Mutations in Connexin-31 (Cx31) are associated with multiple human diseases including erythrokeratodermia variabilis (EKV). The molecular action of Cx31 pathogenic mutants remains largely elusive. We report here that expression of EKV pathogenic mutant Cx31R42P induces cell death with necrotic characteristics. Inhibition of hemichannel activity by a connexin hemichannel inhibitor or high extracellular calcium suppresses Cx31R42P-induced cell death. Expression of Cx31R42P induces ER stress resulting in reactive oxygen species (ROS) production, in turn, to regulate gating of Cx31R42P hemichannels and Cx31R42P induced cell death. Moreover, Cx31R42P hemichannels play an important role in mediating ATP release from the cell. In contrast, no hemichannel activity was detected with cells expressing wildtype Cx31. Together, the results suggest that Cx31R42P forms constitutively active hemichannels to promote necrotic cell death. The Cx31R42P active hemichannels are likely resulted by an ER stress mediated ROS overproduction. The study identifies a mechanism of EKV pathogenesis induced by a Cx31 mutant and provides a new avenue for potential treatment strategy of the disease.

Gap junctions, consisting of connexins, allow the exchange of small molecules (<1 kD) between adjacent cells, thus providing a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is a member of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearing impairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseases are still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cells stably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells were treated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or α-glycyrrhetinic acid, suggesting that Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin 31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, α-glycyrrhetinic acid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assay revealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstrated that in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cells were treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed, and possibly degraded by both proteasomal and lysosomal pathways.

The preparation, approximate composition, degree of hydrolysis (DH), lipase activity and antioxidant capacity (including the ability of scavenging DPPH and ABTS free radicals) were determined for various of aqueous enzymatic extract (AEE) obtained from rice bran (RB) using alcalase (A), trypsin (T), papain (Pn), neutrase (N) and protamex (Px). Peptides and γ-oryzanol are the major functional components of AEE. Generally, the protein yield varied with the type of enzyme used in the order of T>Pn>N>Px>A. Both trypsin and alcalase extracts exhibit a higher level of γ-oryzanol than other enzymes. Lipase activity was measured using the 4-Nitrophenyl Laurate (p-NPL) substrate method, and significantly different (P<0.05) lipase activity values ranging from 0.55 to 2.38 mU/mL were observed from five hydrolysates. Furthermore, two synthetic free radicals were used to evaluate the antioxidant activity of AEE. The alcalase extract (at 4.35±0.15mg protein/mL AEE) shows the greatest scavenging rate (28.42%) of DPPH, but trypsin extract has highest ABTS radical-scavenging activity of 94.05%. Data revealed trypsin was possible the most effective extractant under their optimal conditions. These results demonstrate that the product obtained from rice bran by different enzymatic digestions may contain bioactive components and might potentially be natural antioxidants. Meanwhile, the aqueous enzymatic extraction method can also improve the stability of the hydrolysates.