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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.

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.

The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant

PLoS Genet. 10 (10): e1004765.

Quereda JJ, Ortega ÁD, Pucciarelli MG, García-del Portillo F.

PLoS Genet. 10 (10): e1004765Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized.

Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (~6-fold). Rapid amplification of 5′-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5′-untranslated regions (5′-UTR) of 28 and 234 nucleotides. The transcript containing the long 5′-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5′-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5′-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5′-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria.

In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5′-UTR. These findings emphasize how 5′-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5′-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

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.

Arid3b is essential for second heart field cell deployment and heart patterning

Development. 2014; 141 (21): 4168-4181.

Uribe V, Badía-Careaga C, Casanova JC, Domínguez JN, de la Pompa JL, Sanz-Ezquerro JJ.

Development. 2014; 141 (21): 4168-4181Arid3b, a member of the conserved ARID family of transcription factors, is essential for mouse embryonic development but its precise roles are poorly understood. Here, we show that Arid3b is expressed in the myocardium of the tubular heart and in second heart field progenitors.

Arid3b-deficient embryos show cardiac abnormalities, including a notable shortening of the poles, absence of myocardial differentiation and altered patterning of the atrioventricular canal, which also lacks epithelial-to-mesenchymal transition. Proliferation and death of progenitors as well as early patterning of the heart appear normal. However, DiI labelling of second heart field progenitors revealed a defect in the addition of cells to the heart. RNA microarray analysis uncovered a set of differentially expressed genes in Arid3b-deficient tissues, including Bhlhb2, a regulator of cardiomyocyte differentiation, and Lims2, a gene involved in cell migration. Arid3b is thus required for heart development by regulating the motility and differentiation of heart progenitors.

These findings identify Arid3b as a candidate gene involved in the aetiology of human congenital malformations.