Age-Related Differences in Plasma Proteins: How Plasma Proteins Change from Neonates to Adults

The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.GE Healthcare Life Sciences Australia funded Sherif Tawfilis' time in the initial laboratory aspects of this project, some aspects of data analysis and preparation of the manuscript. This study was funded by internal Haematology Research Team funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order and molecular chaperone activity

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer–monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.This work was supported in part by Australian National Health and Medical Research Council Project Grant 1068087 (to J. A. C.), National Health and Medical Research Council Program Grant 1071897 (to A. F. L.), and the Fay Fuller Foundation

Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells

The misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous publishedmethodswere adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate ofα-synuclein fibrillization and produces species that harbourenhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live nontransgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfoldedα-synucleincauses hyperactive mitochondrial respiration without causing any functional deficit.These datagive strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies

Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation

The consolidated European synthesis of CO2emissions and removals for the European Union and United Kingdom : 1990-2018

Acknowledgements FAOSTAT statistics are produced and disseminated with the support of its member countries to the FAO regular budget. Philippe Ciais acknowledges the support of the European Research Council Synergy project SyG-2013-610028 IMBALANCE-P and from the ANR CLAND Convergence Institute. We acknowledge the work of the entire EDGAR group (Marilena Muntean, Diego Guizzardi, Edwin Schaaf and Jos Olivier). We acknowledge Stephen Sitch and the authors of the DGVMs TRENDY v7 ensemble models for providing us with the data. Financial support This research has been supported by the H2020 European Research Council (grant no. 776810).Peer reviewedPublisher PD

Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608–621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a ‘back-to-back’ dimer of dimers compared to the ‘head-to-head’ architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a ‘back-to-back’ homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter