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.

This work investigated the subcellular dynamics of the plant elicitor peptide AtPep1 and its interplay with plant defense responses. First, an introduction of the plant innate immunity system is provided with emphasis on pattern trigger immunity (PTI), which is based on the recognition of \"non-self\" and \"self\" elicitor molecules by surface-localized patternrecognition receptors (PRRs). Then, the Arabidopsis endogenous peptides that act as selfelicitor molecules are presented, with details on AtPep1 and its PEPR receptors. Plant endomembrane trafficking is described, encompassing endocytic pathways, clathrin mediated endocytosis (CME) and receptor-mediated endocytosis (RME). In the next chapter, we explored strategies for the in vivo study of the subcellular behavior of AtPep1; to this end, we fused the precursor protein of AtPep1 (PROPEP1) to GFP and assessed its localization. We found that PROPEP1 was associated with the tonoplast and accumulated in the vacuole, suggesting that this organelle could work as the station where PROPEP1 is stored and later released, only in a danger situation, hence initiating AtPep1. Moreover, we generated AtPep1 versions labeled with fluorescent dyes and demonstrated that this peptide could be fluorescently tagged without loss of its biological activity. In chapter 3, we combined classical and chemical genetics with life imaging to study the behavior of a bioactive fluorescently labeled AtPep1 in the Arabidopsis root meristem. We discovered that the labeled AtPep1 was able to bind the plasma membrane very quickly in a receptor-dependent manner. Subsequently, the PEPR-AtPep1 complex was internalized via CME and transported to the lytic vacuole, passing through early and late endosomal compartments. Impairment of CME compromised the AtPep1 responses. Our findings provide for the first time an in vivo visualization of a signaling peptide in plant cells, thus giving insights into its intracellular fate and dynamics. The role of the coregulatory receptor BRI1-associated kinase 1 (BAK1) in AtPep1-responses was also investigated (chapter 4). Our results confirmed that BAK1 interacts with PEPRs in a ligand-dependent manner and indicate that BAK1 modulates AtPep1 signaling and endocytosis, but that, when absent, it might be replaced by homologous SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) proteins that could have additional functions during the AtPep1 signaling. Furthermore, phosphorylation events after the formation of PEPR-BAK1 complexes seem to dictate the molecular bases of AtPep1 internalization and signaling. Finally, we discussed our findings in a more general perspective, highlighting the important findings for the plant endomembrane trafficking field, the potential use of fluorescently labeled ligands as a tool to study ligand-receptors pairs, the availability of AtPep1-PEPRs as an excellent model to study endocytosis and its interplay with signaling, and the future challenges in the field. / Neste trabalho, foi investigada a dinâmica subcelular do peptídeo elicitor de planta AtPep1 e suas implicações nas respostas de defesa. Primeiramente, é fornecida uma introdução do sistema imune inato de plantas com ênfase na imunidade ativada por moléculas elicitoras derivadas de organismos invasores ou da mesma planta, após seu reconhecimento por receptores localizados na membrana plasmática (PTI responses). Peptídeos endógenos que têm sido reportados em Arabidopsis como ativadores de PTI são descritos, dando especial destaque para o peptídeo AtPep1 e seus receptores PEPRs. O tráfego de endomembranas em plantas é introduzido, abrangendo as vias de internalização, endocitose mediada por proteínas clathrinas (CME) e endocitose mediada por receptor (RME). No capítulo seguinte, foram avaliadas estratégias para o estudo in vivo da dinâmica subcelular do AtPep1. Para isso a proteína precursora do AtPep1 (PROPEP1) foi fusionada a GFP e sua localização visualizada, encontrando que PROPEP1 é associado com o tonoplasto e acumula dentro do vacúolo, fato que sugere uma função de armazenamento do PROPEP1 para esta organela, desde onde é liberado em caso de uma situação de perigo dando origem ao AtPep1. Adicionalmente, foram produzidas versões biologicamente ativas do AtPep1 marcado com fluróforos. No capítulo três foram combinados genética clássica e genética química com visualizações in vivo para estudar o comportamento de um AtPep1 bioativo e marcado fluorescentemente na células meristemática da ponta da raiz de Arabidopsis, sendo encontrado que AtPep1 se liga rapidamente na membrana plasmática numa forma dependente de receptor. Em seguida, o complexo AtPep1-PEPR foi internalizado via CME e transportado para o vacúolo, passando através do endossomo primário e secundário. Quando o funcionamento da CME foi comprometido, as respostas ao AtPep1 também foram afetadas. Estes resultados fornecem a primeira visualização in vivo de um peptídeo de sinalização em plantas, mostrando sua dinâmica e destino intracelular. O papel regulatório durante as respostas induzidas pelo AtPep1 do co-receptor BRI1-associated kinase 1 (BAK1) foram investigadas (Capítulo quatro). Nossos resultados confirmaram que BAK1 interage com PEPRs numa forma dependente do ligante e indicam que BAK1 modula sinalização e endocitose do AtPep1, no entanto quando ausente, BAK1 pode ser substituído por seus homólogos SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE os quais poderiam ter funções adicionais durante as repostas induzidas pelo AtPep1. Eventos de fosforilação após a formação do complexo PEPR-BAK1 parecem ditar as bases moleculares da internalização e sinalização do AtPep1. Finalmente, são discutidos os resultados encontrados nesta pesquisa numa perspectiva geral, destacando a relevância destas descobertas na área de pesquisa em que estão inseridos, o potencial que representa o uso de ligantes marcados fluorescentemente como ferramenta para o estudo de complexos entre ligante-receptor, a disponibilidade do sistema AtPep1-PEPRs como modelo de estudo da endocitose em plantas e sua relação com sinalização, e os futuros desafios na área.

<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>