Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein

Even though the Duchenne muscular dystrophy (DMD) gene product Dystrophin Dp71d is involved in various key cellular processes through its role as a scaffold for structural and signalling proteins at the plasma membrane as well as the nuclear envelope, its subcellular trafficking is poorly understood. Here we map the nuclear import and export signals of Dp71d by truncation and point mutant analysis, showing for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1. Binding was confirmed in cells using pull-downs, while in vitro binding assays showed direct, high affinity (apparent dissociation coefficient of c. 0.25 nM) binding of Dp71d to IMPα/β. Interestingly, treatment of cells with the microtubule depolymerizing reagent nocodazole or the dynein inhibitor EHNA both decreased Dp71d nuclear localization, implying that Dp71d nuclear import may be facilitated by microtubules and the motor protein dynein. The role of Dp71d in the nucleus appears to relate in part to interaction with the nuclear envelope protein emerin, and maintenance of the integrity of the nuclear architecture. The clear implication is that Dp71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.     

1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture

We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.     

Gene tree parsimony of multilocus snake venom protein families reveals species tree conflict as a result of multiple parallel gene loss

The proliferation of gene data from multiple loci of large multigene families has been greatly facilitated by considerable recent advances in sequence generation. The evolution of such gene families, which often undergo complex histories and different rates of change, combined with increases in sequence data, pose complex problems for traditional phylogenetic analyses, and in particular, those that aim to successfully recover species relationships from gene trees. Here, we implement gene tree parsimony analyses on multicopy gene family data sets of snake venom proteins for two separate groups of taxa, incorporating Bayesian posterior distributions as a rigorous strategy to account for the uncertainty present in gene trees. Gene tree parsimony largely failed to infer species trees congruent with each other or with species phylogenies derived from mitochondrial and single-copy nuclear sequences. Analysis of four toxin gene families from a large expressed sequence tag data set from the viper genus Echis failed to produce a consistent topology, and reanalysis of a previously published gene tree parsimony data set, from the family Elapidae, suggested that species tree topologies were predominantly unsupported. We suggest that gene tree parsimony failure in the family Elapidae is likely the result of unequal and/or incomplete sampling of paralogous genes and demonstrate that multiple parallel gene losses are likely responsible for the significant species tree conflict observed in the genus Echis. These results highlight the potential for gene tree parsimony analyses to be undermined by rapidly evolving multilocus gene families under strong natural selection.     

Domain loss facilitates accelerated evolution and neofunctionalization of duplicate snake venom metalloproteinase toxin genes

Gene duplication is a key mechanism for the adaptive evolution and neofunctionalization of gene families. Large multigene families often exhibit complex evolutionary histories as a result of frequent gene duplication acting in concordance with positive selection pressures. Alterations in the domain structure of genes, causing changes in the molecular scaffold of proteins, can also result in a complex evolutionary history and has been observed in functionally diverse multigene toxin families. Here, we investigate the role alterations in domain structure have on the tempo of evolution and neofunctionalization of multigene families using the snake venom metalloproteinases (SVMPs) as a model system. Our results reveal that the evolutionary history of viperid (Serpentes: Viperidae) SVMPs is repeatedly punctuated by domain loss, with the single loss of the cysteine-rich domain, facilitating the formation of P-II class SVMPs, occurring prior to the convergent loss of the disintegrin domain to form multiple P-I SVMP structures. Notably, the majority of phylogenetic branches where domain loss was inferred to have occurred exhibited highly significant evidence of positive selection in surface-exposed amino acid residues, resulting in the neofunctionalization of P-II and P-I SVMP classes. These results provide a valuable insight into the mechanisms by which complex gene families evolve and detail how the loss of domain structures can catalyze the accelerated evolution of novel gene paralogues. The ensuing generation of differing molecular scaffolds encoded by the same multigene family facilitates gene neofunctionalization while presenting an evolutionary advantage through the retention of multiple genes capable of encoding functionally distinct proteins.     

Matrix metalloproteinases: protective roles in cancer

The original notion that matrix metalloproteinases (MMPs) act as tumour and metastasis-promoting enzymes by clearing a path for tumour cells to invade and metastasize has been challenged in the last decade. It has become clear that MMPs are involved in numerous steps of tumour progression and metastasis, and hence are now considered to be multifaceted proteases. Moreover, more recent experimental evidence indicates that some members of the MMP family behave as tumour-suppressor enzymes and should therefore be regarded as anti-targets in cancer therapy. The complexity of the pro- and anti-tumorigenic and -metastatic functions might partly explain why broad-spectrum MMP inhibitors failed in phase III clinical trials. This review will provide a focussed overview of the published data on the tumour-suppressive behaviour of MMPs.