Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells
María BjarnadóttirBirgitte S. WulffMansoureh SameniBonnie F. SloaneDietrich KepplerAnders GrubbMagnus Abrahamson
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AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.Keywords:
3T3 cells
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Cystatin C, the full name of cystatin C, is one of the most potent cathepsin inhibitors currently known, which can strongly inhibit cathepsin in lysosomes and regulate the level of intracellular proteolysis. Cystatin C plays a very broad role in the body. High temperature-induced brain injury leads to very serious damage to brain tissue, such as cell inactivation, brain tissue edema, etc. At this time, cystatin C can play a crucial role. Based on the research on the expression and role of cystatin C in high temperature-induced brain injury in rats, this paper draws the following conclusions: high temperature can cause very serious damage to the brain tissue of rats, which can seriously lead to death. Cystatin C has a protective effect on brain cells and cerebral nerves. When the brain is damaged by high temperature, cystatin C can relieve the damage of high temperature to the brain and protect brain tissue. In this paper, a detection method for cystatin C with more outstanding performance is proposed, and compared with the traditional detection method, the detection method in this paper is verified to have more accurate accuracy and excellent stability through comparative experiments. Compared with traditional detection methods, it is more worthwhile to use and is a better detection method.
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Cystatin C is a protease inhibitor produced in a constant manner by nucleated cells.This molecule is freely filtrated by the glomerule and quite completely catabolized in the proximal tubules.Its plasmatic concentration might be endogenous maker of glomerular filtration rate(GFR).Many studies show that serum cystatin C is at least as good as serum creatinine to estimate GFR,and researchers induced some formulaes for estimating GFR based on cystatin C.In addition,urinary cystatin C has been found to be specific maker of renal tubular early dysfunction.This article reviews applications of cystatin C in the field of kidney disease.
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Cystatin C, an endogenous inhibitor of cathepsin proteases has emerged as a biomarker of cardiovascular risk and reduced renal function. Epidemiological studies indicate that serum cystatin C increased in human obesity. Here, we evaluated the contribution of adipose tissue to this elevation, based on our previous observation that cystatin C is produced by in vitro differentiated human adipocytes. We measured serum cystatin C in 237 nonobese (age: 51 +/- 0.8 years; BMI: 22.8 +/- 0.11 kg/m(2)) and 248 obese subjects (age: 50 +/- 0.8 years; BMI: 34.7 +/- 0.29 kg/m(2)). Creatinine-based estimated glomerular filtration rate (eGFR) was calculated to account for renal status. Cystatin C gene expression and secretion were determined on surgical adipose tissue biopsies in a distinct group of subjects. Serum cystatin C is elevated in obese subjects of both genders, independently of reduced eGFR. Cystatin C mRNA is expressed in subcutaneous and omental adipose tissue, at twice higher levels in nonadipose than in adipose cells. Gene expression and cystatin C release by adipose tissue explants increase two- to threefold in obesity. These data confirm elevation of serum cystatin C in human obesity and strongly argue for a contribution of increased production of cystatin C by enlarged adipose tissue. Because cystatin C has the potential to affect adipose tissue and vascular homeostasis through local and/or systemic inhibition of cathepsins, this study adds a new factor to the list of adipose tissue secreted bioactive molecules implicated in obesity and obesity-linked complications.
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Cystatin C is a constitutively expressed and abundant cysteine protease inhibitor within the cerebrospinal fluid (CSF). Recent studies have reported a significant reduction in cystatin C concentration in the CSF of patients with amyotrophic lateral sclerosis (ALS) and several other neurodegenerative diseases, relative to healthy controls. Cystatin C can exhibit both neuroprotective and neurotoxic properties, suggesting that altered CSF cystatin C concentrations could potentially impact the pathogenesis or progression of these disorders. However, it is unclear if alterations in cystatin C concentration result in physiologically relevant differences in its functional activity within the CSF. Measurements of the cysteine protease inhibitory activity of cystatin C within the CSF have not been reported, and the relationship between CSF cystatin C concentration and activity levels in different disease contexts has not been investigated.We used a papain inhibition assay to evaluate the total cystatin C activity in CSF samples from 23 ALS patients, 23 healthy controls, and 23 neurological disease controls. Cystatin C concentrations in these samples were previously measured by ELISA. Correlations between cystatin C concentration and activity were assessed with nonparametric statistics. Activity ratios were compared among diagnostic groups using both one-way ANOVA and repeated measures statistics.Total cystatin C activity was found to be directly proportional to its protein concentration in all subjects, and cystatin C activity was not altered in ALS patients. In addition, our data suggest that cystatin C is the predominant cysteine protease inhibitor in human CSF.Our data demonstrate the successful measurement of the functional activity of cystatin C in the CSF, and show that total cystatin C activity can be inferred from its total protein concentration. Our results also suggest that cystatin C is the major cysteine protease inhibitor in human CSF and altered CSF cystatin C concentration may play a role in the pathobiology of ALS and other neurological diseases.
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Hereditary cystatin C amyloid angiopathy (HCCAA) is a disorder characterised by multiple strokes in young adults, resulting in paralysis and dementia. The disease is caused by a mutation in the gene coding for the peptidase inhibitor cystatin C. The mutation causes an amino acid substitution Leu68 -> Gln in cystatin C (L68Q cystatin C) which results in deposition of the protein in the blood vessels in the brain. The overall goal of this study was to elucidate the pathogenic mechanisms leading to amyloid deposition in patients suffering from HCCAA. This was addressed by studying factors involved in the regulation of cystatin C expression and the behaviour of the L68Q variant in different cellular systems and clinical samples. When studying factors involved in cystatin C gene regulation we found that the steroid dexamethasone causes a significant and dose-dependent increase in cystatin C secretion from cultivated HeLa cells, up to a maximal increase of 80% at biological levels of the steroid. When looking at monocytes isolated from HCCAA patients and transfected mouse and human neuroblastoma cells we saw, in all cells tested, that the mutated cystatin C was secreted, but at a significantly lower rate than the wildtype protein. An increased accumulation of cystatin C was seen in all transfected cells expressing the gene encoding L68Q cystatin C. The accumulated L68Q cystatin C is insoluble and located mainly in the endoplasmic reticulum. When studying human neuroblastoma cell-lines we saw that they expressed and secreted very high levels of cystatin C compared to other human cell-lines tested. The neuroblastoma cells were also different from other cell types in that they showed intracellular insoluble wildtype cystatin C. Finally we investigated the turnover of cystatin C in plasma and cerebrospinal fluid (CSF) from HCCAA patients. Both L68Q and wildtype cystatin C were found in plasma from these patients but only the wildtype cystatin C could be detected in CSF.
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