REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. Here we show that at Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1Brelated protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, we show that Baz and Par-6 are also expressed at synapses and that their synaptic localization depends on aPKC activity. Our findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory.     

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.     

Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue

Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6−10 cells, traversing a network of large germ line–derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.