Protein import machineries in endosymbiotic organelles

Abstract.: Chloroplast and mitochondria, the two organelles with an accepted endosymbiotic origin, have developed multiple translocation pathways to ensure the subcellular allocation of proteins synthesized by cytosolic ribosomes, and to guarantee their assembly into functional complexes in coordination also with organellar-encoded subunits. The evolution of different protein import machineries was thus essential for the development of these two organelles within cells. A general overview of the translocation machineries in chloroplast and mitochondrial membranes involved in targeting and import of nuclearencoded proteins, with special focus on plant cells where the two organelles coexist, is expounde

Structural Stability of the PsbQ Protein of Higher Plant Photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (GD) at 20 C extrapolated from GdnHCl (4.0 ± 0.6 kcal mol-1) or thermal unfolding (4.4 ± 0.8 kcal mol-1) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 C, the PsbQ protein is a monomer with an elongated shape.Spanish Minitry of Science and Technology (PB1998-0480 and AGL2004-00045)This work was funded by the Spanish Ministry of Science and Technology (project references PB1998-0480 and AGL2003-0045). M.B. holds a fellowship from the Spanish Ministry of Science and Technology.Peer reviewe

Ligand-Receptor Interactions

The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the interest of biologists to the kinetic and mechanical properties of cell membrane receptors. The aim of this review is to give a description of these advances that benefitted from a largely multidisciplinar approach

Isolation of novel PSII-LHCII megacomplexes from pea plants characterized by a combination of proteomics and electron microscopy

This work was supported by the Italian Ministry of Education, University and Research, “Futuro in Ricerca 2013” program RBFR1334SB to CP

New insights into the neolithisation process in southwest Europe according to spatial density analysis from calibrated radiocarbon dates

The agricultural way of life spreads throughout Europe via two main routes: the Danube corridor and the Mediterranean basin. Current archaeological literature describes the arrival to the Western Mediterranean as a rapid process which involves both demic and cultural models, and in this regard, the dispersal movement has been investigated using mathematical models, where the key factors are time and space. In this work, we have created a compilation of all available radiocarbon dates for the whole of Iberia, in order to draw a chronological series of maps to illustrate temporal and spatial patterns in the neolithisation process. The maps were prepared by calculating the calibrated 14C date probability density curves, as a proxy to show the spatial dynamics of the last hunter-gatherers and first farmers. Several scholars have pointed out problems linked with the variability of samples, such as the overrepresentation of some sites, the degree of regional research, the nature of the dated samples and above all the archaeological context, but we are confident that the selected dates, after applying some filters and statistical protocols, constitute a good way to approach settlement spatial patterns in Iberia at the time of the neolithisation process

Inhibition of adenovirus transport from the endosome to the cell nucleus by rotenone

Regardless of the clinical impact of human adenovirus (HAdV) infections in the healthy population and its high morbidity in immunosuppressed patients, a specific treatment is still not yet available. In this study, we screened the CM1407 COST Action's chemical library, comprising 1,233 natural products to identify compounds that restrict HAdV infection. Among them, we identified rotenolone, a compound that significantly inhibited HAdV infection. Next, we selected four isoflavonoid-type compounds (e.g., rotenone, deguelin, millettone, and tephrosin), namely rotenoids, structurally related to rotenolone in order to evaluate and characterized in vitro their antiviral activities against HAdV and human cytomegalovirus (HCMV). Their IC50 values for HAdV ranged from 0.0039 mu M for rotenone to 0.07 mu M for tephrosin, with selective indices ranging from 164.1 for rotenone to 2,429.3 for deguelin. In addition, the inhibition of HCMV replication ranged from 50% to 92.1% at twice the IC50 concentrations obtained in the plaque assay for each compound against HAdV. Our results indicated that the mechanisms of action of rotenolone, deguelin, and tephrosin involve the late stages of the HAdV replication cycle. However, the antiviral mechanism of action of rotenone appears to involve the alteration of the microtubular polymerization, which prevents HAdV particles from reaching the nuclear membrane of the cell. These isoflavonoid-type compounds exert high antiviral activity against HAdV at nanomolar concentrations, and can be considered strong hit candidates for the development of a new class of broad-spectrum antiviral drugs

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteinsparticipateinawidevarietyofphysiologicallyrelevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmoleculespermonomerinredoxcommunicationwithanactive disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-basedtransferofreducingequivalentsinbacterialmembranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.Spanish Ministerio de Economía, Industria y Competitividad BFU2016-80343-P, BIO2016-75634-

Long range molecular dynamics study of regulation of eukaryotic glucosamine-6-phosphate synthase activity by UDP-GlcNAc

Glucosamine-6-phosphate (GlcN-6-P) synthase catalyses the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5’ diphospho N-acetyl-D-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes it a potential target for antifungal, antibacterial and antidiabetic therapy. The crystal structure of the isomerase domain of GlcN-6-P synthase from human pathogenic fungus Candida albicans, in complex with UDP-GlcNAc has been solved recently but it has not revealed the molecular mechanism of inhibition taking place under UDP-GlcNAc influence, the unique feature of the eukaryotic enzyme. UDP-GlcNAc is a physiological inhibitor of GlcN-6-P synthase, binding about 1 nm away from the active site of the enzyme. In the present work, comparative molecular dynamics simulations of the free and UDP-GlcNAc-bounded structures of GlcN-6-P synthase have been performed. The aim was to complete static X-ray structural data and detect possible changes in the dynamics of the two structures. Results of the simulation studies demonstrated higher mobility of the free structure when compared to the liganded one. Several amino acid residues were identified, flexibility of which is strongly affected upon UDP-GlcNAc binding. Importantly, the most fixed residues are those related to the inhibitor binding process and to the catalytic reaction. The obtained results constitute an important step toward understanding of mechanism of GlcN-6-P synthase inhibition by UDP-GlcNAc molecule