cAMP promotes the synthesis in early G1 of gp115, a yeast glycoprotein containing glycosyl-phosphatidylinositol.

The glycoprotein gp115 (Mr = 115,000, pI 4.8-5) is localized in the plasma membrane of Saccharomyces cerevisiae cells and maximally expressed during G1 phase. To gain insight on the mechanism regulating its synthesis, we have examined various conditions of cell proliferation arrest. We used pulse-labeling experiments with [35S]methionine and two-dimensional gel electrophoresis analysis, which allow the detection of the well characterized 100-kDa precursor of gp115 (p100). In the cAMP-requiring mutant cyr1, p100 synthesis is active during exponential growth, shut off by cAMP removal, and induced when growth is restored by cAMP readdition. The inhibition of p100 synthesis also occurs in TS1 mutant cells (ras1ras2-ts1) shifted from 24 to 37 degrees C. During nitrogen starvation of rca1 cells, a mutant permeable to cAMP, p100 synthesis is also inhibited. cAMP complements the effect of ammonium deprivation, promoting p100 synthesis, even when added to cells which have already entered G0. Experiments with the bcy1 and cyr1bcy1 mutants have indicated the involvement of the cAMP-dependent protein kinases in the control of p100 synthesis. Moreover, the synthesis of p100 was unaffected in A364A cells, terminally arrested at START B by alpha-factor. These results indicate that the switch operating on p100 synthesis is localized in early G1 (START A) and is one of the multiple events controlled by the cAMP pathway

Contact Lens Wear Induces Alterations of Lactoferrin Functionality in Human Tears

The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

The intrinsically disordered and amyloidogenic protein α-synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nanoelectrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as oxidation

Disulfide driven folding for a conditionally disordered protein

Altres ajuts: ICREA, ICREA-Academia 2015 to S.V.Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity

α-Synuclein is a Novel Microtubule Dynamase.

α-Synuclein is a presynaptic protein associated to Parkinson's disease, which is unstructured when free in the cytoplasm and adopts α helical conformation when bound to vesicles. After decades of intense studies, α-Synuclein physiology is still difficult to clear up due to its interaction with multiple partners and its involvement in a pletora of neuronal functions. Here, we looked at the remarkably neglected interplay between α-Synuclein and microtubules, which potentially impacts on synaptic functionality. In order to identify the mechanisms underlying these actions, we investigated the interaction between purified α-Synuclein and tubulin. We demonstrated that α-Synuclein binds to microtubules and tubulin α2β2 tetramer; the latter interaction inducing the formation of helical segment(s) in the α-Synuclein polypeptide. This structural change seems to enable α-Synuclein to promote microtubule nucleation and to enhance microtubule growth rate and catastrophe frequency, both in vitro and in cell. We also showed that Parkinson's disease-linked α-Synuclein variants do not undergo tubulin-induced folding and cause tubulin aggregation rather than polymerization. Our data enable us to propose α-Synuclein as a novel, foldable, microtubule-dynamase, which influences microtubule organisation through its binding to tubulin and its regulating effects on microtubule nucleation and dynamics

Insights on peptide topology in the computational design of protein ligands: the example of lysozyme binding peptides

Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Sudden Unexpected Deaths and Vaccinations during the First Two Years of Life in Italy: A Case Series Study

Background The signal of an association between vaccination in the second year of life with a hexavalent vaccine and sudden unexpected deaths (SUD) in the two days following vaccination was reported in Germany in 2003. A study to establish whether the immunisation with hexavalent vaccines increased the short term risk of SUD in infants was conducted in Italy. Methodology/Principal Findings The reference population comprises around 3 million infants vaccinated in Italy in the study period 1999–2004 (1.5 million received hexavalent vaccines). Events of SUD in infants aged 1–23 months were identified through the death certificates. Vaccination history was retrieved from immunisation registries. Association between immunisation and death was assessed adopting a case series design focusing on the risk periods 0–1, 0–7, and 0–14 days after immunisation. Among the 604 infants who died of SUD, 244 (40%) had received at least one vaccination. Four deaths occurred within two days from vaccination with the hexavalent vaccines (RR = 1.5; 95% CI 0.6 to 4.2). The RRs for the risk periods 0–7 and 0–14 were 2.0 (95% CI 1.2 to 3.5) and 1.5 (95% CI 0.9 to 2.4). The increased risk was limited to the first dose (RR = 2.2; 95% CI 1.1 to 4.4), whereas no increase was observed for the second and third doses combined. Conclusions The RRs of SUD for any vaccines and any risk periods, even when greater than 1, were almost an order of magnitude lower than the estimates in Germany. The limited increase in RRs found in Italy appears confined to the first dose and may be partly explained by a residual uncontrolled confounding effect of age

Liquid-Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus-Host Interactions

International audienceIntrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid-liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections