EKV mutant connexin 31 associated cell death is mediated by ER stress

The epidermis expresses a number of connexin (Cx) proteins that are implicated in gap junction-mediated cell communication. Distinct dominantly inherited mutations in Cx31 cause the skin disease erythrokeratoderma variabilis (EKV) and hearing loss with or without neuropathy. Functional studies reveal tissue-specific effects of these Cx31 disease-associated mutations. The Cx31 mutants (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 are associated with EKV and the mutant (66delD)Cx31 with peripheral neuropathy and hearing loss, however the mechanisms of pathogenesis remain to be elucidated. Expression of (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 in vitro, but not (WT)Cx31 or (66delD)Cx31, cause elevated levels of cell-type specific cell death. Previous studies suggest that Cx-associated cell death may be related to abnormal ‘leaky’ hemichannels but we produced direct evidence against that being the major mechanism. Additionally, our immunocytochemistry showed upregulation of components of the unfolded protein response (UPR) in cells expressing the EKV-associated Cx31 mutants but not (WT)Cx31 or (66delD)Cx31. We conclude that the endoplasmic reticulum (ER) stress leading to the UPR is the main mechanism of mutant Cx31-associated cell death. These results indicate that, in vivo, ER stress may lead to abnormal keratinocyte differentiation and hyperproliferation in EKV patient skin

Pathogenic Connexin-31 Forms Constitutively Active Hemichannels to Promote Necrotic Cell Death

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

Expression and characterisation of modified human cytochrome P450 (CYP1B1)

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Expression and Characterisation of Modified Human Cytochrome P450 IB l (CYPIBI)

Cytochromes P450 (CYPs, P450s) are a superfamily of haem-containing enzymes, responsible for the metabolism of a diverse collection of both endogenous compounds and xenobiotics. An increased level of CYPIBI protein in tumour tissues makes this particular isoform a potential target in cancer therapeutics. In order to guide development of new compounds, CYPIBI must be fully characterised and the structure of the active site defined. CYPIBI is expressed naturally at low levels, thus a method to generate recombinantly expressed protein as a more abundant source was developed. The CYPIBI gene was amplified from a cDNA clone, using PCR. The PCR primers were designed to modify the CYPIBI gene sequence so that expression was facilitated in E. coli. Two engineered forms of CYPIBI were cloned since CYPs are integral membrane proteins and purification and solubilisation is difficult. One generated a modified, membrane bound protein (CYP1B1"MEM) and the other generated a membrane anchor- free protein (CYPIBIoSOL). CYPlBl"SOL was developed in order to facilitate purification as a soluble protein. Removal of the membrane anchor region resulted in a membrane associated protein, which was removed from the membranes by washing with 0.01% Triton X-100, and was further isolated using metal chelate chromatography. Activity analysis was carried out using the 7-ethoxyresorufin O-deethylation assay, initially with resuspended bacterial membranes contining either the membrane associated CYP1B1 *SOL or intrinsic CYP1B1 *MEM. Despite the membrane anchor region being removed, the activity of CYPlBl*SOL was not affected when compared with the activity of CYP1B1*MEM. The activity of CYP1B1"S0L was comparable to both CYPIBI oMEM and the commercially available native CYPIBI. The N-terminal membrane-spanning region is therefore, not vital for the activity of CYPIBI since membrane-associated CYPIBI oSOL retained 100% of the membrane bound activity