Lucerna CDK inhibitor fehérje poszttraszlációs módosítása és ennek hatásai = Post-translational regulation of an alfalfa CDK inhibitor protein

Megállapítottuk, hogy az MsCPK3 a KRPMt 91. szerin aminosavát foszforilálja. Kísérleteink során bebizonyosodott, hogy az A-típusú CDK-k ugyanezen az aminosavon foszforilálják a lucerna CDK inhibitor fehérjét. A B-típusú CDK foszforiláció különbözőséget vagy azonosságát technikai problémák nem tették lehetővé, lévén ezen kinázokkal nem tudtunk megfelelően erős kináz aktivitást detektálni a KRPMt foszforilációja esetén. Többszöri kísérletek ellenére sem sikerült ubiqitinálást kimutatni sem a vad típusú sem a foszforilációt mimikáló rekombináns fehérjék esetében. Elkészítettük a 91. szerin aminosav foszforilációt mimikáló glutamát valamint aszpartát mutánsait. Valamint a nem foszforilálható alanin mutánst is. Ezen rekombináns fehérjék egyike sem volt foszforilálható sem az MsCPK rekombináns fehérjével, sem az A-típusú CDK-kal. Kináz gátlási kísérleteink során a foszforilációt mimikáló szerin aszpartát csere jelentös mértékben (kétszeresére) fokozta az inhibitor fehérje gátló képességét. Elkészítettük a mutáns fehérjék növényi expressziójához szükséges vektorokat, melyekkel dohány levél korongokat transzformáltunk. A GUS pozitív kalluszok regenerációja folyamatban van, azonban sajnálatos módon a legígéretesebb, legerősebb expressziót mutató kalluszok esetében jelentős degenerációt tapasztaltunk nem képesek gyökeret létrehozni. A kutatás idő előtt lezárásra kerül, mert külföldre távoztam és a halasztási kérelmemet nem támogatta az OTKA Bizottság Elnöke. | We found that the MsCPK3 Ca-dependent protein kinase could phosphorylate the KRPMt CDK inhibitor on a serine residue at position 91. Our experiments demonstrated that the A-type CDKs phosphorylate the same amino acid residue in alfalfa CDK inhibitor proteins. We were not able to reveal the kinase activity of the B-type CDK on KRPMt due to the fact that we could not purify sufficiently strong activity for clarifying KRPMt phosphorylation event. We made 91 serine phosphorylation mimicking mutants: 91Ser-91Glu and 91Ser-91Asp, and phosphoinactive 91Ser-91Ala mutants as well. None of those recombinant proteins were phosphorylated by MsCPK3 or A-type CDKs. We could not detect ubiqitination and the subsequent degradation of either the wild-type or the phosphorylation mimicking and phosphoinactive recombinant proteins. In kinase inhibition experiments, the 91Ser-91Asp recombinant protein showed significantly (twice) increased inhibitory effect on A-type CDKs. We have created the plant expression vectors with the wild type and mutant KRPMts, which were used for tobacco leaf discs transformation. The regeneration of whole plants from GUS positive calli is in progress, but unfortunately, the most promising calli -showing the strongest expression of GUS - have significant degeneration: they can not form roots. The time before the research is completed, I left for abroad and the application for postponing the project was not supported by the president of the OTKA committee

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

The DREAM complex represses growth in response to DNA damage in Arabidopsis

The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage. © 2021 Rockefeller University Press. All rights reserved

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

Drosophila small ovary gene is required for transposon silencing and heterochromatin organization, and ensures germline stem cell maintenance and differentiation

Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organization. In our study, we describe a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. We demonstrate that soy is required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. soy maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Soy colocalizes with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position-effect variegation assay, we found a dominant genetic interaction between soy and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. We propose a model in which soy maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation

PP7L is essential for MAIL1-mediated transposable element silencing and primary root growth

The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells

Contribution of syndecans to cellular internalization and fibrillation of amyloid-β (1–42)

Intraneuronal accumulation of amyloid-beta(1-42) (A beta 1-42) is one of the earliest signs of Alzheimer's disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of A beta 1-42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of A beta 1-42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of A beta 1-42 the most. Kinetics of A beta 1-42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased A beta 1-42 uptake from the earliest time points, while other syndecans facilitated A beta 1-42 internalization at a slower pace. Internalized A beta 1-42 colocalized with syndecans and flotillins, highlighting the role of lipid-rafts in syndecan-mediated uptake. Syndecan-3 and 4 also triggered fibrillation of A beta 1-42, further emphasizing the pathophysiological relevance of syndecans in plaque formation. Overall our data highlight syndecans, especially the neuron-specific syndecan-3 isoform, as important players in amyloid pathology and show that syndecans, regardless of cell type, facilitate key molecular events in neurodegeneration

Widely conserved AHL transcription factors are essential for NCR gene expression and nodule development in Medicago

Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids which can be reversible or terminal. The latter is controlled by the plant, it is more beneficial and has evolved in multiple clades of the Leguminosae family. The plant effectors of terminal differentiation in inverted repeat-lacking clade legumes (IRLC) are nodule-specific cysteine-rich (NCR) peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCR s co-evolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR -specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in its nodule-specific expression, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear Localized (AHL) transcription factors from both M. truncatula and soybean nodules that bound to AT-rich sequences in the NCR169 promoter inducing its expression. Whereas mutation of NCR169 arrested bacteroid development at a late stage, the absence of MtAHL1 or MtAHL2 completely blocked bacteroid differentiation indicating that they also regulate other NCR genes required for the development of nitrogen-fixing nodules. Regulation of NCR s by orthologous transcription factors in non-IRLC legumes opens up the possibility of increasing the efficiency of nitrogen fixation in legumes lacking NCR s

E2FB interacts with RETINOBLASTOMA RELATED and regulates cell proliferation during leaf development

Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in e2fb mutant and overexpression lines during leaf development in Arabidopsis thaliana, we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels either by expression driven by its own promoter or ectopically together with DIMERISATION PARTNER A, further elevates the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in e2fb mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression leading to the reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses

Heat shock factors regulate responses to high temperatures, salinity, water deprivation or heavy metals. Their function in stress combinations is however not known. The Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature and combination of these conditions. Fast translocation of HSFA4A-YFP protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by MAP kinases MPK3/6 but also by MPK4 and Ser309 is the dominant MAPK phosphorylation site. In vivo data suggest that HSFA4A can be substrate of other kinases as well. Changing Ser309 to Asp or Ala has altered intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes