Módszerfejlesztés fehérje-glikoziláció analízisére = Method development for protein glycosylation analysis

Az extracelluláris glikoziláció tanulmányozása némiképp elhanyagolt kutatási terület. Ennek az egyik oka az elképesztő heterogenitás: egy adott pozíció hol módosított, hol nem, és számtalan különböző cukor-szerkezetet viselhet, így a glikopeptidek többnyire szubsztöchiometrikus mennyiségben fordulnak elő. Ráadásul a poszt-transzlációs módosítások vizsgálatára általában használatos tömegspektrometria is nehezebben boldogul a glikopeptidekkel. Mi szérum-fehérjék Ser és Thr oldalláncát módosító gyakori és egyszerű cukrok vizsgálatára fókuszáltunk. Marhaszérummal dolgoztunk. Egy dúsítási eljárást dolgoztunk ki egy cukorkötő-fehérje (lektin) segítségével. A glikopeptid-elegyet egy új MS/MS technika: elektron-transzfer disszociáció (ETD) segítségével analizáltuk. Ennek sikeréhez az adatbázis-lekereső szoftvert is optimalizálni kellett az ETD adatokhoz. Kutatásunk során kb. 40 új glikozilációs helyet azonosítottunk. Ennyit eddig senkinek nem sikerült egyetlen kísérlet-sorozatból. Sejten belül is előfordul O-glikoziláció, egyetlen GlcNAc kerül a Ser/Thr oldalláncokra, regulációs és jelátviteli szerepe van. Bár biológiai szempontból nagyon eltér az extracelluláris rokonságtól, hasonló analitikai kihívást jelent. Erre a módosításra is kidolgoztunk egy dúsítási eljárást. | Studying extracellular glycosylation is a somewhat neglected research area. Partly because the incredible heterogeneity glycoproteins feature both in site occupancy and in the number of different sugar structures modifying the same site. Thus, glycopeptides almost always are present in substoichiometric quantities. In addition, these modifications are a bit difficult to tackle with mass spectrometry that is generally used for the analysis of post-translational modifications. We focused on some simple and frequently occurring sugars modifying the side-chains of Ser and Thr residues of serum proteins. We worked with bovine serum. We developed an enrichment method using a carbohydrate-binding protein (lectin). We characterized the glycopeptide mixtures utilizing a novel MS/MS technique, electron-transfer dissociation (ETD). For this purpose the softwer used for database searching also had to be optimized. We identified ~40 novel glycosylation sites, more than anybody ever assigned in a single study. O-glycosylation occurs within the cell too: a single GlcNAc is deposited on Ser/Thr side chains. It fulfills a regulatory, signaling function. Though biologically very distant from its extracellular relatives, it represents a similar analytical challenge. We developed an enrichment method for this modification too

Alzheimer-kór elleni béta-amiloid antagonista peptidek előállítása és vizsgálata = Synthesis and investigation of beta-amyloid peptide antagonists against Alzheimer`s disease

Szilárdfázisú peptid szintézis módszer és Boc-stratégia alkalmazásával állítottuk elő az új, β-amiloid antagonista peptideket. Tisztítás után igazoltuk a szerkezetüket és tisztaságukat. Előállítottuk: 1) a korábban neuroprotektív hatást mutató vegyületünk, a propionil-RIIGL-NH2 pentapeptid 11 szabad N-terminálisú és 22 acilezett, valamint 23 tripeptid és 3 dipeptid származékát és 2) az irodalomban „beta sheet breaker” (BSB) hatásúnak leírt LPFFD pentapeptid analógjait: 19 penta- és 75 tetrapeptid ill peptidomimetikum származékát. A származékok D-aminosavakat, nem természetes aminosavakat, aminokat és karbonsav származékokat tartalmaznak. Kidolgoztuk az Aß(1-42) racionális szintézisét, amellyel lehetővé vált nagy mennyiségű Aß(1-42) előállítása a biológiai vizsgálatok számára. A β-amiloid antagonista peptidek neuroprotektív hatását differenciált humán neuroblasztóma sejt tenyészeten (SH-SY5Y) és primer sejtkultúrán MTT teszttel és mikrofluoreszcenciás imaging technikával (in vitro) és elektrofiziológiai módszerrel in vivo körülmények között vizsgáltuk meg. Mivel voltak közöttük kiemelkedő neuroprotektív hatású vegyületek, a 75 új tetrapeptid- és peptidomimetikumra a Biogal-TEVA gyárral közös szabadalmat jelentettünk be. A P29 és P59 kódszámú vegyületek bármelyike az Alzheimer-kór potenciális gyógyszere lehet, amely a kór progresszióját megállítani vagy legalább lassítani képes. | The new antagonistic Aß peptides were synthesized by solid phase method using Boc-strategy. After purification with RP-HPLC, their structure and purity was confirmed by analytical HPLC, amino acid analysis, and ESI-MS measurements. We synthesized: 1) eleven pentapeptides with free N-terminus, 22 acylated analogues, 23 tri-, and 3 dipeptides. These peptides are derivatives of propionyl-RIIGL-NH2 which previously showed neuroprotective effect against Aß(1-42). 2) the analogues of the known "beta sheet breaker" (BSB) LPFFD: 19 penta- and 75 tetrapeptides and peptidomimetics. In order to increase resistance against enzymes, the derivatives had D- and unnatural amino acids, amines, and derivatives of carboxylic acids. We worked out the rationale synthesis of Aß(1-42) to get large quantity of this peptide for biological tests. The neuroprotective effects of the new antagonistic Aß peptides were tested by MTT test and microfluorescence imaging assay (in vitro) on differentiated human neuroblastoma cells (SH-SY5Y) and on primary cell culture and by electrophysiological mesurements in vivo. Together with Biogal-Teva Company, we filed a patent application for the 75 tetrapeptide and peptidomimetics as some of them were highly active both in vitro and in vivo. Compounds P29 and P59 are potential drugs against Alzheimer's disease since they might stop or delay the progression of the disease

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

The very existence of extracellular phosphorylation has been questioned for a long time, although casein phosphorylation was discovered a century ago. In addition, several modification sites localized on secreted proteins or on extracellular or lumenal domains of transmembrane proteins have been catalogued in large scale phosphorylation analyses, though in most such studies this aspect of cellular localization was not considered. Our review presents examples when additional analyses were performed on already public datasets that revealed a wealth of information about this "neglected side" of the modification. We also sum up accumulated knowledge about extracellular phosphorylation, including the discovery of Golgi-residing kinases and the special difficulties encountered in targeted analyses. We hope future phosphorylation studies will not ignore the existence of phosphorylation outside of the cell, and further discoveries will shed more light on its biological role

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (lmax = 730 nm) and inactive Pr (lmax = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of lightdependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyBSer86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyBSer86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling

The Arabidopsis Rho of Plants GTPase ROP1 is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test

Differential phosphorylation of the N-terminal extension regulates phytochrome B signaling

Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants' light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr-Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N-terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro

AbstractPlants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta

Differential phosphorylation of the N - terminal extension regulates phytochrome B signaling

Viczián, András. Institute of Plant Biology. Biological Research Centre. Szeged, Hungary.Ádám, Éva. Institute of Plant Biology. Biological Research Centre. Szeged, Hungary.Staudt, Anne Marie. University of Freiburg. Institute of Biology II. Freiburg, Germany.Lambert, Dorothee. University of Freiburg. Institute of Biology II. Freiburg, Germany.Klement, Eva. Biological Research Centre. Laboratory of Proteomics Research. Szeged, Hungary.Romero Montepaone, Sofia. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura (IFEVA). Buenos Aires, Argentina.Hiltbrunner, Andreas. University of Freiburg. Institute of Biology II. Freiburg, Germany.Casal, Jorge José. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura (IFEVA). Buenos Aires, Argentina.1635–1650Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants’ light sensitivity. In this study we identified several serine/threonine residues on the N-terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr–Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling

The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone