Engineering a Dimeric Caspase-9: A Re-evaluation of the Induced Proximity Model for Caspase Activation

Caspases are responsible for the execution of programmed cell death (apoptosis) and must undergo proteolytic activation, in response to apoptotic stimuli, to function. The mechanism of initiator caspase activation has been generalized by the induced proximity model, which is thought to drive dimerization-mediated activation of caspases. The initiator caspase, caspase-9, exists predominantly as a monomer in solution. To examine the induced proximity model, we engineered a constitutively dimeric caspase-9 by relieving steric hindrance at the dimer interface. Crystal structure of the engineered caspase-9 closely resembles that of the wild-type (WT) caspase-9, including all relevant structural details and the asymmetric nature of two monomers. Compared to the WT caspase-9, this engineered dimer exhibits a higher level of catalytic activity in vitro and induces more efficient cell death when expressed. However, the catalytic activity of the dimeric caspase-9 is only a small fraction of that for the Apaf-1-activated caspase-9. Furthermore, in contrast to the WT caspase-9, the activity of the dimeric caspase-9 can no longer be significantly enhanced in an Apaf-1-dependent manner. These findings suggest that dimerization of caspase-9 may be qualitatively different from its activation by Apaf-1, and in conjunction with other evidence, posit an induced conformation model for the activation of initiator caspases

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer

BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. RESULTS: The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn(2+), or Mg(2+), which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the K(m )value shows an acidic pK(a )of ~6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. CONCLUSION: The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins

Exercise intervention and sexual function in advanced prostate cancer: A randomised controlled trial

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. Objectives: Treatments for prostate cancer such as androgen deprivation therapy (ADT), surgery and radiation therapy can adversely affect sexual, urinary and bowel function. Preliminary research has demonstrated the efficacy of exercise to preserve sexual function in men with localised prostate cancer receiving ADT, though this has yet to be investigated in a metastatic setting. We examined the effects of a 12-week exercise programme comprising resistance, aerobic and flexibility training on sexual health and function in men with advanced prostate cancer. Methods: Patients with prostate cancer (70.0±8.4 year; body mass index 28.7±4.0 kg/m2) with bone metastases (rib/thoracic spine, 66.7%; lumbar spine, 43.9%; pelvis, 75.4%; femur, 40.4%; humerus, 24.6%; other sites, 70.2%) were randomly assigned to supervised exercise 3 days/week (n=28) or usual care (n=29). Sexual health and function were assessed using the International Index of Erectile Function, the Expanded Prostate Cancer Index Composite and the EORTC-PR25 at baseline and 12 weeks. Results: Patients attended 89% of planned sessions and there were no adverse events. After adjusting for baseline values, there was no significant difference between groups for any measure of sexual function and activity (p \u3e 0.05). Additionally, there was no significant difference between groups for urinary and bowel function assessed by the EORTC-PR25 (p \u3e 0.05). Conclusions: A short-Term programme of supervised exercise does not appear to enhance indices of sexual health and function in men with advanced prostate cancer. Limitations of the intervention included the conservative modular exercise programme, which deliberately avoided loading bone metastatic sites