Preliminary Crystallographic Study Of D-2-hydroxyglutarate Dehydrogenase And ER-resident Reticulocalbin-1

BackgroundBiomacromolecules,based on proteins,support almost all the activities of a healthy organism.A comprehensive understanding of these proteins and their complexes,the three-dimensional structure of their assemblies,their interactions and modes of movement,and their relationship to normal biological function and abnormal pathological phenomena is strategically critical in structural genomics.An important feature of tumors is abnormal cell metabolism.The research objects of this thesis are two proteins that play a key role in metabolism,D-2-hydroxyglutarate dehydrogenase and endoplasmic reticulum protein Reticulocalbin-1.According to literature reports and previous research results of this laboratory,both proteins affect tumorigenesis through metabolism.D-2-hydroxyglutaric acid(D-2-HG)is a low-concentration metabolite of the body,which is mainly derived from ?-ketoglutarate(?-KG),an important metabolic intermediate in the tricarboxylic acid cycle.Recently,a large number of studies have shown that D-2-HG is a cancer-promoting metabolite,and the abnormal accumulation of D-2-HG has been found in glioma,acute myeloid leukemia,and many other cancers,which may lead to the occurrence of cancer by competitively inhibiting ?-KG-dependent enzyme.At present,the relevant studies on D-2-HG metabolic pathway have attracted much attention in the field of tumor research.As the main catabolic enzyme of D-2-HG,D2HGDH not only maintains the low level of D-2-HG,but also participates in the regulation of ?-KG,and plays an important role in the body's metabolism.However,the molecular mechanism by which D2HGDH performs catalytic functions is still poorly understood,so this part was carried out.Endoplasmic reticulum(ER)is an important organelle of cells,which directly participates in and influences various life activities of cells.The chaperones and enzymes in the endoplasmic reticulum are essential to ensure normal endoplasmic reticulum function.Therefore,many studies have attempted to reveal the role and regulatory mechanism of these endoplasmic reticulum resident proteins.Reticulocalbin-1(RCN1)is a calcium binding protein located in the lumen of the endoplasmic reticulum and belongs to the CREC family.Recent studies have revealed that abnormal expression of RCN1 is closely related to the occurrence of diseases,especially tumors.RCN1 is highly expressed in cancer cells such as highly invasive breast cancer cells and colorectal cancer cells,but low in prostate cancer cells.However,the exact function of RCN1 is still unknown,and its mechanism is more unclear,and further research is urgently needed.In addition,our research group preliminarily found that RCN1 protein may play a role in regulating glucose metabolism.In order to explore its mechanism,the crystal structure of RCN1 is preliminarily studied in this part,so as to promote the emerging functional research.ResultPart? Preliminary crystallographic study of D-2-hydroxyglutarate dehydrogenase(1)We screened the expression conditions of full-length protein and truncated protein of human D2HGDH in the prokaryotic expression system,and obtained a small amount of soluble protein with enzyme activity,but not enough for subsequent crystallographic studies.(2)We expressed and purified two bacteria-derived D2HGDH homologous proteins,Homologuel and Homologue2.A diffractive single crystal of Homologue2 was obtained,and a crystal structure with a resolution of 2.5OA was obtained.(3)The overall structure of Homologue2 includes two domains:a cap domain and a FAD binding domain,and a slit is formed between the two domains.The cofactor FAD is mostly wrapped in the FAD binding domain.The isoprazine ring is located at the slit formed by the FAD binding domain and the cap domain,and together with the loop between ?12 and ?13 constitutes the enzyme catalytic active center.(4)Homolugue2 forms a functional unit of dimer with its symmetric molecule.Each monomer molecule has a random loop inserted into the groove of the FAD binding domain of the symmetric molecule.In vitro enzyme activity experiments suggest that the C-terminal mediates the dimerization of D2HGDH,and the dimerization is to maintain the high affinity of FAD and does not directly participate in catalysis.(5)In vitro enzyme activity experiments also initially verified the active center of Homologue2,and the specific mechanism of enzyme activity catalysis needs further study.Part ? Preliminary crystallographic study of ER-resident Reticulocalbin-1(1)We expressed the full-length and truncated protein of hRCNl using prokaryotic expression system,and purified the protein by affinity chromatography,ion exchange chromatography and molecular sieve chromatography.(2)According to the stability experiment,we obtained a stable truncated protein hRCN166-331,and achieved its high-efficiency expression and purification.(3)Based on the truncated protein hRCN166-331,the crystals of hRCN166-331 were obtained through crystallization screen,but the diffraction force of the crystal were very weak,which still need to be optimized.(4)We preliminary explored the external factors that can affect the aggregation state of hRCN1 protein and found that Ca2+,DTT and Mn2+promote the formation of monomers.These results lay a solid foundation for further crystallographic study of RCN1.Innovation and deficiency1.Innovation points(1)We first reported the crystal structure of D2HGDH family.The overall structure of D2HGDH family protein included two domains:the cap domain and the FAD binding domain.The active center was located in the slit formed by the two domains.(2)We first reported the formation of homodimer of D2HGDH was in order to maintain high affinity of FAD and not involved in catalysis.(3)We first explored the crystal structure of RCN1,and obtained the initial screening crystals of RCN1.2.Deficiency(1)In the first part of this paper,the structure of a protein of D2HGDH family was reported.However,the substrate of this protein and the metal ions it binds were not clear,and the catalytic mechanism of this protein was not explored in depth,so it still needs to be further studied.(2)In the second part of this paper,a preliminary crystallographic study on RCN1 protein was carried out,and the initial screening crystals of the protein were obtained,but the crystals were small,which still needs to be optimized by subsequent experiments.