Szfingozin-1-foszfát: Intracelluláris célfehérjék és plazma-membrán transzport azonosítása = Sphingosine-1-phosphate: Identification its intracellular targets and plasma membrane transport

A jelátvitelben jelentős szerepet játszó lizofoszfolipidek, a szfingozin-1-foszfát (S1P), a szfingozin (Sph), a szfingozilfoszforilkolin (SPC) és a lizofoszfatidsav (LPA) sejten belüli célfehérjéinek azonosítása során kimutattuk, hogy a sejtek fő kalcium-érzékelő fehérjéje, a kalmodulin (CaM) szelektíven köti az aggregált SPC-t, melynek hatására a CaM funkciója gátlódik, mind apoCaM, mind Ca2+CaM esetén (Biochem J 2008). A CaM-SPC kölcsönhatás mechanizmusát és a komplex kristályszerkezetét meghatároztuk (J Biol Chem 2010, FASEB J 2010). Kimutattuk, hogy a Sph hasonló kölcsönhatásra lép a CaM-nal, és vizsgáltuk a CaM-SPC kötődés hatását a rianodin-receptor (RyR1)-általi intracelluláris kalcium mobilizációra. Kimutattuk, hogy a beta-2-mikroglobulin (b2M), amely vesedialízises betegekben amiloid lerakódásokat képez, szelektíven kölcsönhat az aggregált LPA-val, melynek hatására az egyébként stabil fehérje amiloidogén konformációt vesz fel (Biochemistry 2009). A CaM-SPC és a b2M-LPA kölcsönhatások a lipid-fehérje kötődés újfajta modelljeinek tekinthetők. Kimutattuk, hogy sejttípustól függően mind az ABCA1, mind az ABCC1 transzporterek képesek átjuttatni az S1P-t a plazmamembránon, legalábbis részlegesen, de ezeket az eredményeket kísérleteink befejezése előtt a nemzetközi szakirodalomban más kutatócsoportok közölték. A pályázati időszak végén ezért elkezdtük a Sph, S1P, SPC és LPA szállítófehérjéinek azonosítását egyes lipoproteinek szelektív kötési képességeinek vizsgálatával. | This work aimed at identifying the intracellular target proteins of important lysophospholipid mediators (sphingosine-1-phosphate, S1P, sphingosine, Sph, sphingosylphosphorylcholine, SPC, and lysophosphatidic acid, LPA) in signal transduction. We showed that calmodulin (CaM), the ubiquitous calcium sensor of cells, binds selectively to the aggregated SPC, resulting in the inhibition of CaM function in its apo as well as Ca2+-saturated forms (Biochem J 2008). We determined the crystal structure and the mechanism of formation of the complex (J Biol Chem 2010, FASEB J 2010). We showed that Sph similarly binds CaM and studied the role of CaM-SPC interaction in the intracellular Ca2+-mobilization through ryanodine receptor RyR1. We also identified the mechanism of interaction of beta-2-microglobulin (b2M) with aggregated LPA, inducing the amyloidogen conformation of the otherwise very stabile protein, leading to amyloid fibril deposits in patients on long-term dialysis (Biochemistry 2009). The CaM-SPC and b2M-LPA interactions represent a novel lipid-protein binding model. We showed that ABCA1 and ABCC1 proteins, depending on the cell-type, are able to partially transport S1P through plasma membrane. Unfortunately, these results had been published by other groups before we could finish our experiments. We have lately started the identification of carrier proteins of Sph, S1P, SPC, and LPA by investigating their selective binding to different lipoproteins

A fehérjeaggregáció és amiloidképződés szerkezeti alapjai; a különféle morfológiájú aggregátumok kialakulásának körülményei és in vivo hatásuk vizsgálata = Structural basis and thermodynamics of protein aggregation and amyloid formation; in vivo effect of aggregates of different morphologies

Fehérjeaggregációra, amiloidképződésre vezethető vissza számos neurodegeneratív betegség. Az újabb eredmények szerint e betegségek kialakulásában az amiloid formán kívül szerepet játszanak az egyéb aggregátumok, pl. az oligomerek, szférikus aggregátumok, protofilamentumok, amelyek toxikusabbak lehetnek az élő sejtek számára. Míg a natív, globuláris fehérjék foldingja jól ismert, keveset tudunk az amiloid szálak szerkezetéről, kialakulásuk mechanizmusáról, és az őket stabilizáló tényezőkről. Az általunk kidolgozott izotermális titrációs kalorimetriai eljárással és a fehérjeaggregátumok magas hőmérséklet indukálta disszociációjának vizsgálatával lehetővé vált az amiloidképződés termodinamikai jellemzése, a stabilitást meghatározó tényezők felderítése. Az amiloid-? peptid fragmentumaiból és más fehérjékből különféle morfológiájú aggregátumokat képeztünk és vizsgáltuk, a környezeti feltételek hatását, a morfológia és a szerkezet közötti kapcsolatot. Nem tisztázott, hogy az Alzheimer-kór korai szakaszában milyen aggregátumok felelősek a memóriafunkciók zavaráért és azok hogyan fejtik ki hatásukat. In vivo mérésekkel tanulmányoztuk a különféle aggregátumok idegrendszeri hatását. Eljárást dolgoztunk ki az aggregátumok oligomerizációs fokának fluoreszcens jelöléssel való detektálására. Felderítettük a lizofoszfolipidek szerepét az amiloidképződésben. A terv megvalósításához komplex biofizikai, molekuláris biológiai és elektrofiziológiai módszereket használtunk. | Protein aggregation is associated with several neurodegenerative diseases. It has become obvious recently that besides the amyloid form, other type of aggregates (oligomers, spheric aggregates, protofilaments) also play role in the development of the disease bearing increased toxicity for the living cell. While the folding of globular proteins has been well characterized, our knowledge is limited on the structure, mechanism of formation and the stabilizing interactions of protein aggregates. With our previously developed method using isothermal titration calorimetry and the novel heat-induced depolymerization technique, we studied the thermodynamics of amyloid formation and characterized the effect of the different conditions and factors on the stability of protein aggregates and amyloid fibrils. It has not been known well what type of aggregates is responsible for the memory impairment in the early stages of Alzheimer’s disease. We studied the in vivo effect of amyloid-beta peptides at various oligomerization levels on the brain by electrophysiology and proteomics. We developed a method for the detection of the oligomerization level of aggregates by fluorescence labeling. We characterized the role of lysophospholipids in amyloid formation. Our plan was achieved by using the complex repertoire of biophysics, molecular biology and electrophysiology

Lizofoszfolipid mediátorok receptorai. Szfingo- és glicerolipid növekedési faktorokat kötő fehérjék azonosítása és jellemzése. = Identification and characterization of specific proteins for sphingo- and glycerolipid growth factors

A legegyszerűbb foszfolipidek, a lizofoszfatidsav (LPA) és a szfingozin-1-foszfát (S1P) jelátvivő molekulák, amelyek részt vesznek a sejtek túlélésének, osztódásának és mozgásainak szabályozásában az egyszerű organizmusoktól az emberig. Kutatásaink célja ezen foszfolipidek hatásmechanizmusának megismerése, a kölcsönható fehérjék azonosítása. Az LPA és az S1P hatását elsősorban sejtfelszíni receptorokon keresztül fejti ki. Az LPA-receptortípusokra szelektív aktiváló és gátló molekulák két új csoportját azonosítottuk és jellemeztük: a zsíralkohol-foszfátokat és az oligoprenil-foszfátokat. Sikeresen azonosítottuk az S1P1-es típusú receptor ligandkötésében résztvevő apoláros aminosavakat, teljessé téve ezzel korábbi munkánkat a kötőhely feltérképezésére. Eredményeink a lizofoszfolipid receptorok farmakológiai vizsgálatának új lehetőségeit teremtik meg. Az S1P különlegessége, hogy másodlagos hírvivő is. Egyértelmű bizonyítékot szolgáltattunk arra, hogy az S1P valóban intracellulárisan is képes a kalcium-ionok felszabadítására. Legújabban kimutattuk, hogy az S1P-rokon szfingozilfoszforilkolin köti és gátolja a kalcium hatását univerzálisan közvetítő kalmodulint. Az S1P a sejtben keletkezik, tehát sejtfelszíni receptorai aktiválásához ki kell jutnia onnan. Vizsgáltuk egyes ABC-transzporterek szerepét ebben a folyamatban. Kimutattuk, hogy az MRP1-fehérje az S1P egyik lehetséges kipumpálója. E fehérje működése során fellépő kooperatív kölcsönhatásokat is azonosítottunk. | The simplest phospholipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lipid mediators regulating survival, proliferation, and migration of cells. Our aim is to characterize the mechanisms of action of these phospholipids and to identify their interacting proteins. LPA and S1P exert their actions mainly through cell surface G protein-coupled receptors. We identified two new sets of LPA receptor subtype selective ligands: fatty alcohol phosphates and oligoprenyl phosphates. We also successfully completed the mapping of the ligand binding pocket of the S1P1 receptor, by determining the amino acids lining the hydrophobic part of the binding site. Our results make new pharmacologic interventions possible at lysophospholipid receptors. S1P is unique because it also acts as a second messenger. We provided strict evidence for the action of S1P on the intracellular calcium mobilization from endoplasmic reticulum, independent of cell surface receptors. Very recently we have shown that the related lipid sphingosylphosphorylcholine binds to and inhibits the actions of calmodulin, the ubiquitous calcium sensor of cells. S1P is formed inside cells by the action of sphingosine kinases. It should leave the cell to act as an autocrine/paracrine mediator. We investigated the role of ABC transporters in this process and identified MRP1 as a potential efflux pump for S1P. We also identified cooperative interactions between the ATP and drug binding sites of MRP1

Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate

Functional analysis on a naturally occurring variant of the Staphylococcus Aureus uracil DNA Glycosylase inhibitor

Repair of DNA damage relies on various pathways including the base excision repair (BER) which targets erroneous bases in the DNA. Here, Uracil-DNA glycosylases (UDGs) are responsible for recognition and removal of uracil base from the DNA. Here, we characterize the interaction of Staphylococcus aureus UDG (SAUDG) with a naturally occurring variant of S. aureus uracil-DNA glycosylase inhibitor (SAUGI). This variant contains a histidine instead of a glutamate at the 24th position which affects the SAUDG:SAUGI interaction surface. We assessed the complex formation of SAUDG with these two SAUGI variants by independent biophysical methods. Our data reveal that the residue difference at the 24th position does not have a marked effect on the binding affinity, yet it confers alteration of the thermodynamics of the interaction. We propose that the E24H variant of SAUGI allows efficient complex formation, and consequently, inhibition of SAUDG. © 2018, Budapest University of Technology and Economics. All rights reserved

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging

Accumulation of the PX domain mutant Frank-ter Haar syndrome protein Tks4 in aggresomes

BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization. RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells. CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging