EGF regulates tyrosine phosphorylation and membrane-translocation of the scaffold protein Tks5

Background: Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.Results: Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.Conclusions: Our results identify Tks5 as a novel component of the EGF signaling pathway. © 2013 Fekete et al.; licensee BioMed Central Ltd

Különböző antibiotikumok emissziós tulajdonságainak analízise csont-transzplantátumokból és csontpótló anyagokból = Analysis of antibiotic emission properties from different bone transplants and bone substitute materials

Kidolgoztunk egy standard, in vitro vizsgálómódszert, mellyel vizsgáltuk különböző csontcementek és antibiotikumok kioldódási tulajdonságait. Az általunk használt csontcementből antibakteriális hatással rendelkező antibiotikum áramlik ki, mely exponenciális módon csökken ugyan, de in vitro körülmények között még egy év után is mérhető mennyiségű antibiotikumot bocsát ki. A kiáramlásban nem találtunk szignifikáns különbséget a vizsgált különböző típusú csontcementek, antibiotikumok között, azonban szignifikánsan jelentősebb antibiotikum kiáramlást észleltünk az összekeverés módozatát vizsgálva a "házi", manuális technikával összekevert antibiotikum-cement komplex esetén, a gyári komplexel összehasonlítva. A sebváladékban az átlagos antibiotikum mennyiség 24 órán át a terápiás mennyiség felett volt. A revíziók során eltávolított csontcement darabok is képesek antibiotikumot kibocsátani magukból évek múltán. Megállapítottuk az ortopédiában rutinszerűen transzplantációra használt különböző allogén szövetek antibiotikus abszorpciós és emissziós képességeit. Meghatároztuk a nálunk előforduló sebfertőzések és mélyinfekciók valódi arányáról, a kórokozók típusáról és azok megoszlásáról. Azt találtuk, hogy a láz az operált betegeink mintegy 35%-ánál fellépett, bár valódi igazolt mélyinfekció csak 2.3%-ban fordult elő. Más infekciós jelek jelenléte nélkül tehát az antibiotikumos kezelések nem szükségesek. | W examined the storing and emitting parameters of different bone-cement - antibiotic complexes. We managed to evaluate a standard, reproducible in vitro method that can be used to assess the emission properties of different bone-cements. We have found that the bone-cement is capable of emitting antibiotics. However, the release decreases exponentially but a detectable amount of antibiotics is present even a year after mixing in vitro. In the in vivo experiments we concluded that the amount of antibiotics in the wound exudates were above the therapeutic range after 24 hours, and in large proportion of cases this level was reached even after 48 hours. The bone-cement fragments removed during revision are capable of emitting antibiotics. We captured the absorption and emission characteristics of antibiotics in different allogenic tissues routinely used in the orthopaedic tissue transplantation, which formerly was not addressed in the literature. Since the treatment of periprosthetic infections is particularly difficult, the importance of decreasing the infection rate is extremely significant. The concept of antibiotic mixed with the implanted bone-cement goes back to the seventies and has been a well-accepted method in orthopaedics ever since. Our experiments provided additional data for the better understanding and the use of every day routine of such a prevention

Lehetőségek és buktatók a SARS-CoV-2-antigén és az ellene termelt antitestek kimutatásában = Possibilities and pitfalls of virus antigen and antibody detection of SARS-CoV-2

Összefoglaló. A koronavírus-betegség 2019 (COVID–19)-pandémia komoly kihívás elé állította nemcsak a mikrobiológiai laboratóriumokat, hanem az eredmények interpretálásában a klinikumban dolgozó kollégákat is. Az orvostudomány specializált világában az immunológiai és a fertőző betegségekkel kapcsolatos ismeretek az antimikrobás terápiás megoldások sikeressége, valamint a széles körű vakcináció miatt az idők folyamán számos szakterületen háttérbe szorultak, felfrissítésük sürgető és elengedhetetlen része a pandémiával való megküzdésnek. A diagnosztikai vizsgálatok fontos eszközei a járvány megfékezésének, illetve a betegek ellátásának, azonban a vírus és az emberi szervezet interakciójának megértése elengedhetetlenül szükséges a korrekt epidemiológiai és gyógyászati véleményalkotáshoz. Jelen cikkünk az orvosi gyakorlat számára foglalja össze a súlyos akut légzőszervi szindrómát okozó koronavírus-2 (SARS-CoV-2) kimutatására, valamint az immunrendszer specifikus immunválaszának szerológiai vizsgálatára irányuló, gyakorlatban használatos módszereket, azok helyét, szerepét és értékelésük szempontjait a tudomány jelen állása szerint. Orv Hetil. 2021; 162(15): 563–570. Summary. The coronavirus disease 2019 (COVID-19) pandemic posed a serious challenge not only for microbiology laboratories, but also for the clinicians in interpretation of the results. In the specialized world of medicine, knowledge of immunological and infectious diseases has been relegated to the background in many disciplines over time due to the success of antimicrobial therapies and widespread vaccination, so updating them is an urgent and essential part of the fight against the pandemic. Diagnostic tests are important tools for controlling the epidemic and caring for patients, but understanding the interaction between the virus and the human body is essential to form a correct epidemiological and medical opinion. This paper summarizes the medical methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the serological testing of the specific immune response of the immune system, their place, role and criteria of their evaluation according to current scientific knowledge. Orv Hetil. 2021; 162(15): 563–570

Reaktív oxigén származékok szerepe a fibrózis kialakulásában = Reactive oxygen species in the development of organ fibrosis

Kutatásaink legfontosabb eredményei a következők: 1. Kimutattuk, hogy a peroxidazin fehérje expressziója fokozódik a miofibroblasztok differenciálódása folyamán és a fehérje szekretálódik a sejtek közötti térbe. Azt is kimutattuk, hogy a vese fibrotikus átalakulása során a peroxidazin felszaporodik a tubulus hámsejtek közötti térben. A peroxidazin sejtek közötti térbe történő szekréciója fontos, eddig ismeretlen eleme lehet a szöveti fibrózisnak. 2. Kimutattuk, hogy az emlős peroxidázok családjába tartozó laktoperoxidáz enzim hatékonyan katalizálja tirozin aminosavak összekapcsolását. Az emlős peroxidázok ditirozin-képző aktivitásának szerepe lehat a sejtek közötti állomány módosításában. 3. A NADPH oxidáz enzimcsalád Duox1 tagjáról kimutattuk, hogy szerepet játszhat a húgyhólyag hámsejtjeinek jelátviteli folyamataiban. 4. Elsőként mutattuk ki élő sejtekben, hogy az endoplazmás retikulum lumenében magas a H2O2 szintje, ami elsősorban az Ero-1L enzim aktivitásának köszönhető és független a Nox enzimek aktivitásától. 5. Genetikai modellekkel alátámasztva kimutattuk, hogy a fibroblaszt-miofibroblaszt átalakulás közben megfigyelhető H2O2 termelés a Nox4-p22phox enzimkomplex aktivitásának köszönhető. | The most important results of the research project are the followings: 1. We demonstrated the increased expression and secretion of peroxidasin during myofibroblastic differentiation. We showed that during the course of kidney fibrosis, peroxidasin accumulates in the peritubular space. The secretion of peroxidasin represents a previously unknown mechanism in tissue fibrosis. 2. We showed that lactoperoxidase, a member of the mammalian peroxidase family, efficiently catalyzes the formation of dityrosine residues. Dityrosine formation by mammalian peroxidases may play a role in the modification of the extracellular matrix. 3. We showed that the NADPH oxidase Duox1 has a role in the signaling mechanisms of urothelial cells. 4. We were the first to show in live cells that lumen of the mammalian endoplasmic reticulum is highly oxidative. The high level of H2O2 in the lumen is mainly due to Ero-1L activity and seems to be independent of Nox enzymes. 5. Using genetic models we showed that H2O2 production during myofibroblastic differentiation is due to the activity of the Nox4-p22phox enzyme complex

Interaction between p22(phox) and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22(phox) protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22(phox) in the TGF-beta1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-beta1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22(phox). Interestingly, the p22(phox) protein was present in the absence of any detectable Nox/Duox expression, and the p22(phox) level was unaffected by TGF-beta1. On the other hand, Nox4 expression was dependent on the presence of p22(phox), establishing an asymmetrical relationship between the two proteins. Nox4 and p22(phox) proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-beta1. We used a chemically induced protein dimerization method to study the orientation of p22(phox) and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle

The Homolog of the Five SH3-Domain Protein (HOFI/SH3PXD2B) Regulates Lamellipodia Formation and Cell Spreading

Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin

Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking

Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking. ©2015 Published by Elsevier Inc

Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically-encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, named MetSOx and MetROx for their ability to detect S and R-forms of MetO, respectively, were utilized for targeted analysis of protein oxidation, regulation and repair, as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO, and examining responses to physiological stimuli

Peroxidasins: novel players in tissue genesis

Stabilization of extracellular matrix by protein crosslinking is a universal and essential process in multicellular organisms. Recent findings revealed that peroxidasin, a unique heme-peroxidase, produces hypohalides to support matrix synthesis. Unexpectedly, the highly reactive and potentially damaging hypohalides mediate the formation of sulfilimine bonds between adjacent collagen IV protomers. This crosslink is a fundamental feature of basal membranes, defining peroxidasin-dependent oxidant generation and sulfilimine crosslink formation as an elemental mechanism of tissue biogenesis. © 2014 Elsevier Ltd. All rights reserved