Biosintesi dell’acido δ1-pirrolin-5-carbossilico e sua riduzione a prolina: meccanismi di regolazione in specie vegetali modello e in altri eucarioti, e possibili applicazioni

Stress is a factor that can negatively affect plants’ functions, altering their growth and productivity. Due to their sessile nature, plants are unable to escape this situation. For this reason, evolution has led to the development of some defense mechanisms to limit the damage caused by biotic and abiotic stress conditions. Among these strategies, many plants accumulate in the cell high concentrations of the so-called compatible osmolytes, such as proline, a very soluble imino acid involved in protein synthesis. Proline metabolism is a conserved process within almost all organisms, both prokaryotes and eukaryotes, which uses glutamate or ornithine as precursors. In most higher eukaryotes, biosynthesis proceeds mainly from glutamate that is reduced in the cytosol to pyrroline-5- carboxylate (P5C) by P5C synthetase (P5CS); P5C is then reduced in turn to proline by P5C reductase (P5CR). The catabolic process takes place in the mitochondrion by the action, in order, of proline dehydrogenase (PRODH) and P5C dehydrogenase (P5CDH), which oxidize proline to P5C and P5C to glutamate. Proline metabolism enzymes use a series of cofactors such as NAD(P)+, NAD(P)H and FAD needed to provide or acquire electrons during redox reactions. It is therefore easy to understand how these processes can reflect on the redox balance and the energy state of the cells. Furthermore, in animals and very probably also in plants, a cycle is established between mitochondria and cytosol in which PRODH and P5CR catalyse the interconversion of proline to P5C. This apparently futile cycle allows for the regulation of the levels of pyridine nucleotides, the synthesis of ATP, the production of ROS, and the exchange of reducing equivalents between the two compartments, resulting very advantageous for the cellular economy. The experiments performed in the frame of this Ph.D. project were carried out on both plant and animal model systems. To better understand the functions and implications of proline, in fact, it is important to compare different systems, identifying all possible similarities and differences. For example, yeast represents an exception compared to most other organisms since it does not accumulate proline following the exposure to stress and, to date, there is no evidence related to the existence of the proline-P5C cycle in this species. Therefore, the biochemical characterization of the P5CR of Saccharomyces cerevisiae was performed, with particular attention to its post-translational regulation. The analysis highlighted the fact that the enzyme seems to prefer the use of NADH as the electron donor, unlike the P5CR from Arabidopsis thaliana (A. thaliana) and rice, and that in the presence of millimolar concentrations of exogenous proline, the catalytic activity is inhibited. Furthermore, the lack of stimulation by salts is consistent with the fact that osmotic stress does not induce proline accumulation in yeast. Further aspects have been investigated for human P5CR. In this case, in addition to the biochemical characterization, the ability of molecules of phosphonic nature to inhibit the enzyme was evaluated. These substances had already been tested and proved to be active against plant and bacterial P5CRs. The decision to extend the investigation to the human enzyme derives from the fact that in many types of cancers, there is an increase in the expression of P5CR, which leads to an increase in the activity of the cycle between proline and P5C, favoring the development of neoplasia. Therefore, through this inhibitory strategy, it was possible to observe the repercussions resulting from the interference with the cycle and the importance that the latter covers. Testing the inhibitor on some human tumor cell lines showed in fact a reduction in cell proliferation and viability. In the synthesis of proline from glutamate, the limiting step is the one catalyzed by the P5CS enzyme. In many higher plant species, including A. thaliana and Oryza sativa (O. sativa), the gene coding for this enzyme has undergone a duplication process, giving rise to the isoenzymes P5CS1 and P5CS2. To date, the studies carried out to understand the differences between them have mainly focused on the analysis of their transcriptional regulation. However, the biochemical characteristics of these two isoforms should also be investigated, therefore in this project the enzymes P5CS1 of A. thaliana and P5CS2 of rice were cloned, expressed in bacterial cells and characterized. Through enzymatic assays conducted in vitro, it was possible to determine the kinetic properties of both enzymes and the mechanisms for their post-translational regulation. Investigations on the second isoform of rice were completed, highlighting that exogenous proline, even at low concentrations, inhibits the enzyme, while the presence of chlorides has a positive effect on the activity. It has been hypothesized that both rice isoenzymes are involved in the accumulation of proline during osmotic stress, while in A. thaliana they should play a more distinct functional role. For this reason, it was decided to continue the experiments on AtP5CS1 and AtP5CS2 enzymes. In the present project some preliminary results related to AtP5CS1 are reported that show some relevant details. Also this isoform, as expected, is positively modulated by the presence of salts. More interestingly, proline does not exert feedback inhibition, supporting the hypothesis that this isoenzyme allows the plant to accumulate the amino acid during exposure to osmotic stress. The last aspect taken into consideration concerns the role of proline within the symbiotic process between legumes and rhizobia. Some studies reported that the levels of some enzymes involved in proline metabolism increase inside the nodules during symbiosis. To explain this phenomenon, some hypotheses have been formulated on the implications that proline can have in the process, during which it could act as an energy substrate, support symbiosis in stressful situations, or play a role in the differentiation of bacteroids. None of these theories has been conclusively proven, but understanding these mechanisms could prove fundamental to maximize the yields obtainable from the symbiotic process. For this reason, information relating to the topic was collected and presented in a mini-review, where informations relating to a third isoform of P5CS identified in Medicago truncatula were also reported, which could represent a specific leguminous variant involved in the symbiotic process. Within this doctoral project, therefore, some research lines have been carried out to broaden our knowledge relating to the roles of proline and the regulation of the enzymes involved in its metabolism, with the aim to provide new ideas toward the development of new plant varieties more able to cope with stressful situations.