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Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases

J Biol Chem. 2014; 289 (33):22614-22629.

López-Perrote A, Alatwi HE, Torreira E, Ismail A, Ayora S, Downs JA, Llorca O.

J Biol Chem. 2014; 289 (33):22614-22629Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood.

Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1.

Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.



Rewiring of genetic networks in response to modification of genetic background

Genome Biol Evol. 2014; pii: evu255.

Bajić D, Moreno C, Poyatos JF.

Genome Biol Evol. 2014; pii: evu255Genome-scale genetic interaction networks are progressively contributing to map the molecular circuitry that determines cellular behaviour. To what extent this mapping changes in response to different environmental or genetic conditions is however largely unknown.

Here we assembled a genetic network using an in silico model of metabolism in yeast to explicitly ask how separate genetic backgrounds alter network structure. Backgrounds defined by single deletions of metabolically active enzymes induce strong rewiring when the deletion corresponds to a catabolic gene, evidencing a broad redistribution of fluxes to alternative pathways. We also show how change is more pronounced in interactions linking genes in distinct functional modules, and in those connections that present weak epistasis. These patterns reflect overall the distributed robustness of catabolism.

In a second class of genetic backgrounds, in which a number of neutral mutations accumulate, we dominantly observe modifications in the negative interactions that together with an increase in the number of essential genes indicate a global reduction in buffering. Notably, neutral trajectories that originate considerable changes in the wild-type network comprise mutations that diminished the environmental plasticity of the corresponding metabolism, what emphasizes a mechanistic integration of genetic and environmental buffering. More generally, our work demonstrates how the specific mechanistic causes of robustness influence the architecture of multiconditional genetic interaction maps.

Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop

Nat Commun. 2014; 5: 5484.

Alvira S, Cuéllar J, Röhl A, Yamamoto S, Ito H, Alfonso CB, Rivas G, Buchner J, Valpuesta JM.

Nat Commun. 2014; 5: 5484In eukarya, chaperones Hsp70 and Hsp90 act coordinately in the folding and maturation of a range of key proteins with the help of several co-chaperones, especially Hop. Although biochemical data define the Hop-mediated Hsp70–Hsp90 substrate transfer mechanism, the intrinsic flexibility of these proteins and the dynamic nature of their complexes have limited the structural studies of this mechanism. Here we generate several complexes in the Hsp70/Hsp90 folding pathway (Hsp90:Hop, Hsp90:Hop:Hsp70 and Hsp90:Hop:Hsp70 with a fragment of the client protein glucocorticoid receptor (GR-LBD)), and determine their 3D structure using electron microscopy techniques.

Our results show that one Hop molecule binds to one side of the Hsp90 dimer in both extended and compact conformations, through Hop domain rearrangement that take place when Hsp70 or Hsp70:GR-LBD bind to Hsp90:Hop. The compact conformation of the Hsp90:Hop:Hsp70:GR-LBD complex shows that GR-LBD binds to the side of the Hsp90 dimer opposite the Hop attachment site.

Cancer stem cell-like phenotype and survival are coordinately regulated by Akt/FoxO/Bim pathway

Stem Cells. 2014; doi: 10.1002/stem.1904.

Gargini R, Cerliani JP, Escoll M, Antón IM, Wandosell F.

Stem Cells. 2014; doi: 10.1002/stem.1904Many solid tumors contain a subpopulation of cells with stem characteristics and these known as cancer stem cell (CSCs) or tumor-initiating cells (TICs). These cells drive tumor growth and appear to be regulated by molecular pathway different from other cells in the tumor bulk.

Here we set out to determine if elements of the PI3K-AKT pathway are necessary to maintain the CSC-like phenotype in breast tumour cells and for these cells to survive, bearing in mind that the identification of such elements is likely to be relevant to define future therapeutic targets. Our results demonstrate a close relationship between the maintenance of the CSC-like phenotype and the survival of these TICs. Inhibiting PI3K activity, or eliminating AKT activity, mostly that of the AKT1 isoform, produces a clear drop in TICs survival, and a reduction in the generation and growth of CD44High/CD24Low mammospheres. Surprisingly, the apoptosis of these TICs that is triggered by AKT1 deficiency is also associated with a loss of the stem cell/mesenchymal phenotype and a recovery of epithelial–like markers. Finally, we define downstream effectors that are responsible for controlling the CSC-phenotype, such as FoxO-Bim, and the death of these cells in the absence of AKT1.

In summary, these data closely link the maintenance of the stem cell-like phenotype and the survival of these cells to the AKT-FoxO-Bim pathway.

Probing DNA helicase kinetics with temperature-controlled magnetic tweezers

Small. 2014; doi: 10.1002/smll.201402686.

Gollnick B, Carrasco C, Zuttion F, Gilhooly NS, Dillingham MS, Moreno-Herrero F.

Small. 2014; doi: 10.1002/smll.201402686Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions.

In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair.

Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21–24 kBT) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.