Lactobacillus plantarum WCSF1 has two putative prolinases (PepR1 and PepR2), and they share only 48.5% amino acid sequence identity. To investigate the differences in enzymatic characters between two enzymes, the genes are cloned and expressed in E. coli using non-tagged pKK223-3 and His-tagged pET32b(+) systems. Culture conditions of overexpressed recombinant prolinases (r-PepR1 and r-PepR2) are optimized as pH7.0-7.5 LB media at 16°C with 1 mM IPTG induction. Recombinant prolinases with His-tag give higher yields and are more cost-efficient over non-tagged recombinant prolinases. After purification, these recombinant enzymes show similar hydrolysis activities towards Pro-Gly substrate, proving their nature as prolinases. Structural analyses using CD spectrum and computer modelling show that r-PepR1 and r-PepR2 share structural similarity in their secondary structure having the highest β-sheets over other components; and dynamic light scattering and gel filtration chromatography analyses indicate their quaternary structure being homotetrameric. Structural similarity can be linked to enzyme function feature. The two enzymes have the same enzymatic functionality may be due to their structural similarity. Despite for their structural similarities and the same enzymatic functionality, they show differences in their substrate specificity, optimum temperature and pH, kinetic parameters (Km and kcat values), thermal stability, and proteolysis mode. r-PepR1 has its optimal activity at 25°C pH7.5 against substrate Pro-Met, whereas r-PepR2 is most active at 30°C pH8.0 against Pro-Gly. r-PepR1 has a low thermal stability with a TM (the midpoint temperature of the unfolding transition) at 29°C, whereas r-PepR2 has a higher TM at 48°C. However, r-PepR1 is aggregated and inactivated at near physiological temperature (42°C). The catalytic mode of r-PepR1 could be a metallo-protease since its activity reduces by 38% with a metal-chelating agent EDTA; while the activity of r-PepR2 is inhibited by 47% with a serine protease inhibitor PMSF, suggesting it is a serine protease. These isozymes cooperatively and complementarily work together to hydrolyze proline-containing peptides, showing broader specificity, broader range of working pH and temperature, and higher efficiency, suggesting that the proline recycling are mediated through these two enzymes to adapt a wide rage of environmental conditions.

Τα επισωματικά οχήματα γονιδιακής μεταφοράς αποτελούν ελκυστική εναλλακτική πειραματική προσέγγιση της γονιδιακής θεραπείας σε σχέση με τα ιϊκά οχήματα. Στην παρούσα εργασία και στο πλαίσιο της ανάπτυξης επισωματικών οχημάτων για την γονιδιακή θεραπεία των αιμοσφαιρινοπαθειών, μελετάται το όχημα hβ-SMAR(Α), ένα κυκλικό πλασμίδιο, που φέρει το γονίδιο της ανθρώπινης β-σφαιρίνης και το μLCR και βασίζεται σε ανθρώπινα χρωμοσωμικά στοιχεία, το S/MAR στοιχείο από την περιοχή 5’ του γονιδίου της ανθρώπινης ιντερφερόνης β. Διαπιστώθηκε ότι το hβ-SMAR(Α) συγκρατήθηκε εντός της διαμολυσμένης κυτταρικής σειράς MEL για περισσότερες από 300 γενιές με διαπιστωμένη την επισωματική του κατάσταση για τουλάχιστο 180 γενιές. Επιπλέον, διατήρησε υψηλά επίπεδα έκφρασης του διαγονιδίου, τα οποία θα αντιστοιχούσαν σε θεραπευτικά επίπεδα, αν αναπαράγονταν in vivo. / Episomal vehicles for gene transfer are an attractive alternative experimental approach to gene therapy in the place of viral vectors. The vehicle hβ-SMAR(Α) studied here, within the context of developing efficient episomal vectors for the gene therapy of hemoglobinopathies, is a circular plasmid bearing the human β globin gene and the μLCR and is based on human chromosomal elements, the S/MAR element from the region 5’ of the human interferon β gene. It was established that hβ-SMAR(Α) was retained within the transfected MEL cell line for more than 300 generations, with its episomal state ascertained for at least 180 generations. Furthermore, it retained high level expression of the transgene, which would be therapeutic, if reproduced in vivo.

<p>Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons, where mitochondrial dysfunction and neuroinflammation are implicated in this process. However, the exact mechanisms of mitochondrial dysfunction, oxidative stress and neuroinflammation leading to the onset and development of Parkinson’s disease are not well understood. There is a lack of tools necessary to dissect these mechanisms, therefore we engineered genetically encoded fluorescent biosensors to monitor redox status and an inflammatory signal peptide with high spatiotemporal resolution. To measure intracellular redox dynamics, we developed red-shifted redox sensors and demonstrated their application in dual compartment imaging to study cross compartmental redox dynamics in live cells. To monitor extracellular inflammatory events, we developed a family of spectrally diverse genetically encoded fluorescent biosensors for the inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced dopaminergic disruption in a zebrafish animal model and evaluated a behavioral assay as a method to screen for dopaminergic dysfunction. Pairing our intracellular redox sensors and our extracellular bradykinin sensors in a Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will prove useful for dissecting the role of mitochondrial dysfunction and inflammation in Parkinson’s disease. </p>