Investigating the role of Hsp90 and LRP1 in FN matrix dynamics

Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells

Operon mRNAs are organized into ORF-centric structures that predict translation efficiency

Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggest that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF

Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2 weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress.journal articleresearch support, non-u.s. gov't2016 Jan2015 11 13importe

Increasing crop rotational diversity can enhance cereal yields

9 Pág.Diversifying agriculture by rotating a greater number of crop species in sequence is a promising practice to reduce negative impacts of crop production on the environment and maintain yields. However, it is unclear to what extent cereal yields change with crop rotation diversity and external nitrogen fertilization level over time, and which functional groups of crops provide the most yield benefit. Here, using grain yield data of small grain cereals and maize from 32 long-term (10–63 years) experiments across Europe and North America, we show that crop rotational diversity, measured as crop species diversity and functional richness, enhanced grain yields. This yield benefit increased over time. Only the yields of winter-sown small grain cereals showed a decline at the highest level of species diversity. Diversification was beneficial to all cereals with a low external nitrogen input, particularly maize, enabling a lower dependence on nitrogen fertilisers and ultimately reducing greenhouse gas emissions and nitrogen pollution. The results suggest that increasing crop functional richness rather than species diversity can be a strategy for supporting grain yields across many environments.G.V., R.B. and S.H. acknowledge FORMAS grants 2018-02872 and 2018-02321. TMB acknowledges USDA AFRI grant 2017-67013-26254. LTEs managed by SRUC were supported by the Scottish Government RESAS Strategic Research Programme under project D3-, Healthy Soils for a Green Recovery. Swedish LTEs were funded by the Swedish University of Agricultural Sciences (SLU). We thank the Lawes Agricultural Trust and Rothamsted Research for data from the e-RA database. The Rothamsted Long-term Experiments National Capability (LTE-NC) was supported by the UK BBSRC (Biotechnology and Biological Sciences Research Council, BBS/E/C/000J0300) and the Lawes Agricultural Trust. The Woodslee site was supported by the Agro-Ecosystem Resilience Program (Agriculture & Agri-Food Canada) and field management provided by field crews over 6 decades is appreciated. La Canaleja LTE (Spain) was supported by RTA2017-00006-C03-01 project (Ministry of Science and Innovation. El Encín LTEs were supported by Spanish Ministry of Economy and Competitiveness funds (projects AGL2002-04186-C03-01.03, AGL2007-65698-C03-01.03, AGL2012-39929-C03-01 of which L. Navarrete was the P.I). R.A., A.G.D. and E.H.P. are also grateful to all members of the Weed Science Group from El Encín Experimental Station for their technical assistance in managing the experiments. The Brody/Poznan University of Life Sciences long-term experiments were funded by the Polish Ministry of Education and Science. We acknowledge the E-Obs dataset from the EU-FP6 project UERRA (http://www.uerra.eu) and the Copernicus Climate Change Service, and the data providers in the ECA&D project (https://www.ecad.eu/).Peer reviewe

Crop rotational diversity can mitigate climate-induced grain yield losses

Diversified crop rotations have been suggested to reduce grain yield losses from the adverse climatic conditions increasingly common under climate change. Nevertheless, the potential for climate change adaptation of different crop rotational diversity (CRD) remains undetermined. We quantified how climatic conditions affect small grain and maize yields under different CRDs in 32 long-term (10-63 years) field experiments across Europe and North America. Species-diverse and functionally rich rotations more than compensated yield losses from anomalous warm conditions, long and warm dry spells, as well as from anomalous wet (for small grains) or dry (for maize) conditions. Adding a single functional group or crop species to monocultures counteracted yield losses from substantial changes in climatic conditions. The benefits of a further increase in CRD are comparable with those of improved climatic conditions. For instance, the maize yield benefits of adding three crop species to monocultures under detrimental climatic conditions exceeded the average yield of monocultures by up to 553 kg/ha under non-detrimental climatic conditions. Increased crop functional richness improved yields under high temperature, irrespective of precipitation. Conversely, yield benefits peaked at between two and four crop species in the rotation, depending on climatic conditions and crop, and declined at higher species diversity. Thus, crop species diversity could be adjusted to maximize yield benefits. Diversifying rotations with functionally distinct crops is an adaptation of cropping systems to global warming and changes in precipitation.</p

Structural diversity of ABC transporters

ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity.</p

RNase E and the High-Fidelity Orchestration of RNA Metabolism.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.Wellcome Trus

Genomic Characterization of the Taylorella Genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus