarding the above discovery, the following hypothesis is proposed: The aggregation of pathogenic proteins intensifies with AD progression, then proteasome becomes more active and facilitates the UPS selectively targets the degradation of abnormal proteins to maintain CNS proteostasis. In this paper, bioinformatics and support vector machine learning methods are applied and combined with multivariate statistical analysis of microarray data. Additionally, the concept of entropy was used to detect the disorder of proteasome system, it was discovered that entropy is down-regulated continually with AD progression against system chaos caused by AD. Another conception of the matrix determinant was used to detect the global coordination of proteasome, it was discovered that the coordination is enhanced to maintain the efficiency of degradation. The features of entropy and determinant suggest that active proteasomes resist the attack caused by AD like defenders, on the one hand, to protect themselves (entropy reduces), and on the other hand, to fight the enemy (determinant reduces). It is noted that these are results from biocomputing and need to be supported by further biological experiments.</p

Table_2_The coherence between PSMC6 and α-ring in the 26S proteasome is associated with Alzheimer’s disease.XLSX

Alzheimer’s disease (AD) is a heterogeneous age-dependent neurodegenerative disorder. Its hallmarks involve abnormal proteostasis, which triggers proteotoxicity and induces neuronal dysfunction. The 26S proteasome is an ATP-dependent proteolytic nanomachine of the ubiquitin-proteasome system (UPS) and contributes to eliminating these abnormal proteins. This study focused on the relationship between proteasome and AD, the hub genes of proteasome, PSMC6, and 7 genes of α-ring, are selected as targets to study. The following three characteristics were observed: 1. The total number of proteasomes decreased with AD progression because the proteotoxicity damaged the expression of proteasome proteins, as evidenced by the downregulation of hub genes. 2. The existing proteasomes exhibit increased activity and efficiency to counterbalance the decline in total proteasome numbers, as evidenced by enhanced global coordination and reduced systemic disorder of proteasomal subunits as AD advances. 3. The synergy of PSMC6 and α-ring subunits is associated with AD. Synergistic downregulation of PSMC6 and α-ring subunits reflects a high probability of AD risk. Regarding the above discovery, the following hypothesis is proposed: The aggregation of pathogenic proteins intensifies with AD progression, then proteasome becomes more active and facilitates the UPS selectively targets the degradation of abnormal proteins to maintain CNS proteostasis. In this paper, bioinformatics and support vector machine learning methods are applied and combined with multivariate statistical analysis of microarray data. Additionally, the concept of entropy was used to detect the disorder of proteasome system, it was discovered that entropy is down-regulated continually with AD progression against system chaos caused by AD. Another conception of the matrix determinant was used to detect the global coordination of proteasome, it was discovered that the coordination is enhanced to maintain the efficiency of degradation. The features of entropy and determinant suggest that active proteasomes resist the attack caused by AD like defenders, on the one hand, to protect themselves (entropy reduces), and on the other hand, to fight the enemy (determinant reduces). It is noted that these are results from biocomputing and need to be supported by further biological experiments.</p

Table_1_Case Report: A Novel Truncating Variant of NR0B1 Presented With X-Linked Late-Onset Adrenal Hypoplasia Congenita With Hypogonadotropic Hypogonadism.docx

Nuclear receptor subfamily 0 group B member 1 gene (NR0B1) encodes an orphan nuclear receptor that plays a critical role in the development and regulation of the adrenal gland and hypothalamic–pituitary–gonadal axis. In this study, we report a novel mutation in NR0B1 that led to adult-onset adrenal hypoplasia congenita (AHC) and pubertal development failure in a male adult. Clinical examinations revealed hyponatremia, elevated adrenocorticotropic hormone levels, reduced testosterone and gonadotropin levels, and hyper-responses to gonadotropin-releasing hormone and human chorionic gonadotropin stimulation tests. Whole-exome sequencing and Sanger sequencing were performed to identify the potential causes of AHC. Candidate variants were shortlisted based on the X-linked recessive models. Sequence analyses identified a novel hemizygous variant of c.1034delC in exon 1 of NR0B1 at Xp21.2, resulting in a frameshift mutation and premature stop codon formation. The c.1034delC/p.Pro345Argfs*27 in the NR0B1 gene was detected in the hemizygous state in affected males and in the heterozygous state in healthy female family carriers. These results expand the clinical features of AHC as well as the mutation profile of the causative gene NR0B1. Further studies are needed to elucidate the biological effects of the mutation on the development and function of the adrenal gland and the hypothalamic–pituitary–gonadal axis.</p

DataSheet1_Honokiol Inhibits HIF-1α-Mediated Glycolysis to Halt Breast Cancer Growth.PDF

Background: Hypoxia-inducible factor-1α (HIF-1α) induces the expression of glycolysis-related genes, which plays a direct and key role in Warburg effect. In a recent study, honokiol (HNK) was identified as one of the potential agents that inhibited the HIF-1α signaling pathway. Because the HIF- 1α pathway is closely associated with glycolysis, we investigated whether HNK inhibited HIF-1α-mediated glycolysis.Methods: The effects of HNK on HIF-1α-mediated glycolysis and other glycolysis-related genes’ expressions, cancer cells apoptosis and tumor growth were studied in various human breast cancer models in vitro and in vivo. We performed the following tests: extracellular acidification and oxygen consumption rate assays, glucose uptake, lactate, and ATP assays for testing glycolysis; WST-1 assay for investigating cell viability; colony formation assay for determining clonogenicity; flow cytometry for assessing cell apoptosis; qPCR and Western blot for determining the expression of HIF-1α, GLUT1, HK2 and PDK1. The mechanisms of which HNK functions as a direct inhibitor of HIF-1α were verified through the ubiquitination assay, the Co-IP assay, and the cycloheximide (CHX) pulse-chase assay.Results: HNK increased the oxygen consumption rate while decreased the extracellular acidification rate in breast cancer cells; it further reduced glucose uptake, lactic acid production and ATP production in cancer cells. The inhibitory effect of HNK on glycolysis is HIF-1α-dependent. HNK also downregulated the expression of HIF-1α and its downstream regulators, including GLUT1, HK2 and PDK1. A mechanistic study demonstrated that HNK enhanced the self-ubiquitination of HIF-1α by recruiting two E3 ubiquitin ligases (UFL1 and BRE1B). In vitro, HNK inhibited cell proliferation and clonogenicity, as well as induced apoptosis of cancer cells. These effects were also HIF1α-dependent. In vivo, HNK inhibited tumor growth and HIF-1α-mediated glycolysis.Conclusion: HNK has an inhibitory effect on HIF-1α-mediated glycolysis in human breast cancer. Our research revealed a new mechanism of HNK as an anti-cancer drug, thus representing a novel strategy to improve the prognosis of cancer.</p

Data_Sheet_1_Carbon, Nitrogen, and Phosphorus Stoichiometry and Plant Growth Strategy as Related to Land-Use in Hangzhou Bay Coastal Wetland, China.docx

Ecological stoichiometry can not only instruct soil nutrient stocks and availability, but also indicated plant growth strategy and adaptability to environmental changes or stress. This study was carried out to examine the plant–soil Carbon (C), Nitrogen (N), and Phosphorus (P) stoichiometry distributions and patterns in three tidal wetlands [mudflat (MF), native Phragmites australis-dominated community wetland (NW), invasive Spartina alterniflora-dominated community wetland (IW)], and one reclaimed P. australis-dominated community wetland (RW) in Hangzhou Bay coastal wetland. The results showed that land-uses have more effect on C and N contents, and C:N and N:P ratios in plant than in soil, P content and C:P ratios more affected by plant organ and soil depth. Compared to land-use, both plant organ and soil depth have stronger effects on C, N, and P stoichiometry. Among tidal wetlands, plant N content and C:P, N:P ratios were significantly higher in NW than in IW. In contrast, plant C, N, and P contents and C:P and N:P ratios were significantly lower in RW, and plant C:N was higher. Soil C, N, and P stocks were similar between tidal wetlands, and were significant higher than those of RW, indicating that reclamation were not beneficial to soil nutrient storage. In the NW, soil N availability was relatively high, and P availability was relatively low; and leaf N:P was 15.33, which means vegetation was co-limited by N and P nutrients. In addition, plants in the NW mainly adopted a conservative growth strategy, with a significantly low aboveground biomass of 1469.35 g·m2. In the RW, soil N availability was relatively low, P availability was relatively high, and leaf N:P was 3, which means vegetation was limited by N nutrient. In addition, plants in the RW mainly adopted a rapid growth strategy, with a significantly high aboveground biomass of 3261.70 g·m2. In the IW, soil N availability was relatively low, soil P availability was relatively high, and leaf N:P was 5.13, which means vegetation was limited by N nutrient. The growth strategy and aboveground biomass (2293.67 g·m2) of the IW were between those of the NW and RW. Our results provide a reference for nutrient management and evaluating the impacts of land-use types on coastal wetland ecosystems.</p

Additional file 1 of S100A6 inhibits MDM2 to suppress breast cancer growth and enhance sensitivity to chemotherapy

Additional file 1. Raw data for western blot and colony formation assay