Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making crosslinks between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine in the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein– cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high molecular mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesincoated slides. These results indicate that the pollen tube TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-crosslinking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein:His6– Xpr–GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activitywas observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization

Protein-Glutamine Gamma-Glutamyltransferase

info:eu-repo/semantics/publishedVersio

Proteine modificate post-traduzionalmente da transglutaminasi durante la morte cellulare programmata

Le conoscenze sulla PCD nelle piante sono molto meno avanzate di quelle acquisite negli organismi animali. Frequentemente, la morte programmata nelle piante rappresenta lo stadio terminale del differenziamento. Nel caso dei fiori la fecondazione pu\uf2 determinare la senescenza e l\u2019abscissione dei petali oppure questi eventi possono essere programmati indipendentemente. Uno dei fattori rilevanti nell\u2019apoptosi delle cellule animali \ue8 risultata essere la modificazione post-traduzionale delle proteine e in particolare quella catalizzata dalle transglutaminasi, la cui presenza e l\u2019attivit\ue0 sono state riscontrate in diversi tessuti in apoptosi. L\u2019enzima catalizza la coniugazione di lisina o ammine primarie a glutamine. Con le poliamine si possono formare derivati mono- e bis \u2013sostituiti dando origine a ponti inter- od intra-proteici a significato strutturale. La conformazione dei substrati pu\uf2 esserne modificata e si possono formare generate reti sopramolecolari. Le poliamine alifatiche libere hanno un ruolo ben stabilito nella stimolazione della divisione cellulare, dello sviluppo e nel dilazionare la senescenza Si intende determinare quali siano le proteine, coinvolte nei fenomeni di morte cellulare programmata, modificate post-traduzionalmente, applicando nuove tecnologie informatiche e morfo-funzionali. Si studieranno gli effetti di tali modificazioni sui metabolismi cellulari La ricerca \ue8 volta a determinare il ruolo della transglutaminasi nelle cellule dei petali di Nicotiana durante la senescenza e la PCD. A tal fine applicando approcci di tipo bio-informatico e morfo-funzionale si intende approfondire l\u2019analisi sia degli enzimi che dei loro substrati proteici. Gli obbiettivi sono quelli di: (1) purificare la transglutaminasi vegetale; (II) microsequenziare l\u2019enzima; (III) produrre un modello strutturale della proteina e compararlo con quello di TGasi note; (IV) comparare le funzioni dell\u2019enzima vegetale con la TGasi di Nematode, con cui vi sono indizi che condivida sequenze omologhe, e con quelle di mammifero con cui sembra invece che non ci siano omologie di sequenza; (V) valutare se l\u2019enzima ha attivit\ue0 deamidasica; (VI e VII) studiare la localizzazione, in relazione a senescenza e PCD, dell\u2019enzima e dei substrati e studiare le modificazioni di proteine: (VIII) citoscheletriche, (IX) di parete (X) plastidiali. Un altro obbiettivo con scopi applicativi consiste nel comprendere il meccanismo molecolare di \u201cringiovanimento\u201d della spermina su fiori recisi o altri prodotti freschi

Twenty years of plant transglutaminases. Specificity and similarity with animal transglutaminases.

Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect plant growth and differentiation. Among the myriad of biological reactions exerted in animals, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied in plants. Transglutaminase activity is ubiquitous: it has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. Specificity and similarity with animal transglutaminases are discussed in the light of their biochemical characteristics and functional roles. Possible roles concern the structural modification of specific proteins. In the cytosol, they modify actin and tubulin, also influencing motor proteins, thus exerting a role on cell growth and cell structure. In chloroplasts, transglutaminases appear to stabilise the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photoprotection. Preliminary reports suggest an involvement in construction/organisation of the cell wall and extracellular matrix in relationship with fertilisation. Other roles appear to be related to stresses, senescence and programmed cell death, including the hypersensitive reaction caused by TMV virus. The cross recognition of substrates between plant and animal enzymes suggest both similarities and differences. The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: 1. their ability to produce glutamyl-polyamine derivatives, 2. their recognition by animal transglutaminase antibodies, 3. biochemical features such as calcium- and GTP-dependency, etc. 4. Inhibition by specific inhibitors of animal transglutaminases. However, many of their fundamental physiological properties still remain elusive. At present, it is not possible to classify this enzyme family in plants owing to the scarcity of information on genes encoding them

Transglutaminases of higher, lower plants and fungi

No abstract availabl