Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis

The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity

The role of the exocyst complex in polarized exocytosis

Regulation of exocytosis is fundamental to many biological processes such as hormone secretion and epithelial cell polarization. More than thirty proteins involved in membrane trafficking have been identified in budding yeast, most of which are evolutionarily conserved in human. The exocyst, an octamenc protein complex, plays an essential role in late stages of post-Golgi secretion. However, the molecular mechanism of exocyst function in polarized exocytosis remains unclear. For the first part of my research, I found that Exo84p, a member of the exocyst complex, directly interacts with Sro7p/Sro77p, the yeast homologues of a tumor suppressor Lgl, which plays critical role in epithelial cell polarization. Evidence from biochemical and genetic studies suggested that although Sro7p/Sro77p are broadly distributed in the cell, their localized interaction with the exocyst and kinetic activation are important for SNARE assembly and polarized exocytosis. In the second part of my research, I investigated the regulation of exocytosis mediated by Exo84p and Sro7p/Sro77p. Yeast cells have at least 2 different types of exocytic vesicles that are typified by their cargos, Bgl2p and invertase. We found that, unlike other exocyst mutants that block both the Bgl2p vesicles and the invertase vesicles, exo84-202 preferentially exhibited Bgl2p vesicle accumulation. Further disruption of Exo84p-Sro7p/Sro77p interaction aggravates the Bgl2p secretion defect but had no effect on invertase vesicles secretion. In addition, secretion defects in cells with disruption of Exo84p-Sro7p/Sro77p interaction are pronounced at early stages of the cell cycle. Furthermore, we found a similar vesicle-specific and cell cycle-specific secretory defect in sro7 sro77 null cells and in exo70 sro7Δ double mutants. This study suggests that the interaction of Exo84p with Sro7p/Sro77p is important for Bgl2p vesicle secretion at the early stages of the cell cycle, which is critical for polarized cell growth

The role of the exocyst complex in polarized exocytosis

Regulation of exocytosis is fundamental to many biological processes such as hormone secretion and epithelial cell polarization. More than thirty proteins involved in membrane trafficking have been identified in budding yeast, most of which are evolutionarily conserved in human. The exocyst, an octamenc protein complex, plays an essential role in late stages of post-Golgi secretion. However, the molecular mechanism of exocyst function in polarized exocytosis remains unclear. For the first part of my research, I found that Exo84p, a member of the exocyst complex, directly interacts with Sro7p/Sro77p, the yeast homologues of a tumor suppressor Lgl, which plays critical role in epithelial cell polarization. Evidence from biochemical and genetic studies suggested that although Sro7p/Sro77p are broadly distributed in the cell, their localized interaction with the exocyst and kinetic activation are important for SNARE assembly and polarized exocytosis. In the second part of my research, I investigated the regulation of exocytosis mediated by Exo84p and Sro7p/Sro77p. Yeast cells have at least 2 different types of exocytic vesicles that are typified by their cargos, Bgl2p and invertase. We found that, unlike other exocyst mutants that block both the Bgl2p vesicles and the invertase vesicles, exo84-202 preferentially exhibited Bgl2p vesicle accumulation. Further disruption of Exo84p-Sro7p/Sro77p interaction aggravates the Bgl2p secretion defect but had no effect on invertase vesicles secretion. In addition, secretion defects in cells with disruption of Exo84p-Sro7p/Sro77p interaction are pronounced at early stages of the cell cycle. Furthermore, we found a similar vesicle-specific and cell cycle-specific secretory defect in sro7 sro77 null cells and in exo70 sro7Δ double mutants. This study suggests that the interaction of Exo84p with Sro7p/Sro77p is important for Bgl2p vesicle secretion at the early stages of the cell cycle, which is critical for polarized cell growth

Polysaccharide from Edible Alga Enteromorpha clathrata Improves Ulcerative Colitis in Association with Increased Abundance of Parabacteroides spp. in the Gut Microbiota of Dextran Sulfate Sodium-Fed Mice

Polysaccharide from the edible alga Enteromorpha clathrata has been demonstrated to exert beneficial effects on human health. However, what effect it has on inflammatory bowel diseases has not been investigated. Here, using a mouse model of dextran sulfate sodium (DSS)-induced ulcerative colitis, we illustrate that Enteromorpha clathrata polysaccharide (ECP) could alleviate body weight loss, reduce incidences of colonic bleeding, improve stool consistency and ameliorate mucosal damage in diseased mice. 16S rRNA high-throughput sequencing and bioinformatic analysis indicated that ECP significantly changed the structure of the gut microbiota and increased the abundance of Parabacteroides spp. in DSS-fed mice. In vitro fermentation studies further confirmed that ECP could promote the growth of Parabacteroides distasonis F1-28, a next-generation probiotic bacterium isolated from the human gut, and increase its production of short-chain fatty acids. Additionally, Parabacteroides distasonis F1-28 was also found to have anti-ulcerative colitis effects in DSS-fed mice. Altogether, our study demonstrates for the first time a beneficial effect of ECP on ulcerative colitis and provides a possible basis for understanding its therapeutic mechanisms from the perspective of symbiotic gut bacteria Parabacteroides distasonis

Synthesis of Novel Tylophorine Derivatives and Evaluation of Their Anti-Inflammatory Activity

We have previously demonstrated that DCB-3503, a tylophorine analogue, has an anti-inflammatory property in murine models for autoimmune diseases. However, its mechanism remains unknown. Here, we have synthesized 34 derivatives of DCB-3503 and investigated their effects on T cells differentiation and TNF-α production. Six derivatives (4, 9, 13, 19, 31, and 32) could significantly promote the expression of Foxp3. Among these, the IC₅₀ of 31 and 32 was about 500 μM. Eight analogues (1, 2, 4, 9, 12, 18, 19, and 21) showed anti-TNF-α effect in Raw 264.7 cells and murine splenocytes, of which 18 and 19 were most significant. Moreover, 31 and 18 showed a better activity and cell survival ratio when compared with DCB-3503 at various concentrations. In summary, we have demonstrated the anti-inflammatory characteristics of 34 novel tylophorine derivatives and discussed their structure–activity relationship in order to explore their therapeutic potentials for inflammatory diseases