BMP4 Regulates EMT to be Involved in non-Syndromic Cleft lip With or Without Palate.

OBJECTIVE: In the previous study, we identified bone morphogenetic protein 4 (BMP4) responsible for non-syndromic cleft lip with or without cleft palate (NSCL/P). We aimed to elucidate the effects and mechanisms of BMP4 on epithelial-mesenchymal transition (EMT) through Smad1 signaling pathway to be involved in NSCL/P. METHODS: The human oral epidermoid carcinoma cells (KBs) were transfected with plasmids or small interfering RNA (siRNA) to build the models. The migration of the cells was evaluated by transwell assay. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the expressions of BMP4, E-cadherin, N-cadherin, EMT-related transcription factors snal1 and snal2, matrix metalloproteinase 2 (MMP2), MMP9, Smad1, and phosphorylated Smad1. RESULTS: In the overexpression group, the migration number of cells was increased significantly. The protein expression of E-cadherin was decreased significantly, while the protein expression level of the N-cadherin was increased significantly. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly higher. The expression level of Smad1 was not significantly changed, while the phosphorylation of Smad1 was significantly increased. In the BMP4-siRNA group, the migrating number cells was significantly decreased. The protein expression of E-cadherin was increased significantly, while the expression of N-cadherin was significantly decreased. The protein and mRNA expressions of MMP2, MMP9, snal1, and snal2 were significantly lower than that of the control group. The expressions of Smad1 and phosphorylation of Smad1 were not significantly changed. CONCLUSIONS: BMP4 enhances cell migration and promotes cell EMT through Smad1 signaling pathway. Abnormal BMP4 mediates migration and EMT through other relevant signaling pathways resulting in NSCL/P. The study provides new insight into the mechanisms of NSCL/P associated with BMP4.n.

Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode.

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Quantitative estimate of the effect of cellulase components during degradation of cotton fibers.

A comprehensive mechanistic kinetic model for enzymatic degradation of cotton fibers has been established based on a complete factorial experiment in combination with multivariate stepwise regression analysis. The analysis of the statistical parameter value in the model suggests that the enzymatic degradation of cotton fiber is a progressive and heterogeneous process that includes, at least, two courses that occur sequentially and then progress in parallel. Cellulose fibers were first depolymerized or solubilized by the synergism between cellobiohydrolase I (CBHI) and endoglucanase I (EGI), and then the oligomers obtained were randomly hydrolyzed into glucose by EGI and beta-glucosidase. The proposed model can be applied to the quantitative estimation of the effects of three cellulase components, CBHI, EGI, and beta-glucosidase separately, or in combination during the entire process of cellulose degradation. The validity of the proposed model has been verified by a filter paper activity assay. Its other applicability was also discussed.