Understanding Cystatin C: Structure, Function And Its Role In Cerebral Amyloid Angiopathy

Cystatin C belongs to the cystatin superfamily

Question:

Discuss about the Genetic Regulation of Gene Expression.

The protein cystatin C is proteinase inhibitor. This ubiquitous protein is found in the body fluids of all the mammals. The primary function of Protein Cystatin C is to regulate the activity of cysteine via inhibiting its vital functions. Cysteine plays an important role in regulating the intracellular catabolism of proteins and peptides. Protein Cystatin C shows active inhibitory effect on several cysteine protease namely cathepsins B, H, K, L and S. This ubiquitous protein undergoes proteolytic degradation under the influence of cathepsin D and elastase. Cathepsins are lysosomal proteases and are popularly known as housekeeping genes and they differ in their primary structural symmetry, substrate binding specificities and biochemical characteristics (1).

Cystatin C belongs to the cystatin superfamily. It interacts reversibly with target peptidases and such interactions is seemingly independent and depends only on the affinity contributions from a wedge-shaped binding region that is constructed with the help of the two loop-forming inhibitor segments and a corresponding binding region corresponding to the N-terminal segment of that particular inhibitor (2). 

Protein Cystatin C is present as a single copy in the human genome with a size of 5 kb (approx.) It is a non-glycosylated polypeptide chain of 120-residue. cystatin family exhibits low molecular weight and shares homology in their amino-acid sequences. In spite of their sequential homology, there exists a difference in the post translational modification. The nature of the post-translational modification of cystatin gene (alpha, beta and gamma) is different from each other. The modification is dependent on their localizations. This difference in post translational modification helps in the generation of diverse biological functions (2).

The two Cystatin molecules (as displayed in picture A and B) has canonical cystatin fold, (N)–b1– a–b2–L1–b3–AS–b4–L2–b5–(C), with a five-stranded antiparallel b-sheet gripped around a long alpha helix. The appending structure (AS) is broad and irregular. It is positioned at the opposite end of the beta sheet relative to the N-terminus⁄ loop L1 ⁄ loop L2 edge. Such specialized structural symmetry is known as papain-binding epitope. Out of the rest standard five antiparallel beta platted sheet, b1 is one of the shortest elements and It comprises of two residues. In both the molecules displayed in the above picture, the first 11 residues are completely disordered. The following two AS residues, which differ in each molecule are found to be Proline78-Leucine79 in A and Leucine80-Aspartate81 in B. Some of the disulfide bridges exists in a partially broken form in the tertiary structure (3).

Protein Cystatin C and gene CST3

Cerebral Amyloid Angiopathy (CAA) occurs due to the mutation in one of the three exons of cystatin protein. The luciene residue at 68th position of the gene is the principal target for gene mutation. Generally, heterogeneous point mutation occurs at this particular site which results in the formation of truncated protein from the amino terminal domain. The post translational pathway of protein folding (elastase or a serine protease) fails to recognise this truncated protein, leading to protein misfolding. Moreover, this luciene variant at 68th position (Leu68Gln) of cystatin C gene is the major building block of amyloid fibrils. The misfolded protein gets deposited over the cerebral/spinal arteries as amyloid fibrils leading to disease manifestation (4).

Human Cystatin C Gene or CST3 produce Cystatin Protein upon translation. The same gene is also responsible for the generation of cystatin protein found in mammals. It is located in chromosome number 20 in humans. Protein Cystatin C falls under the group of proteinase inhibitors and manipulates the mode of action of cysteine proteinases via inhibiting their activity (2). Three cystatin loci namely CST1 (cystatin SN), CST2 (cystatin SA) and CSTP1 (a cystatin pseudo gene) together constitute CST3. CST3 gene is made up of two introns (non-coding region) and three exons (coding region). The introns in the gene cover a base air of 2252 and 1254 bp respectively. Introns here are located in between the nucleotide triplets encoding the amino acid residues 55-56 and 93-94 of the mature protein respectively. The respective positions of the introns are identical with cystatin SN and cystatin SA genes. However, the first intron of CST3 gene is comparatively bigger than the first introns present in the cystatin SN and cystatin SA genes and thus creating a structural gene difference. The approximate size of the gene though remains similar amounting to about is 4.3 kb (2).

Cerebral Amyloid Angiopathy (CAA), an autosomal dominant disease and is commonly known as hereditary cystatin C amyloid angiopathy (HCCAA). The disease is a combination of amyloidosis with hereditary cerebral haemorrhage. The disease causes amyloid deposition in the spinal or cerebral arteries along with arterioles. The deposited amyloid fibrils block the proper neuronal function, leading to recurrent haemorrhagic strokes. Such shocks cause severe brain damage, and even lead to fatal stroke (5).

This dominant disease results from a point mutation. The truncated version of the cystatin c gene is a result of heterogeneous point mutation. Elastase or a serine protease fails to recognise the truncated protein, leading to protein misfolding. The misfolded or unfolded protein gets deposited over the cerebral/spinal arteries and is then known as amyloid fibrils (2).

Cerebral Amyloid Angiopathy (CAA)

Cystatin C, a Leu68Gln variant is the principal component of the amyloid fibrils. CAA, an autosomal dominant disorder, results out of heterozygous point mutation, in the cystatin C gene (7).

CST3 is commonly known as human cystatin C gene. It is located in chromosome 20 and its mutation is responsible for hereditary cystatin C amyloid angiopathy (6). The misfolded protein fails to functions properly and leading to the generation of complications.

In CAA, blood leaks out from several vessels of the brain. This leakage of the blood outside the blood vessels leads to the formation of blood clot and the brain cells or neurons suddenly stops working. Due to the malfunction of the neurons, the primary symptoms, which are expressed in case of CAA, are weakness or paralysis of the limbs. These symptoms are gradually followed by difficulty in speaking, loss of sensation, loss of body balance. In extreme cases, patients pass into coma. Sudden leakage of the blood vessels through the sensitive tissues results in the generation of unbearable headache. In rare cases there occurs seizures, short spells and other temporary neurologic symptoms like tingling of face and weakness in limbs (8).

Patients with CAA have focal neurological deficit with large parenchymal hematomas. Symptoms like intracranial pressure are common along with alterations in consciousness. CAA also causes mass lesion and intimal vessel thickening leading to transient neurological symptoms like leukoencephalopathy and seizures. Such symptoms may last for a minutes or an hour (9).

CAA is defined as a condition of vasculopathy. It causes deposition of amyloid fibrils inside the arterioles and the arteries of the cerebral cortex. Staining of the affected brain tissue with hematoxylin and eosin revealed hyaline thickening in the blood vessels of the brain along with luminal narrowing. CAA is also detected in progressive dementia after a course of few weeks or months. Dementia which occurs in association with CAA is characterized by severe deposition of vascular amyloid fibrils along with cortical haemorrhages. This haemorrhages causes destruction of the white matter. In case of Alzheimer disease, the amyloid fibrils are deposited in the parenchyma leading to neuritic dystrophy loss of synaptic activity (9). 

CAA is largely untreated because in the majority of the cases, it remains unidentified.  In order to treat haemorrhage associated with CAA, the therapy is limited to withdrawal of antiplatelet agents and anticoagulant agents. Here anticoagulants like warfarin are used. However, prolong used of warfarin is not safe and such treatments may give rise of ischemic heart attack (10).

Clearance of the amyloid plaques occurs via antiamyloid therapy. Current research is trying to employ gantenerumab as the principal drug for the removal of amyloid plaques. Gantenerumab is a fully human anti-A antibody derived from human IgG1. This potent antibody has high affinity towards fibrilar amyloid beta fibrils and binds specifically to amyloid plaques and thereby promoting their clearance (11).

References

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Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, Turk D. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2012 Jan 31;1824(1):68-88.

Kolodziejczyk R, Michalska K, Hernandez?Santoyo A, Wahlbom M, Grubb A, Jaskolski M. Crystal structure of human cystatin C stabilized against amyloid formation. The FEBS journal. 2010 Apr 1;277(7):1726-37.

Rajagopalan P, Refsum H, Hua X, Toga AW, Jack CR, Weiner MW, Thompson PM, Alzheimer's Disease Neuroimaging Initiative. Mapping creatinine-and cystatin C-related white matter brain deficits in the elderly. Neurobiology of aging. 2013 Apr 30;34(4):1221-30.

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Lamontagne M, Timens W, Hao K, Bossé Y, Laviolette M, Steiling K, Campbell JD, Couture C, Conti M, Sherwood K, Hogg JC. Genetic regulation of gene expression in the lung identifies CST3 and CD22 as potential causal genes for airflow obstruction. Thorax. 2014 Sep 2:thoraxjnl-2014.

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Cerebral Amyloid Angiopathy [FAQ] [Internet]. Angiopathy.org. 2017 [cited 26 September 2017]. Available from: https://www.angiopathy.org/faq.html#symptoms

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