Egy növényi G2/M fázis specifikus promóter izolálása és jellemzése: Hogyan szabályozódik a Cdc2Ms F kináz? = Isolation and Characterization of a G2/M Phase-Specific Plant Promoter: How is the Cdc2MsF kinase regulated?

Csak a növények rendelkeznek sejtciklus-specifikusan, a G2/M fázisban expresszálódó ciklin függő kinázokkal (CDKB-k). Klónoztuk és funkcionálisan jellemeztük a lucerna CDKB2;1 kináz előtti kb. 360 bp hosszú upstream régiót. Bizonyítottuk, hogy ez a szakasz képes a riporter gének expresszióját az osztódó merisztematikus sejtekre korlátozni transzgén lucerna növényekben, és G2/M fázis specifikus expressziót biztosítani szinkronizált, lucerna transzgén növényekből generált sejtszuszpenzióban. Immunohisztokémiai festéssel igazoltuk, hogy transzgén növény gyökerében a riporter gének az endogén kináznak megfelelő mintázatban voltak jelen. Megállapítottuk, hogy az eddig szigorúan sejtciklus-specifikus szabályozásúnak hitt CDKB2;1 kináz a sebzés hatására is expresszálódik. Az etilén, amely egyik lehetséges sebzés-válasz mediátor, szintén bekapcsolta a promotert, de sem a jázminsav, sem a szalicilsav nem hatott a promoter szabályzására, amelyek még lehetséges szignálátvivői lehetnek a sebzésnek. A sebzés, és az etilén mellett az auxin indukció is hatott a CDKB2;1 kináz expressziójára. A sebzéssel indukált transzkripciós válasz gyorsabb volt az auxin indukált transzkripciónál, és sejtciklus- gátlószer jelenlétében csak a sebzés kapcsolta be a promotert. Továbbá, a promoter in silico analízisével mind a sejtciklus szabályozó elemeket, mind sebzés- indukciós faktorokat sikerült azonosítani a promoter szekvenciájában. | Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases, however, their exact functions are still enigmatic. We describe the functional characterization of a 360 bp promoter region of the alfalfa CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analysed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter, indeed, revealed the presence of a set of cis-elements indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific cyclin-dependent kinase genes in plants

A syndecan és decorin proteoglikánok kóroki szerepének modellezése májbetegségekben = Experimental models to study the significance of syndecan-1 and decorin in the development of liver diseases

Az elmúlt 4 évben olyan modellrendszereket dolgoztunk ki, melyekben a proteoglikánok kifejeződését megváltoztattuk. Kialakitottunk egy decorin -/- TGF béta-1 +/- kettős transzgén egértörzset, melyen a cirrhosis és a hepatokarcinogenezis eseményeit tanulmányoztuk. Eredményeiket a vad típusú és egyszeres transzgén állatokével hasonlítottuk össze. Megállapítottuk, hogy a decorin hiányában a thioacetamid indukált májcirrhosis súlyosabb és nehezebben gyógyul, és az állatokban több daganat alakul ki. A DEN indukált májrákok kialakulását szintén elősegíti a decorin hiánya, de kisebb mértékben. A syndecan-1 fehérje szerepét in vitro rendszerben hepatóma és fibroszarkóma sejteken és in vivo szájüregi laphámrákokon tanulmányoztuk. Megállapítottuk, hogy a hepatomákban a csonkolt syndecan a tumorvonal differenciációját idézi elő, melynek hátterében az Ets-1 protoonkogén gátlása áll. Ugyanakkor a fibroszarkóma sejtek agresszivitását a syndecan-1 növeli, egérbe oltva gyorsabban nő és tüdőáttéteket ad. Itt Ets-1 érintettséget nem találtunk.. Adataink szerint tehát a syndecan-1 hatása attól függ, milyen jelátviteli utakkal lép kapcsolatba a fehérje. A molekuláris mechanismus további tanulmányozására folyamatban van egy a syndecan-1-t a májban kifejező transzgén egér kialakítása. Kerestük, milyen más proteoglikánok játszhatnak szerepet májbetegségekben. Felfedeztük, hogy a bazális membrán heparánszulfát agrin alkalmas a májrákok és az epeútrákok kimutatására és ekülönítésére. | In the last four years we attempted to establish experimental models help to explain our observations foind on human material. We created decorin -/- TGF beta-1 +/- double transgenic mice and used them to induce cirrhosis and cancer. They were compared with wild type and single transgenic animals. We found that decorin KO animals are much more prone to develop cirrhosis and heal slower after the withdrawal of the toxic agent thioacetamide. Furthermore we found much more cancer in their liver. The lack of decorin promotes the development of DEN induced cancer, as well, but in a less extent. The role of syndecan-1 was studied in an in vitro system, where the wild type and truncated versions of syndecan-1 was cloned into expression vector and transfected into hepatoma and fibrosarcoma cell lines. The truncated form of syndecan-1 induced differentiation of hepatoma cell lines, and the central factor in this phenomenon was the downregulation of Ets-1 protooncogen. We proved that the Ets-1 regulates the amount of syndecan and syndecan-1 exerts negative feedback to the transcription factor. This is not true for the HT1080 fibrosarcoma, where syndecan-1 increased the aggressivity of the tumor. To study the role of this proteoglycan a syndecan-1 transgenic mice is in progress. We were proceeding with the characterization of proteoglycan pattern of liver diseases. A new marker the heparan sulfate proteoglycan agrin was was descibed as a useful tool to detect liver cancer

Comparative approach for the isolation of genes involved in the osmotolerance of wheat

Improving drought tolerance of wheat is of great agronomical importance. Gene isolation techniques based on expression properties may provide new tools for breeders both in early characterization of new cultivars and in improving drought tolerance via molecular breeding. The main aim of our project is to isolate new drought activated genes which may serve both purposes. As a first step, a subtracted cDNA library was prepared, which represents the difference in the mRNA populations of wheat plantlets grown under 400 mOsm polyethylene-glycol derived osmotic stress versus plantlets grown in optimal conditions. By applying the subtraction approach, the resulted cDNA library becomes enriched in clones of differentially expressed genes including the ones of rare messages as well. This allows us to clone these genes, sequence them and, later, perform in silico analysis. Our first results indicate that the resulted library is enriched in clones coding for membrane associated channel proteins as well as abscisic acid and stress responsive ones

Contribution of syndecans to the cellular entry of SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here, we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans, facilitate the cellular entry of SARS-CoV-2. Among syndecans, the lung abundant syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus’s interactions with syndecans. Besides the polyanionic heparan sulfate chains, other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing the cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offer molecularly precise yet simple strategies to overcome the complex nature of SARS-CoV-2 infection

The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro , p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis

Contribution of syndecans to cellular uptake and fibrillation of alpha-synuclein and tau

Scientific evidence suggests that alpha-synuclein and tau have prion-like properties and that prionlike spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of alpha-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of alpha-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both alpha-synuclein and tau. Syndecan-mediated internalization of alpha-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monomeric alpha-synuclein and tau, yet exerts a fibril forming effect on both proteins. Data obtained from syndecan overexpressing cellular models presents syndecans, especially the neuron predominant syndecan-3, as important mediators of seeding and spreading of alpha-synuclein and tau and reveal how syndecans contribute to fundamental molecular events of a-synuclein and tau pathology

Syndecan-4 Mediates the Cellular Entry of Adeno-Associated Virus 9

Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9’s cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of syndecans in AAV9’s cellular entry. The ubiquitously expressed isoform, syndecan-4 proved its superiority in facilitating AAV9 internalization among syndecans. Introducing syndecan-4 into poorly transducible cell lines enabled robust AAV9-dependent gene transduction, while its knockdown reduced AAV9’s cellular entry. Attachment of AAV9 to syndecan-4 is mediated not just by the polyanionic heparan-sulfate chains but also by the cell-binding domain of the extracellular syndecan-4 core protein. Co-immunoprecipitation assays and affinity proteomics also confirmed the role of syndecan-4 in the cellular entry of AAV9. Overall, our findings highlight the universally expressed syndecan-4 as a significant contributor to the cellular internalization of AAV9 and provide a molecular-based, rational explanation for the low gene delivery potential of AAV9 into the CNS

Syndecan-4 influences mammalian myoblast proliferation by modulating myostatin signalling and G1/S transition

Myostatin, a TGF-beta superfamily member, is a negative regulator of muscle growth. Here we describe how myostatin activity is regulated by syndecan-4, a ubiquitous transmembrane heparan sulfate proteoglycan. During muscle regeneration the levels of both syndecan-4 and promyostatin decline gradually after a sharp increase, concurrently with the release of mature myostatin. Promyostatin and syndecan-4 co-immunoprecipitate, and the interaction is heparinase-sensitive. ShRNA-mediated silencing of syndecan-4 reduces C2C12 myoblast proliferation via blocking the progression from G1- to S-phase of the cell cycle, which is accompanied by elevated levels of myostatin and p21(Waf1/Cip1), and decreases in cyclin E and cyclin D1 expression. Our results suggest that syndecan-4 functions as a reservoir for promyostatin regulating the local bioavailability of mature myostatin. This article is protected by copyright. All rights reserved

Cell-penetrating peptide exploited syndecans

AbstractCell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies

The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro , p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis