Mechanism Of Human Cystatin C-cathepsin B Axis Regulating Amyoid β Level

Amyloid deposition disease is caused by the accumulation of specific peptides or proteins into highly stable,cytotoxic fibrotic deposits of amyloid.Amyloid β（Aβ）is considered to be the pathogenic protein that causes Alzheimer’s disease（AD）.Human cystatin C（HCC）is a cysteine protease inhibitor widely present in Human body fluid and tissue fluid,which can inhibit the activity of extracellular cysteine protease.HCC can bind to soluble Aβ.Studies have found that HCC and Aβfibrillary co-locate in the brain lesions of AD patients,suggesting that HCC has a certain neuroprotective effect.Cathepsin B（Cat B）is a cysteine protease that exists mainly in the lysosomes of glial cells in the human brain.Increased Aβ content can stimulate the release of Cat B by glial cells to regulate Aβ,but its exact role in Amyloid Precursor Protein processing and Aβ metabolism in the central nervous system remains to be determined.This study aimed to investigate the sensitivity of intracellular Aβ aggregation to HCC expression by studying the intracellular dual overexpression of HCC and Aβ.Molecular dynamics method was used to elucidate the interaction modes and dynamic processes of the three proteins,predict the interaction relationship among the three proteins,and finally clarify the influence mechanism of HCC-CAT B-axis on Aβ fiber formation.Using Pichia pastoris（P.pastoris）as a "cell factory" for expressing exogenous proteins,we first constructed three groups of recombinant P.pastoris strains: yeast strain overexpressing HCC,yeast strain overexpressing Aβ,and yeast strain overexpressing both HCC and Aβ.After the three recombinant strains were successfully constructed,they were induced to express the target proteins respectively.It was found that HCC was mostly secreted out of the yeast with a small amount of protein remained in the cells.Second,.The secretion of Aβwas not detected in culture medium,but certain amount of it resided in the cells.By over-expression of PDI（a kind of molecular chaperone）to increase the expression,a large amount of Aβ resides in the cell in the form of monomer,possibly due to the role of molecular chaperone.Finally,the molecular docking method was used to simulate and analyze the interaction between the three proteins and illustrate the interaction process among the three proteins.The significance of this study is to explore the theoretical basis that HCC may play a role in the early stage of AD and Cat B may play a role in the late stage of AD,by understanding the interaction between HCC,Cat B and Aβ,which may lay a foundation for the early prevention and treatment of Alzheimer’s disease.