Structural and Functional Characterisation of the Human Mitochondrial Pyruvate Carrier

Abstract

The metabolism of pyruvate in the cell is abnormally regulated in several human diseases, such as type-II diabetes, cancer and neurodegenerative disorders, in which the human mitochondrial pyruvate carrier (HuMPC) sits at a pivotal point. It transports pyruvate across the inner mitochondrial membrane and consists of two proteins, MPC1 and MPC2, and is a potential drug target against these diseases. There is currently no three-dimensional structure at atomic resolution nor unambiguity of the biological oligomeric composition of this pyruvate transporting complex, although many studies advocate for a heterodimer of MPC1/2 being the main functional unit. In this study, the pyruvate transport activity of HuMPC complexes was investigated as well as the presence of a MPC1/2 heterocomplex. The pyruvate transport activity of human MPC1 alone, MPC2 alone and MPC1/2 mixed together was investigated with a proteoliposome assay based on the enzymatic activity of lactate dehydrogenase (LDH). In addition, the presence of the MPC1/2 complex in proteoliposomes was examined with nano-differential scanning fluorimetry, as well as with cross-linking trials for further analyses with mass spectrometry. Pure HuMPC protein was obtained by affinity chromatography from solubilised Pichia pastoris membranes. Here it was demonstrated that MPC2 and MPC1/2 displayed higher pyruvate transport activity compared to MPC1. Moreover, the nano-DSF measurement did not give any concrete evidence of the presence of the MPC1/2 heterocomplex. The mass spectrometric analyses did not identify any cross-links between MPC1 and MPC2 in the MPC1/2 proteoliposomes but more peptides of MPC2 than MPC1 could be identified. In conclusion, the true pyruvate transporting complex of HuMPC still remains unclear based on these results, as well as the existence of the MPC1/2 heterocomplex, which requires further investigation.

Popular Abstract

In cancer, type-II diabetes and neurological disorders, the way important molecules are interconverted into one another inside human cells is abnormally altered. An example of such a molecule is pyruvate. It is a central intermediary molecule in the processing of glucose that is present in the food we eat so we can generate energy for several important processes in the cell. A critical step in the continued processing of pyruvate is its transportation from the cytosol into the mitochondria. The protein responsible for this transportation is called the mitochondrial pyruvate carrier (MPC) and in humans, it consists of two different proteins (MPC1 and MPC2) cooperating and forming a complex to perform its function. It has been proposed that the MPC complex can serve as a potential drug target against the abovementioned diseases. Up until this point, there is no detailed information of how this protein is built up at a molecular level, which is required for efficient production of drugs. Moreover, it is also unclear of how many MPC1 and MPC2 proteins the MPC complex is composed of and which of three MPC complexes, MPC1 alone, MPC2 alone and MPC1/2 together is the main functional unit. The aim of this study is to investigate which of these three possible MPC complexes has the highest ability to transport pyruvate in a cell membrane resembling environment, called proteoliposomes, which are vesicles of lipid molecules in which the protein is imbedded in. The existence of a MPC1/2 complex, which several studies point towards is the main functional unit, was also examined with temperature stability tests based on the decomposition of the protein and with experiments in which one attempted to attach the two proteins together, called cross-linking. The detailed appearance of the MPC complex at a molecular level was supposed to be investigated as well, but due to lack of time this was not performed. To obtain these MPC1 and MPC2 proteins, cell membranes containing these proteins were extracted from yeast cells followed by isolation of the proteins with chromatographic methods based on their binding properties. It turned out that both MPC2 alone and MPC1/2 were able to actively transport pyruvate into these proteoliposomes, while MPC1 alone did not. In addition, the attempt to prove the existence of the MPC1/2 complex with the temperature stability tests as well as the attempt of cross-linking MPC1 and MPC2 together were both unsuccessful. In conclusion, although both MPC2 alone and MPC1/2 were able to transport pyruvate, it is still unclear which one of these two different species has the highest pyruvate transporting ability. Moreover, the unsuccessful attempt of the temperature stability tests as well as the cross-linking leaves the presence of the MPC1/2 heterocomplex still unknown and needs to be further investigated.

• author: Gripvall, Simon
• supervisor: Susanna Törnroth-Horsefield
• organization: Department of Chemistry
• course: KEMR30 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: biochemistry, complex, MPC, proteoliposomes, pyruvate, transport
• language: English
• id: 9117646
• date added to LUP: 2023-06-01 08:46:16
• date last changed: 2023-06-01 08:46:16