GRP78 promotes the invasion of pancreatic cancer cells by FAK and JNK

The major characteristics of pancreatic cancer are its excessive local invasion and early systemic dissemination. The glucose-regulated protein is over-expressed in many human cancers including pancreatic cancer and correlated with invasion and metastasis in many cancers. To investigate the effect of Grp78 on the invasion of pancreatic cancer, we used western blot and Transwell assay. We found Grp78 is expressed at lower levels in capan-2 and higher expressed in MiaPaCa-2 cells, and Grp78 expression levels were correlated with the invasion potentials of tumor cells. Then,we increased the expression of Grp78 in capan-2 cells and decreased the expression of Grp78 in MiaPaCa-2 cells. We found that over-expression of Grp78 caused significant increase in the expression of TIMP-1, TIMP-2, MMP-14, MMP-2, and MMP-9 in Capan-2 cells. Consistently, knockdown of Grp78 decreased the expression of them in MiaPaCa-2 cells. Gelatin zymography showed Grp78 over-expression stimulated the activities of MMP-2 and MMP-9, while GRP78 knockdown reduced the activities of MMP-2 and MMP-9. Cytoskeleton staining showed that knockdown of Grp78 caused a marked increase in cytoskeleton F-actin stress fibers in MiaPaCa-2 cells. Consistently, GRP78 knockdown hyperactivated RhoA and inhibited significantly Rac activity. Grp78 over-expression decreases the RhoA and stimulated Rac activity. We also found that Grp78 modulated FAK and JNK signaling pathways. Over-expression of GRP78 in Capan-2 activated FAK and JNK. Finally, we demonstrated that knockdown of FAK by shRNA in combination with blockade of JNK signaling pathway with SP600125 completely inhibited GRP78-induced cancer cell invasion. GRP78 is involved in the regulation of pancreatic cancer invasion. FAK and JNK are the key downstream effectors of GRP78.     

DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.     

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol⁻¹ versus 94.7 U nmol⁻¹) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T_m), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.     

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt‐tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty‐six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt‐secretion capacity in crops.     

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology

Lactobacilli are used widely in food, feed, and health applications. The taxonomy of the genus Lactobacillus, however, is confounded by the apparent lack of physiological markers for phylogenetic groups of lactobacilli and the unclear relationships between the diverse phylogenetic groups. This study used the core and pan-genomes of 174 type strains of Lactobacillus and Pediococcus to establish phylogenetic relationships and to identify metabolic properties differentiating phylogenetic groups. The core genome phylogenetic tree separated homofermentative lactobacilli and pediococci from heterofermentative lactobacilli. Aldolase and phosphofructokinase were generally present in homofermentative but not in heterofermentative lactobacilli; a two-domain alcohol dehydrogenase and mannitol dehydrogenase were present in most heterofermentative lactobacilli but absent in most homofermentative organisms. Other genes were predominantly present in homofermentative lactobacilli (pyruvate formate lyase) or heterofermentative lactobacilli (lactaldehyde dehydrogenase and glycerol dehydratase). Cluster analysis of the phylogenomic tree and the average nucleotide identity grouped the genus Lactobacillus sensu lato into 24 phylogenetic groups, including pediococci, with stable intra- and intergroup relationships. Individual groups may be differentiated by characteristic metabolic properties. The link between phylogeny and physiology that is proposed in this study facilitates future studies on the ecology, physiology, and industrial applications of lactobacilli.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.