Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C.

Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.

High throughput testing of drug library substances and monoclonal antibodies for capacity to reduce formation of cystatin C dimers to identify candidates for treatment of hereditary cystatin C amyloid angiopathy.

OBJECTIVE: To establish a high-throughput system for testing the ability of drugs or monoclonal antibodies to reduce the in vitro formation of cystatin C dimers to identify substances potentially useful for treatment of patients with hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Various combinations of incubation temperature, time period, guanidinium chloride concentration and concentration of cystatin C monomers were tested in low-volume formats to induce dimer formation of recombinant cystatin C. The extent of dimerization was analysed by gel filtration chromatography and agarose gel electrophoresis. RESULTS: A high-throughput system based upon agarose gel electrophoresis was developed and used to test 1040 drugs in a clinical drug library for their capacity to reduce cystatin C dimer formation in vitro. Seventeen substances reducing dimer formation by more than 30% were identified. A similar system for testing the capacity of monoclonal antibodies against cystatin C to reduce the in vitro formation of cystatin C dimers was also developed and used to test a panel of 12 monoclonal antibodies. Seven antibodies reducing dimer formation by more than 30% were identified and the two most potent, Cyst28 and HCC3, reduced dimerization by 75 and 60%, respectively. CONCLUSION: We constructed a simple high-throughput system for testing the capacity of drugs and monoclonal antibodies to reduce the in vitro formation of cystatin C dimers and several candidates for treatment of HCCAA could be identified.

Cystatin C attenuates insulin signaling transduction by promoting endoplasmic reticulum stress in hepatocytes.

It has been reported that cystatin c (Cys C) closely correlates with metabolic disorders such as obesity and diabetes. However, it is still unknown whether Cys C plays a role for these disorders. Our results showed that the insulin signal transduction was largely impaired by Cys C in hepatocytes. In myotubes, however, the insulin signal transduction was not affected. Following experiments revealed that Cys C could induce endoplasmic reticulum stress (ER stress) in hepatocytes, whereas Cys C had no such an effect in myotubes. The alleviation of ER stress by 4-Phenyl butyric acid (4-PBA) restored the impaired insulin signal transduction in Cys C-treated hepatocytes. These results provided direct evidence that, by inducing ER stress, Cys C impairs insulin signal transduction in hepatocytes.

Cystatin C is released in association with exosomes: a new tool of neuronal communication which is unbalanced in Alzheimer's disease.

It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. Multiple lines of research support a neuroprotective role for cystatin C in Alzheimer's disease (AD). Herein we demonstrate that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, is also secreted by mouse primary neurons in association with exosomes. Immunoproteomic analysis using SELDI-TOF MS revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familial AD-associated presenilin 2 mutations (PS2 M239I and PS2 T122R) resulted in reduced levels of all cystatin C forms (native and glycosylated) and of amyloid-ß precursor protein (APP) metabolites within exosomes. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against AD and other neurodegenerative diseases.

P. gingivalis regulates the expression of Cathepsin B and Cystatin C.

Porphyromonas gingivalis is a major etiological agent of periodontitis that could affect the expression of Cathepsins B and C by disrupting the balance between these enzymes and their inhibitor, Cystatin C. We tested this hypothesis by infecting human oral epithelial cells with P. gingivalis or activating solely by its lipopolysaccharide. The mRNA level, the enzymatic activity, and the protein expression of Cathepsin B were increased (three-fold) in a dose-dependent manner, while those of Cystatin C decreased (five-fold). No changes were observed for Cathepsin C. Although activation by lipopolysaccharides led to a delayed imbalance (2 days) between Cathepsin B and Cystatin C, this imbalance took place very rapidly during the infection (< 6 hrs), indicating that the whole bacterium contains components that initiate rapid changes in the transcription rates of Cathepsin B and Cystatin C and selectively modify the molecular pathways that lead to this imbalance.