The problem of identifying genes in eukaryotic genomic sequences by computational methods has attracted considerable research attention in recent years. Many early approaches to the problem focused on prediction of individual functional elements and compositional properties of coding and non coding deoxyribonucleic acid (DNA) in entire eukaryotic gene structures. More recently, a number of approaches has been developed which integrate multiple types of information including structure, function and genetic properties of proteins. Knowledge of the structure of a protein is essential for describing and understanding its function. In addition, subcellular localization of a protein can be used to provide some amount of characterization of a protein. In this study, a method for the prediction of protein subcellular localization based on primary sequence data is described. Primary sequence data for a protein is based on amino acid sequence. The frequency value for each amino acid is computed in one given position. Assigned frequencies are used in a new encoding scheme that conserves biological information based on point accepted mutations (PAM) substitution matrix. This method can be used to predict the nuclear, the cytosolic sequences, the mitochondrial targeting peptides (mTP) and the signal peptides (SP). For clustering purposes, other than well known traditional techniques, principle component analysis (PCA)&quot / and self-organizing maps (SOM)&quot / are used. For classication purposes, support vector machines (SVM)&quot / , a method of statistical learning theory recently introduced to bioinformatics is used. The aim of the combination of feature extraction, clustering and classification methods is to design an acccurate system that predicts the subcellular localization of proteins presented into the system. Our scheme for combining several methods is cascading or serial combination according to its architecture. In the cascading architecture, the output of a method serves as the input of the other model used.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

In this study, the focus is on predicting the subcellular localization of a protein, since subcellular localization is helpful in understanding a protein&rsquo / s functions. Function of a protein may be estimated from its sequence. Motifs or conserved subsequences are strong indicators of function. In a given sample set of protein sequences known to perform the same function, a certain subsequence or group of subsequences should be common / that is, occurrence (frequency) of common subsequences should be high. Our idea is to find the common subsequences through clustering and use these common groups (implicit motifs) to classify proteins. To calculate the distance between two subsequences, traditional string edit distance is modified so that only replacement is allowed and the cost of replacement is related to an amino acid substitution matrix. Based on the modified string edit distance, spectral clustering embeds the subsequences into some transformed space for which the clustering problem is expected to become easier to solve. For a given protein sequence, distribution of its subsequences over the clusters is the feature vector which is subsequently fed to a classifier. The most important aspect if this approach is the use of spectral clustering based on modified string edit distance.

The focus of this study is on predicting the subcellular localization of a protein. Subcellular localization information is important for protein function annotation which is a fundamental problem in computational biology. For this problem, a classification system is built that has two main parts: a predictor that is based on a feature mapping technique to extract biologically meaningful information from protein sequences and a client/server architecture for searching and predicting subcellular localizations. In the first part of the thesis, we describe a feature mapping technique based on frequent patterns. In the feature mapping technique we describe, frequent patterns in a protein sequence dataset were identified using a search technique based on a priori property and the distribution of these patterns over a new sample is used as a feature vector for classification. The effect of a number of feature selection methods on the classification performance is investigated and the best one is applied. The method is assessed on the subcellular localization prediction problem with 4 compartments (Endoplasmic reticulum (ER) targeted, cytosolic, mitochondrial, and nuclear) and the dataset is the same used in P2SL. Our method improved the overall accuracy to 91.71% which was originally 81.96% by P2SL. In the second part of the thesis, a client/server architecture is designed and implemented based on Simple Object Access Protocol (SOAP) technology which provides a user-friendly interface for accessing the protein subcellular localization predictions. Client part is in fact a Cytoscape plug-in that is used for functional enrichment of biological networks. Instead of the individual use of subcellular localization information, this plug-in lets biologists to analyze a set of genes/proteins under system view.

Για τη διατήρηση της γονιδιωματικής σταθερότητας, είναι θεμελιώδους σημασίας η πραγματοποίηση της αντιγραφής του DNA με ακρίβεια και πιστότητα. Στη σωστή χρονικά έναρξη της αντιγραφής σημαντικό ρόλο κατέχει η «αδειοδότηση» της αντιγραφής, η οποία διασφαλίζει ότι η χρωματίνη είναι ικανή για τον επόμενο κύκλο αντιγραφής μόνο αφότου το κύτταρο έχει διέλθει από τη μίτωση. Η αδειοδότηση λαμβάνει χώρα μέσω της διαδοχικής συγκρότησης στις αφετηρίες της αντιγραφής, ενός πολύ-πρωτεϊνικού συμπλόκου, του καλούμενου προ-αντιγραφικού συμπλόκου. Απαραίτητος για τη συγκρότηση του προ-αντιγραφικού συμπλόκου είναι ο παράγοντας Cdt1. Στα μετάζωα, ένα μικρό μόριο, η Geminin, αναστέλλει τη δράση του Cdt1 κατά τις φάσεις S, G2 και Μ. Η Geminin είναι ένα μόριο με δομή σπειροειδούς σπειράματος, το οποίο συμμετέχει σε ευρύ φάσμα μοριακών αλληλεπιδράσεων και ελέγχει διαδικασίες του κυτταρικού κύκλου και της διαφοροποίησης. Η ρύθμιση της Geminin επιτυγχάνεται κύρια σε μεταγραφικό επίπεδο και μέσω πρωτεόλυσης, ενώ πρόσφατα δείχθηκε ότι μετα-μεταφραστικές τροποποιήσεις του μορίου διασφαλίζουν την απενεργοποίησή του κατά την G1. Για την εκδήλωση της δράσης της Geminin απαιτείται η παρουσία της πρωτεΐνης στον πυρήνα. Για την ενδοκυτταρική μελέτη της συμπεριφοράς της Geminin, δημιουργήσαμε υβριδικές μορφές του μορίου με την πράσινη φθορίζουσα πρωτεΐνη (GFP) και κατόπιν παροδικής διαμόλυνσης μελετήσαμε τον ενδοκυτταρικό του εντοπισμό. Η εξωγενής Geminin εντοπίζεται στον πυρήνα (όπως και το ενδογενές μόριο) σε έναν υποπληθυσμό ασύγχρονων κυττάρων αλλά επιπλέον, στα μισά κύτταρα του πληθυσμού, αποκλείεται από τον πυρήνα και συσσωρεύεται στο κυτταρόπλασμα. Η εμφάνιση της Geminin αποκλειστικά εκτός πυρήνα πραγματοποιείται σε κύτταρα που βρίσκονται στη φάση G1 του κυτταρικού κύκλου και συγκεκριμένα φαίνεται να λαμβάνει χώρα στο τέλος της G1 φάσης, πλησίον της μετάβασης από την G1 στην S. Κατά την είσοδο των κυττάρων στον κυτταρικό κύκλο από τη φάση ηρεμίας G0, το ποσοστό των κυττάρων που εκφράζουν την Geminin στο κυτταρόπλασμα αυξάνεται σημαντικά. Το γεγονός αυτό προσδίδει φυσιολογικό ρόλο στο φαινόμενο του αποκλεισμού της Geminin από τον πυρήνα και το συνδέει με την προαγωγή της αδειοδότησης της αντιγραφής πριν την είσοδο του κυττάρου στην S. Ανάλυση των επιμέρους περιοχών του μορίου με δημιουργία προοδευτικών απαλοιφών περιοχών της Geminin ανέδειξε το ρόλο της αμινοτελικής περιοχής του μορίου (aa 1-30) στον κυτταροπλασματικό εντοπισμό του. Ο πυρηνικός εντοπισμός της Geminin επάγεται από την κεντρική περιοχή του μορίου ενώ ένα αμινοτελικό πιθανολογούμενο NLS δε φαίνεται από μόνο του –τουλάχιστον- στην ανθρώπινη Geminin να αρκεί για τον πυρηνικό εντοπισμό της. Από τους παράγοντες που μελετήθηκαν, οι οποίοι θα μπορούσαν να επηρεάζουν in trans τον εντοπισμό της Geminin, το Cdt1, ο μοριακός συνοδός της Geminin, είναι ικανός να κατευθύνει τον αναστολέα του στον πυρήνα μετά από συνδιαμόλυνση σε ανθρώπινα καρκινικά κύτταρα. Συνδιαμόλυνση με μορφές των δύο μορίων που δεν μπορούν να αλληλεπιδράσουν δεν οδηγεί σε πυρηνικό εντοπισμό της Geminin. Συμπεραίνουμε ότι το Cdt1 προκαλεί διαμετατόπιση της Geminin στον πυρήνα μέσω άμεσης αλληλεπίδρασης με αυτήν. Τα αποτελέσματά μας αναδεικνύουν έναν νέο τρόπο ρύθμισης της Geminin μέσω πυρηνικού αποκλεισμού και παρέχουν πληροφορίες για το πότε και πώς λαμβάνει χώρα αυτή η ρύθμιση. Πρόσφατα, στις ηλεκτρονικές βάσεις δεδομένων εντοπίσαμε έναν γονιδιακό τόπο που κωδικοποιεί για πρωτεϊνικό μόριο με μεγάλη ομολογία με το σπειροειδές σπείραμα (coiled-coil) της Geminin (Geminin cc-like ή Castor). Στην παρούσα μελέτη εξετάζεται η χωρο-χρονική έκφραση της Geminin cc-like (Castor), οι αλληλεπιδράσεις με υποψήφια μόρια και η συμπεριφορά του μορίου σε συνθήκες υπερέκφρασης και αποσιώπησης σε ανθρώπινα κύτταρα. Για τη μελέτη αυτή δημιουργήθηκε υβριδική μορφή της πρωτεΐνης με την GFP. Τα αποτελέσματά μας δείχνουν ότι Geminin cc-like (Castor) είναι πυρηνική πρωτεΐνη, όπως και η ενδογενής Geminin και η ενεργότητα σήματος πυρηνικού εντοπισμού φαίνεται να εδράζεται στην καρβοξυτελική περιοχή του μορίου. Η Geminin cc-like (Castor) αλληλεπιδρά με την Geminin και μάλιστα αποτελεί έναν ακόμη παράγοντα, μαζί με το Cdt1, που μπορεί να κατευθύνει in trans την Geminin στον πυρήνα. Πειράματα υπερέκφρασης παρέχουν ενδείξεις για παρεμπόδιση της εισόδου των κυττάρων στη φάση S, ενώ αποσιώπηση της Geminin cc-like (Castor) με τη χρήση RNAi φαίνεται να αυξάνει το ποσό της φωσφορυλιωμένης μορφής της ιστόνης H2Ax, δείκτη διπλών κατατμήσεων στο DNA. Από εξέταση για την ύπαρξη ορθολόγων σε άλλους οργανισμούς, βρέθηκε ορθόλογη πρωτεΐνη στον ιχθύ Oryzias latipes (medaka). Κλωνοποιήθηκε τμήμα του mRNA της O.l Castor πρωτεΐνης, πραγματοποιήθηκε ανάλυση της έκφρασης της Geminin cc-like (Castor) σε έμβρυα medaka καθώς και πειράματα υπερέκφρασης μετά από ένεση τμήματος του mRNA, τα οποία παρέχουν προκαταρκτικές ενδείξεις για ελαττωματικό σχηματισμό της κεφαλής και των οφθαλμών. Συμπερασματικά, μελετήσαμε μία νέα πρωτεϊνη στον άνθρωπο η οποία εμφανίζει μερική ομολογία προς την Geminin (Geminin cc-like ή Castor), και δείξαμε ότι αποτελεί ένα νέο μοριακό συνοδό της Geminin, με πιθανή συμμετοχή σε κυτταρικό κύκλο και διαφοροποίηση. / For genomic stability to be maintained, accurate regulation of replication has to be achieved. Replication licensing takes places during G1 and involves the stepwise formation of a multimeric complex, the pre-replicative complex (pre-RC), onto origins of replication, ensuring that chromatin will be competent for replication only once per cell cycle. Cdt1, a key component of this complex, is accurately regulated throughout cell cycle, through multiple mechanisms. Geminin, a metazoan-specific inhibitor of licensing, binds and inhibits Cdt1, thus preventing ectopic pre-RC formation during S, G2 and M. Geminin is regulated both at the level of transcription and proteolysis. Recently, it was also demonstrated that inactivation of non-proteolysed Geminin during G1 ensures timely formation of the pre-RC. Through its coiled-coil region, many interactions with binding partners are achieved, thus enabling participation in a variety of cellular processes during the cell cycle and differentiation. In order to study the intracellular behaviour of Geminin, fused forms of the molecule with the fluorescent protein GFP were constructed and transfected in MCF7 cells. Exogenous Geminin is located in the nucleus, similar to the endogenous molecule, in a subpopulation of asynchronously growing cells, but in approximately 50% of cells, Geminin is specifically excluded from the nucleus. Cytoplasmic localization of Geminin takes place during G1 and specifically during late G1, close to the G1 to S transition. In addition, upon exit from quiescence and cell cycle re-entry, cytoplasmic localization of Geminin increases notably. This may reflect a physiological role of Geminin exclusion to the cytoplasm upon cell cycle re-entry, in order to permit a ‘window of opportunity’ for licensing to take place prior to S phase onset. Progressive deletions of the Geminin molecule resulted in a variety of mutated forms which were then analysed for their ability to be localized to the nucleus or the cytoplasm. This analysis showed the importance of the aminoteminal region of Geminin (aa 1-30) in the cytoplasmic localization of the molecule. A putative nuclear localization activity was mapped to the central part of the molecule, while a proposed NLS at the aminoterminal part was shown to be neither sufficient nor required for nuclear localization. Amongst the factors tested, which could affect intracellular localization of Geminin, we show that Cdt1 possesses the ability to direct Geminin to the nucleus. Our data provide evidence for a novel means of regulation of Geminin by cell cycle dependant subcellular localization and offer insight on when and how this regulation takes place. Recently, in silico analysis revealed a gene locus in the human genome, coding for a predicted coiled coil protein with homology to Geminin. The gene product was initially named after this similarity as Geminin coiled coil-like (Geminin cc-like) and then renamed ‘Castor’. Present work examines the spatio-temporal expression of a hybrid Castor-GFP molecule, interactions with candidate molecular partners and the consequences for the cell of Castor overexpression or silencing. Our results show that exogenous Castor-GFP is localized in the nucleus and excluded from the nucleoli, similar to endogenous Geminin, whereas evidence for a putative NLS activity at the carboxy-terminus of the molecule is provided. Castor interacts with Geminin in cellular extracts and is sufficient to translocate Geminin-GFP to the nucleus, similar to Cdt1. Overexpression experiments provided evidence for a delayed G1 to S transition. Silensing Castor by siRNA led to an increase in γ-H2Ax staining, a marker of double strand breaks. Further in silico analysis revealed orthologues of Castor in other organisms, from mammals to fish. Part of the putative Castor mRNA in the fish Oryzias latipes (Medaka) was cloned. In vivo analysis of Castor in medaka involved expression pattern analysis by whole mount in situ hybridization and overexpression amalysis by mRNA injections, providing preliminary evidence for impaired forehead and eye formation. In conclusion, we have studied a novel protein with similarity to the coiled-coil region of Geminin (Geminin cc-like/ Castor) and showed that it constitutes a novel molecular partner of Geminin with possible roles during the cell cycle and in development.

Η παρούσα εργασία είχε ως αντικείμενο, αρχικά, τη δημιουργία υβριδικών μορφών της πρωτεΐνης Geminin- ενός αρνητικού ρυθμιστή του κυτταρικού κύκλου- με το φθορίζον μόριο GFP (Green Fluorescent Protein). Πραγματοποιήθηκε τόσο αμινοτελική όσο και καρβοξυτελική σύντηξη της Geminin με το μόριο-ιχνηθέτη, έτσι ώστε να μελετηθεί η συμπεριφορά του νέου, υβριδικού μορίου και στις δύο περιπτώσεις και να εξεταστεί εάν ο προσανατολισμός της GFP πρωτεΐνης δύναται να μεταβάλλει το χαρακτήρα της σημασμένης πρωτεΐνης. Ακολούθησε ανίχνευση και παρακολούθηση καθενός υβριδικού μορίου σε ανθρώπινα καρκινικά κύτταρα μετά από διαμόλυνση για μικρό (παροδική διαμόλυνση διάρκειας 22 ωρών) ή μεγαλύτερο χρονικό διάστημα (σταθερά διαμολυσμένες κυτταρικές σειρές). Ο χαρακτηρισμός των φθοριζουσών μορφών της Geminin περιελάμβανε μελέτη τόσο του υποκυτταρικού εντοπισμού όσο και της χρονικής έκφρασης των μορίων και σύγκριση με τα αντίστοιχα χαρακτηριστικά του ενδογενούς μορίου. Ανάλυση του ενδοκυτταρικού εντοπισμού τόσο της καρβοξυτελικής όσο και της αμινοτελικής σύντηξης της Geminin με τη GFP, αποκάλυψε, εκτός του πυρηνικού φαινοτύπου, που παρατηρείται φυσιολογικά για το ενδογενές μόριο, σημαντικό ποσοστό παροδικά διαμολυσμένων κυττάρων με τα υβριδικά μόρια εκτός του πυρηνικού διαμερίσματος. Ο φαινότυπος αυτός εμφανίζεται – σε μικρότερο ποσοστό- ακόμη και στην περίπτωση σταθερά διαμολυσμένων κυττάρων, όπου τα επίπεδα έκφρασης της πρωτεΐνης Geminin-GFP είναι παρόμοια με τα αντίστοιχα του ενδογενούς μορίου. Στη σειρά αυτή, το μεγαλύτερο ποσοστό κυττάρων, παρά την ισχυρή μεταγραφική δραστηριότητα που προσδίδει ο CMV υποκινητής, εκφράζει το εξωγενές μόριο σύμφωνα με το πρότυπο που παρουσιάζει η ενδογενής Geminin, δηλαδή κατά τις φάσεις S και G1. Επομένως, η ρύθμιση της Geminin-GFP υβριδικής πρωτεΐνης πραγματοποιείται σε μεγάλο βαθμό σε μετα-μεταγραφικό επίπεδο. Ιδιαίτερο ενδιαφέρον παρουσιάζει το γεγονός ότι η αλλαγή στην ενδοκυτταρική κατανομή του υβριδικού μορίου δεν παρουσιάζει τυχαία χρονική εμφάνιση. Το γεγονός ότι εμφανίζεται αποκλειστικά σε αρνητικά για κυκλίνη Α κύτταρα, φανερώνει την ύπαρξη συσχέτισης με τη φάση του κυτταρικού κύκλου και μάλιστα υποδηλώνει την επιλεκτική έξοδό της από τον πυρήνα κατά τη φάση G1. Πιθανόν, η έξοδος της Geminin από τον πυρήνα κατά τη φάση αυτή, να διασφαλίζει τη διεκπεραίωση της διαδικασίας της αδειοδότησης του γενετικού υλικού –αναστολέας της oποίας είναι η Geminin και κατ’ επέκταση την πρόοδο του κυτταρικού κύκλου Το φαινόμενο αυτό χρήζει περαιτέρω διερεύνησης μιας και η αλλαγή στην ενδοκυτταρική κατανομή πολλών ρυθμιστικών μορίων αποτελεί έναν αποδοτικό τρόπο ελέγχου της γονιδιακής έκφρασης στους ευκαρυωτικούς οργανισμούς, ενώ παράλληλα φαίνεται να επηρεάζει πολλές ζωτικής σημασίας λειτουργίες. / The aim of the present study was the construction of fused forms of Geminin- a negative cell cycle regulator-with GFP (Green Fluorescent Protein). Amino- and carboxy terminal fusions of Geminin and GFP were performed, in order to study the behaviour of the hybrid molecule in both cases and to examine if the orientation of the reporter gene can interfere with the character of the new molecule. Each fused molecule was either transiently or stably transfected in human cancer cells and its behaviour was studied. The characterization of the fluorescent molecules of Geminin was performed at the level of subcellular localization and regulation throughout the cell cycle and was compared to the endogenous characteristics. Analysis of the subcellular distribution of both fused forms, revealed -apart from the nuclear phenotype, normally observed- a significant percentage of transiently transfected cells, where the hybrid molecules were excluded from the nucleus. This phenotype was observed- though less strongly- in the case of stably transfected cells, where the expression levels of Geminin-GFP protein are similar to the endogenous molecule. The majority of these cells, despite the strong CMV driven transcriptional activity, express Geminin-GFP during S and G1 phase, like the endogenous protein, suggesting that the regulation of Geminin-GFP is performed mainly at post-transcriptional level. This nuclear exclusion was observed according to a cell cycle- dependent manner. The phenomenon of nucleocytoplasmic shuttling of many proteins seems to be a very efficient way of controlling gene expression in eukaryotes. In order to elucidate the role of sucellular distribution of Geminin-GFP in cell cycle regulation, further examination of this phenomenon is needed.

No description available.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

Orientador: Marcelo Menossi Teixeira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T15:42:58Z (GMT). No. of bitstreams: 1 Vicentini_Renato_D.pdf: 5901500 bytes, checksum: 7c8fe505e4c1675900f96c6643895d32 (MD5) Previous issue date: 2008 / Resumo: As células de plantas são altamente organizadas e muitos processos biológicos estão associados com estruturas subcelulares específicas. A localização subcelular é uma característica chave das proteínas, visto que está relacionada com a função biológica. A determinação da localização subcelular usando a predição é uma estratégia altamente desejável, principalmente porque as abordagens experimentais demandam um tempo considerável. Com o objetivo de desenvolver um método para melhorar a predição de localização subcelular, diversos algoritmos foram integrados visando à exploração ótima do potencial de cada um. O desempenho com 90% de exatidão deste novo método foi claramente superior a todos os métodos utilizados em sua criação. Usando esta estratégia foi realizada a primeira análise em larga escala da localização subcelular do proteoma de cana-de-açúcar (com 11.882 proteínas preditas), sendo encontrado que a maioria das proteínas estão localizadas em quatro compartimentos: núcleo (44%), citoplasma (19%), mitocôndria (12%), e extracelular (11%). Adicionalmente foi observado que cerca de 19% das proteínas são localizadas em múltiplos compartimentos. Outros resultados foram capazes de identificar um conjunto de proteínas de cana-de-açúcar que podem apresentar duplo direcionamento pelo uso de variações na extremidade amino. Utilizando expressão transiente em células da epiderme de cebola, foi investigada a localização subcelular de 96 proteínas de cana com fusão a proteína GFP. As construções contendo fusão amino- e carboxi-terminal dos genes foram expressas, e a localização das proteínas de fusão foi detectada por microscopia de fluorescência. É relatado também a caracterização do gene ScBAK1, um receptor do tipo quinase com repetições ricas em leucina, que apresenta similaridade de seqüência com o gene brassinosteroid insensitive1-associated receptor kinase1. Foi mostrado que transcritos desse gene se acumulam em níveis muito mais altos nas células da bainha do feixe vascular do que nas células do mesófilo, e que a fusão ScBAK1-GFP é localizada na membrana plasmática. Essa distribuição espacial e esse padrão de expressão indicam que a ScBAK1 pode estar potencialmente envolvida em cascatas de sinalização celular intermediadas por altos níveis de açúcar na folha. Ainda considerando estudos de localização subcelular, é conhecido que seqüências de nucleotídeos que flanqueiam o códon de início da tradução afetam a eficiência traducional dos mRNA, e podem indicar a presença de sítios de inicio de tradução (TIS) alternativos. O multi-direcionamento pode ser um reflexo da variabilidade traducional destas outras formas da proteína. Neste estudo foi desenvolvido um método computacional para investigar o uso de TISs alternativos na síntese de novas variantes protéicas que podem apresentar localização subcelular diferente. Visando contribuir para o nosso entendimento da complexidade do genoma da cana-de-açúcar, foi empregada uma análise em larga escala dos TIS nesta espécie. Também é demonstrado que os transcritos com expressão induzida apresentam um forte TIS quando comparados com os reprimidos, e que os transcritos constitutivos possuem uma alta freqüência de TIS alternativos. O mesmo ocorre para os genes com altas taxas evolutivas, e transcritos específicos de folhas e entrenós, levantando a hipótese de que esses genes possam codificar diferentes polipeptídeos / Abstract: Plant cells are highly organized and many biological processes are associated with specialized subcellular structures. Subcellular localization is a key feature of proteins, since it is related to biological function. The determination of subcellular localization using computational prediction is a highly desirable strategy because experimental approaches are time-consuming. In order to develop a method for the enhanced prediction of subcellular localization, the outputs of some prediction tools were integrated so as to optimally exploit the potential of each one. The prediction performance (with 90%of accuracy) of this new method was clearly superior to all the methods used to create the predictor. Using this method, the first in silico genome-wide subcellular localization analysis was performed for sugarcane (with 11,882 predicted proteins). It was found that most of the proteins are localized to four compartments: nucleus (44%), cytosol (19%), mitochondria (12%), and secretory destination (11%). It is also shown that about 19%of the proteins are localized to multiple compartments, and that a potential set of sugarcane proteins can show dual targeting by use of N-truncated form of proteins. The subcellular localization of 96 sugarcane proteins fused with GFP were evaluated using transient expression in onion epidermal cells. Constructs containing the Nand C-terminal fusion of genes encoding both endogen and GFP proteins were transiently expressed, and the localization of the fusion proteins were detected by fluorescent microscopy. It was reported the characterization of ScBAK1, a sugarcane leucine-rich repeat receptor-like kinase, with sequence similarity to brassinosteroid insensitive1-associated receptor kinase1. We have found that ScBAK1 transcripts accumulated at higher levels in bundle-sheath than in mesophyll cells. ScBAK1-GFP fusions were localized to the plasma membrane. This spatial distribution and expression pattern indicates that ScBAK1 might be potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. The nucleotide sequence flanking the translation initiation codon affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this study it was also developed a computational method to investigate the usage of alternative TISs for the synthesis of new protein variants that might have different subcellular localization. To contribute to our understanding of the genome complexity of sugarcane, we undertook a genome wide TIS analysis in sugarcane data. It is demonstrated that up-regulated transcripts show a stronger TIS when compared with the down-regulated, and that ubiquitous transcripts have a high frequency of alternative TIS in the next downstream AUG codon. The same occurs for fast-evolving genes, and leaf and internodes specific transcripts, that may encode different polypeptides by N-terminal polymorphism / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Maspina (mammary serpin) foi identificada em 1994 como uma serpina (serine protease inhibitor) que apresenta atividade de supressão tumoral. Foi classificada como uma serpina devido à homologia na sequência de aminoácidos, porém, maspina não apresenta atividade de inibição de serina proteases. Entre os efeitos biológicos de maspina estão a modulação da adesão, a inibição do crescimento e a invasão tumoral, a inibição da angiogênese, o efeito pró-apoptótico e o controle da resposta ao stress oxidativo, propriedades que contribuem para supressão tumoral. Esta diversidade de funções se reflete nos inúmeros ligantes de maspina e na sua localização subcelular, já que é encontrada na membrana plasmática, no citoplasma, núcleo e mitocôndrias. A localização subcelular de maspina guarda importante relação com sua função, já que foi demonstrado que sua localização nuclear está correlacionada com bom prognóstico em diversos tumores e seu efeito supressor de tumor foi observado somente quando maspina está localizada no núcleo. Entre os ligantes de maspina estão a HDAC1, IRF6, GST, HSP90 e HSP70, β1 integrina, uPAR e colágeno tipo I e III. O mecanismo molecular envolvido na regulação dessas atividades não foi elucidado, e até o momento, somente um gene e uma proteína de maspina foram descritos, desta forma alterações pós-traducionais devem estar envolvidas na regulação dessas atividades. Com objetivo de verificar se há modificações pós-traducionais em maspina, utilizamos células MCF10A, que expressam grande quantidade dessa proteína, e submetemos seu extrato proteico à separação por gel bidimensional seguido de western blot. Identificamos quatro formas de maspina com a mesma massa molecular (42kDa), mas pontos isoelétricos distintos. Três destas formas são sensíveis ao tratamento com fosfatase ácida, o que sugere que estas sejam fosforiladas. Utilizamos ainda peroxidovanadato de sódio, um potente inibidor de tirosina fosfatase para investigar o papel da fosforilação de maspina em resíduos de tirosina. Através de western blot e imunofluorescência, observamos que o tratamento das células com o inibidor resultou no aumento dos níveis celulares de maspina assim como no seu acúmulo no citoplasma. Deste modo, concluímos que existem três diferentes fosfoformas de maspina em células MCF10A e ainda a inibição de tirosinas fosfatases aumentam os níveis de maspina e resultam no acúmulo da proteína no citoplasma. Esses resultados sugerem que a fosforilação pode estar envolvida na localização subcelular de maspina e na regulação dos seus níveis proteicos na célula. / Maspin (mammary serpin) was identified in 1994 as a serpin (serine protease inhibitor) which presents tumor suppressor activity. It was classified as a serpin due to its homology in amino acids sequence; however, maspin doesn\'t exhibit serine protease inhibition activity. Among maspin biological effects are modulation of cell adhesion, inhibition of tumor growth, invasion and angiogenesis, a pro-apoptotic effect and control of oxidative stress response, properties which contribute to tumor suppression. This functional diversity reflects maspin numerous ligands and its subcellular localization, since it is found on the plasma membrane, in the cytoplasm, nucleus and in mitochondria. Maspin subcellular localization is closely related to its function, as its nuclear localization correlates with good prognostic in several tumors and maspin tumor suppressor activity is only observed when it is located in the nucleus. Among maspin ligands are histone H1 deacetylase, IRF6, GST, HSP90 e HSP70, β1 integrin, uPAR and type I and III collagen. The molecular mechanisms involved in the regulation of maspin biological activities are poorly understood. So far, only one gene and one protein have been assigned to maspin, so posttranslational modification should be involved. In order to verify posttranslational modification in maspin, we utilized MCF10A cells, which express great amount of this protein, and we submitted its proteic extract to 2D-SDS-PAGE followed by western blot. We identified four maspin forms with the same molecular mass (42kDa), but different isoelectric point. Three of these forms are sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated maspin forms. We also utilized sodium peroxovanadate, a potent tyrosine phosphatase inhibitor to investigate the role of maspin tyrosine phosphorylation. Through western blot and immunofluorescence analyses, we observed that cell treatment resulted in increase in maspin cellular levels as well as its cytoplasmic accumulation. Thus, we concluded that there are three diferente maspin phosphoforms in MCF10A cells and yet tyrosine phosphatase inhibition increases maspin levels and results in accumulation of the protein in the cytoplasm. These data suggest that phosphorylation may be involved in maspin subcellular localization and regulation of its protein levels in the cell.