Biology subjects

Giardina, G.

Publications and source records attributed to Giardina, G..

3 recordsLinked to original sources

bioRxiv2021

Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Genetic variations expand the conformational landscape of proteins and may underlie cryptic properties that promote environmental adaptability. However, they can also represent modifying factors for disease susceptibility, by changing frustrated regions that in turn affect protein overall intracellular fitness. In this dichotomy between conservation and innovation, understanding at structural level how genetic variations keep the balance to maintain protein fitness represents an unmet need. Herein, we took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By crystallographic studies and molecular dynamics simulations we showed that the polymorphic amino acid substitutions shape the conformational flexibility of AGT1 so that three surface regions that are structured in AGT-Ma become disordered in AGT-Mi, thanks to plasticity effects propagated from the mutation site(s) to the whole structure. In-depth biochemical characterisation of variants from a library encompassing the three regions correlate this plasticity to a fitness window between AGT-Ma and AGT-Mi, and suggest the existence of cryptic functions related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between stability and frustration to expand the potential innovability of the protein.

biochemistry↗

bioRxiv2020

SHMT1-RNA interaction dynamically regulates serine and glycine concentration in cells

Human serine hydroxymethyltransferase (SHMT) regulates the serine-glycine one carbon metabolism and plays a role in cancer metabolic reprogramming. Two SHMT isozymes are acting in the cell: SHMT1 encoding the cytoplasmic isozyme, and SHMT2 encoding the mitochondrial one. Here we present a molecular model built on experimental data reporting the interaction between SHMT1 protein and SHMT2 mRNA, recently discovered in lung cancer cells. Using a stochastic dynamic model, we show that RNA moieties dynamically regulate serine and glycine concentration, shaping the system behaviour. For the first time we observe an active functional role of the RNA in the regulation of the serine-glycine metabolism and availability, which unravels a complex layer of regulation that cancer cells exploit to fine tune amino acids availability according to their metabolic needs. The quantitative model, complemented by an experimental validation in the lung adeno-carcinoma cell line H1299, exploits RNA molecules as metabolic switches of the SHMT1 activity. Our results pave the way for the development of RNA-based molecules able to unbalance serine metabolism in cancer cells.

biochemistry↗

bioRxiv2020

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), the latter two targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result strongly indicates that the role of the dTMP-SC assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, may provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

biochemistry↗