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Focus on Molecules: Cystatin C
Experimental Eye Research 84 (2007) 1019e1020 www.elsevier.com/locate/yexer
Focus on Molecules: Cystatin C Luminita Paraoan*, Ian Grierson School of Clinical Sciences, Unit of Ophthalmology, UCD Building, University of Liverpool, Daulby Street, Liverpool, L69 3GA, UK Available online 14 April 2006
Keywords: cystatin C; proteolysis; retinal pigment epithelium; secretion; age-related macular degeneration
1. Structure Cystatin C was called in early reports g-trace and post-g-globulin. It is encoded by the human gene CST3 (gene map locus 20p11.21; accession number NM_000099; cystatin C precursor accession number NP_000090) and is the main amyloid-forming type 2 member of the cystatin superfamily of structurally homologous cysteine peptidase inhibitors. The CST3 gene presents three KspI polymorphisms that result, due to strong linkage disequilibrium, in only two human haplotypes known as variant A (wild-type) and variant B (in which one of the polymorphisms translates into an amino acid substitution in the signal peptide; see below). The protein is synthesized as a precursor of 146 amino acids residues, with the N-terminal 26 amino acid-long hydrophobic signal sequence bearing an essential role in targeting and processing the protein for secretion. After directing precursor cystatin C (wildtype) to the endoplasmic reticulum, the signal sequence is cleaved and the resulting mature cystatin C is processed through the Golgi apparatus and the secretory pathway. The active, monomeric form of the mature 120 amino acid residues-long cystatin C (molecular mass of w13 kDa) folds into a core structure that defines the topology of cystatins (Fig. 1A) and that is essentially composed of five-stranded antiparallel b-sheets wrapped around a central helix, with two disulphide bonds towards the carboxyl terminus (Janowski et al., 2001, among others). The cysteine peptidase-binding surface is composed of two beta-hairpin loops and the N-terminal segment of the mature protein. Additionally, a distinct site is involved in binding of mammalian legumain, a member of the caspase family of lysosomal peptidases.
promiscuous function in relation to target peptidases are suggestive of a plethora of roles in proteolysis-linked biological processes, most likely underlying tissue-regulatory functions. Cystatin C is present in human body fluids at physiological concentrations and is the dominating cysteine peptidase inhibitor in cerebrospinal fluid where, as a consequence of remarkably high expression in the choroid plexus of the brain, it is over five times more concentrated than in blood plasma. Despite the wealth of biochemical data on its inhibitory characteristics, the specific functions played by cystatin C in physiological processes are documented only to a limited extent. As such, roles for cystatin C are hypothesized in bone resorption, arterial wall remodelling, regulation of neuronal apoptosis triggered by oxidative stress. Further elucidation of cystatin C roles is likely to emerge from characterization of its involvement in various pathologies (see below). In the eye, in non-pathological conditions, cystatin C is localized in various tissues in the anterior segment, with one of the highest concentrations in the ciliary epithelium, which presumably represents the main source of this protein in the aqueous. In the posterior eye, the inhibitor is synthesized almost exclusively by the retinal pigment epithelium (RPE), which presents fairly abundant levels of both its transcript and protein and constitutively secretes the mature form of the protein (Paraoan et al., 2001). RPE secretion of cystatin C appears polarized basally in vitro, suggesting a putative extracellular function, at least in part, in or around Bruch’s membrane (Paraoan et al., 2001).
3. Disease involvement 2. Function Cystatin C is a biochemically well-characterized, strong inhibitor of cysteine peptidases of the papain family, especially cathepsins B, H, L and S, and also of some lysosomal caspases, such as legumain. The broad expression of cystatin C by various tissues coupled with its ubiquitous presence in extracellular fluids and its relatively * Corresponding author. E-mail address: [email protected] (L. Paraoan). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.01.024
Mature cystatin C undergoes dimerization/oligomerization through three-dimensional domain swapping that involves one of the beta-hairpin loops, thus rendering it inactive as a cysteine peptidase inhibitor. The process is enhanced in vivo by the presence of an N-terminally truncated form of the protein and occurs in vitro under pre-denaturing conditions. Oligomerization contributes to the formation of highly stable amyloid fibres that are deposited mainly in brain arteries and are associated with various neuropathologies in the elderly, notably alongside amyloid-beta precursor protein in amyloid plaques in the brain of Alzheimer disease patients. A naturally occurring, albeit relatively
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L. Paraoan, I. Grierson / Experimental Eye Research 84 (2007) 1019e1020
Fig. 1. The three-dimensional cystatin fold is best defined by the structure of chicken cystatin (panel A; reproduced with permission after Janowski et al., 2001), consisting of five antiparallel b-sheets (b1-5) enfolding a long a1 helix. The N-terminus together with loops L1 and L2 define the active site of the mature cystatin C. Trafficking of human variant B A25T precursor cystatin C (panel B) is spectacularly altered compared with that of wild-type form (panel C). Confocal micrographs of living human RPE cells transiently transfected with the respective cystatin C constructs fused to enhanced green fluorescent protein (EGFP) are shown as merged images that were collected on three channels corresponding to the following fluorescent emissions: green fluorescence of the cystatin C-EGFP fusion proteins; red fluorescence of the oxidated Mitotracker Red CM-H2Xros dye (Molecular ProbesÔ, Invitrogen, Paisley, UK) concentrated in the mitochondria of actively respiring cells; blue fluorescence of the endoplasmic reticulum (ER)/Golgi-specific dye ER-TrackerÔ Blue-White DPX (Molecular ProbesÔ, Invitrogen, Paisley, UK). Transfected cells are indicated by arrows. A considerable fraction of the variant B precursor cystatin C colocalizes with mitochondria (yellow/orange colour indicated by thin arrows in panel B), while being significantly excluded from the ER/Golgi (blue). Conversely, wild-type precursor cystatin C is targeted and processed efficiently through the ER/Golgi apparatus (thick arrows in panel C), entirely independent of the mitochondria (red).
rare, L68Q mutant significantly accelerates the formation of cystatin C-containing amyloids and is the causative agent of an autosomal dominant form of hereditary cerebral haemorrhage with amyloidosis that is fatal in early adult life. In addition to such conformation-linked diseases, a role of cystatin C in other degenerative processes in the central nervous system such as Parkinson’s disease, as well as vascular diseases (notably atherosclerosis and aneurysms) can be attributed to an altered expression of the inhibitor, reflected primarily in an imbalance in its ratio to target cathepsins. A surprising association of cystatin C with eye pathology has unveiled a possible novel mechanism of cystatin C involvement in pathogenesis. Homozygosity for haplotype (variant) B of CST3 correlates with increased risk of developing exudative age-related macular degeneration (Zurdel et al., 2002). Variant B precursor cystatin C has an alanine (A) to threonine (T) substitution in the penultimate position of the signal sequence (A25T). The A25T substitution has dramatic consequences in terms of intracellular trafficking of the protein (Fig. 1B and C) by unexpectedly diverting it from the conspicuous Golgi localization to a predominant mitochondrial association (Paraoan et al., 2004). As a corollary, the secretion of cystatin C is significantly impaired. The A25T variant does not appear to interact with the wild-type cystatin C, nor to affect its secretion, thus providing further experimental support for the variant allele acting in a recessive fashion. Further data resulting from characterization of trafficking and processing of other engineered mutants, particularly the A25S precursor cystatin C mutant, a biochemically intermediate between the wild-type and the AMD-associated variant, corroborate the hypothesis that the variant B cystatin C protein is excluded from transport through the endoplasmic reticulum. Possibly this is due to an unfavourable interaction with components of the translocation channel.
4. Future studies Whether the retinal neurodegeneration-related biological effect of the A25T variant precursor cystatin C is due to its impaired secretion or to its mitochondrial association, or to the combination of these,
remains to date an open question. The mitochondrial localization is intriguing and certainly worthy of further investigation, as is the contribution of impaired function of cystatin C to the multifactorial aetiology of age-related macular degeneration. Cystatin C appears instrumental in regulating the deleterious effects of cysteine peptidases implicated in central nervous system-associated pathology. Evidence uncovered steadily over the last few years points towards involvement of cystatin C in the process of neuroregeneration or neurodegeneration of neurons in response to cellular damage, having presumed anti-apoptotic and/or neuroprotective roles. Notably in this respect, the significance of variant B cystatin C association with increased risk for late-onset Alzheimer disease remains to be established. In the light of current knowledge of cystatin C biology, impairment of secretion, in particular involving modulators of proteolysis, alongside subsequent misfolding of soluble proteins in the RPE cells are emerging as critical molecular events underlying macular degeneration. Acknowledgements The authors thank Dr Dave Spiller and Arjuna Ratnayaka for skilful help with cell imaging and flawless transfections.
References Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., Grubb, A., Abrahamson, M., Jaskolski, M., 2001. Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Nat. Struct. Biol. 8, 316e320. Paraoan, L., White, M.R.H., Spiller, D.G., Grierson, I., Maden, B.E.H., 2001. Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway. Mol. Membr. Biol. 18, 229e236. Paraoan, L., Ratnayaka, A., Spiller, D.G., Hiscott, P., White, M.R.H., Grierson, I., 2004. Unexpected intracellular localization of the AMDassociated cystatin C variant. Traffic 5, 884e895. Zurdel, J., Finckh, U., Menzer, G., Nitsch, R.M., Richard, G., 2002. CST3 genotype associated with exudative age related macular degeneration. Br. J. Ophthalmol. 86, 214e219.
Focus on Molecules: Cystatin C Luminita Paraoan*, Ian Grierson School of Clinical Sciences, Unit of Ophthalmology, UCD Building, University of Liverpool, Daulby Street, Liverpool, L69 3GA, UK Available online 14 April 2006
Keywords: cystatin C; proteolysis; retinal pigment epithelium; secretion; age-related macular degeneration
1. Structure Cystatin C was called in early reports g-trace and post-g-globulin. It is encoded by the human gene CST3 (gene map locus 20p11.21; accession number NM_000099; cystatin C precursor accession number NP_000090) and is the main amyloid-forming type 2 member of the cystatin superfamily of structurally homologous cysteine peptidase inhibitors. The CST3 gene presents three KspI polymorphisms that result, due to strong linkage disequilibrium, in only two human haplotypes known as variant A (wild-type) and variant B (in which one of the polymorphisms translates into an amino acid substitution in the signal peptide; see below). The protein is synthesized as a precursor of 146 amino acids residues, with the N-terminal 26 amino acid-long hydrophobic signal sequence bearing an essential role in targeting and processing the protein for secretion. After directing precursor cystatin C (wildtype) to the endoplasmic reticulum, the signal sequence is cleaved and the resulting mature cystatin C is processed through the Golgi apparatus and the secretory pathway. The active, monomeric form of the mature 120 amino acid residues-long cystatin C (molecular mass of w13 kDa) folds into a core structure that defines the topology of cystatins (Fig. 1A) and that is essentially composed of five-stranded antiparallel b-sheets wrapped around a central helix, with two disulphide bonds towards the carboxyl terminus (Janowski et al., 2001, among others). The cysteine peptidase-binding surface is composed of two beta-hairpin loops and the N-terminal segment of the mature protein. Additionally, a distinct site is involved in binding of mammalian legumain, a member of the caspase family of lysosomal peptidases.
promiscuous function in relation to target peptidases are suggestive of a plethora of roles in proteolysis-linked biological processes, most likely underlying tissue-regulatory functions. Cystatin C is present in human body fluids at physiological concentrations and is the dominating cysteine peptidase inhibitor in cerebrospinal fluid where, as a consequence of remarkably high expression in the choroid plexus of the brain, it is over five times more concentrated than in blood plasma. Despite the wealth of biochemical data on its inhibitory characteristics, the specific functions played by cystatin C in physiological processes are documented only to a limited extent. As such, roles for cystatin C are hypothesized in bone resorption, arterial wall remodelling, regulation of neuronal apoptosis triggered by oxidative stress. Further elucidation of cystatin C roles is likely to emerge from characterization of its involvement in various pathologies (see below). In the eye, in non-pathological conditions, cystatin C is localized in various tissues in the anterior segment, with one of the highest concentrations in the ciliary epithelium, which presumably represents the main source of this protein in the aqueous. In the posterior eye, the inhibitor is synthesized almost exclusively by the retinal pigment epithelium (RPE), which presents fairly abundant levels of both its transcript and protein and constitutively secretes the mature form of the protein (Paraoan et al., 2001). RPE secretion of cystatin C appears polarized basally in vitro, suggesting a putative extracellular function, at least in part, in or around Bruch’s membrane (Paraoan et al., 2001).
3. Disease involvement 2. Function Cystatin C is a biochemically well-characterized, strong inhibitor of cysteine peptidases of the papain family, especially cathepsins B, H, L and S, and also of some lysosomal caspases, such as legumain. The broad expression of cystatin C by various tissues coupled with its ubiquitous presence in extracellular fluids and its relatively * Corresponding author. E-mail address: [email protected] (L. Paraoan). 0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2006.01.024
Mature cystatin C undergoes dimerization/oligomerization through three-dimensional domain swapping that involves one of the beta-hairpin loops, thus rendering it inactive as a cysteine peptidase inhibitor. The process is enhanced in vivo by the presence of an N-terminally truncated form of the protein and occurs in vitro under pre-denaturing conditions. Oligomerization contributes to the formation of highly stable amyloid fibres that are deposited mainly in brain arteries and are associated with various neuropathologies in the elderly, notably alongside amyloid-beta precursor protein in amyloid plaques in the brain of Alzheimer disease patients. A naturally occurring, albeit relatively
1020
L. Paraoan, I. Grierson / Experimental Eye Research 84 (2007) 1019e1020
Fig. 1. The three-dimensional cystatin fold is best defined by the structure of chicken cystatin (panel A; reproduced with permission after Janowski et al., 2001), consisting of five antiparallel b-sheets (b1-5) enfolding a long a1 helix. The N-terminus together with loops L1 and L2 define the active site of the mature cystatin C. Trafficking of human variant B A25T precursor cystatin C (panel B) is spectacularly altered compared with that of wild-type form (panel C). Confocal micrographs of living human RPE cells transiently transfected with the respective cystatin C constructs fused to enhanced green fluorescent protein (EGFP) are shown as merged images that were collected on three channels corresponding to the following fluorescent emissions: green fluorescence of the cystatin C-EGFP fusion proteins; red fluorescence of the oxidated Mitotracker Red CM-H2Xros dye (Molecular ProbesÔ, Invitrogen, Paisley, UK) concentrated in the mitochondria of actively respiring cells; blue fluorescence of the endoplasmic reticulum (ER)/Golgi-specific dye ER-TrackerÔ Blue-White DPX (Molecular ProbesÔ, Invitrogen, Paisley, UK). Transfected cells are indicated by arrows. A considerable fraction of the variant B precursor cystatin C colocalizes with mitochondria (yellow/orange colour indicated by thin arrows in panel B), while being significantly excluded from the ER/Golgi (blue). Conversely, wild-type precursor cystatin C is targeted and processed efficiently through the ER/Golgi apparatus (thick arrows in panel C), entirely independent of the mitochondria (red).
rare, L68Q mutant significantly accelerates the formation of cystatin C-containing amyloids and is the causative agent of an autosomal dominant form of hereditary cerebral haemorrhage with amyloidosis that is fatal in early adult life. In addition to such conformation-linked diseases, a role of cystatin C in other degenerative processes in the central nervous system such as Parkinson’s disease, as well as vascular diseases (notably atherosclerosis and aneurysms) can be attributed to an altered expression of the inhibitor, reflected primarily in an imbalance in its ratio to target cathepsins. A surprising association of cystatin C with eye pathology has unveiled a possible novel mechanism of cystatin C involvement in pathogenesis. Homozygosity for haplotype (variant) B of CST3 correlates with increased risk of developing exudative age-related macular degeneration (Zurdel et al., 2002). Variant B precursor cystatin C has an alanine (A) to threonine (T) substitution in the penultimate position of the signal sequence (A25T). The A25T substitution has dramatic consequences in terms of intracellular trafficking of the protein (Fig. 1B and C) by unexpectedly diverting it from the conspicuous Golgi localization to a predominant mitochondrial association (Paraoan et al., 2004). As a corollary, the secretion of cystatin C is significantly impaired. The A25T variant does not appear to interact with the wild-type cystatin C, nor to affect its secretion, thus providing further experimental support for the variant allele acting in a recessive fashion. Further data resulting from characterization of trafficking and processing of other engineered mutants, particularly the A25S precursor cystatin C mutant, a biochemically intermediate between the wild-type and the AMD-associated variant, corroborate the hypothesis that the variant B cystatin C protein is excluded from transport through the endoplasmic reticulum. Possibly this is due to an unfavourable interaction with components of the translocation channel.
4. Future studies Whether the retinal neurodegeneration-related biological effect of the A25T variant precursor cystatin C is due to its impaired secretion or to its mitochondrial association, or to the combination of these,
remains to date an open question. The mitochondrial localization is intriguing and certainly worthy of further investigation, as is the contribution of impaired function of cystatin C to the multifactorial aetiology of age-related macular degeneration. Cystatin C appears instrumental in regulating the deleterious effects of cysteine peptidases implicated in central nervous system-associated pathology. Evidence uncovered steadily over the last few years points towards involvement of cystatin C in the process of neuroregeneration or neurodegeneration of neurons in response to cellular damage, having presumed anti-apoptotic and/or neuroprotective roles. Notably in this respect, the significance of variant B cystatin C association with increased risk for late-onset Alzheimer disease remains to be established. In the light of current knowledge of cystatin C biology, impairment of secretion, in particular involving modulators of proteolysis, alongside subsequent misfolding of soluble proteins in the RPE cells are emerging as critical molecular events underlying macular degeneration. Acknowledgements The authors thank Dr Dave Spiller and Arjuna Ratnayaka for skilful help with cell imaging and flawless transfections.
References Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., Grubb, A., Abrahamson, M., Jaskolski, M., 2001. Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Nat. Struct. Biol. 8, 316e320. Paraoan, L., White, M.R.H., Spiller, D.G., Grierson, I., Maden, B.E.H., 2001. Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway. Mol. Membr. Biol. 18, 229e236. Paraoan, L., Ratnayaka, A., Spiller, D.G., Hiscott, P., White, M.R.H., Grierson, I., 2004. Unexpected intracellular localization of the AMDassociated cystatin C variant. Traffic 5, 884e895. Zurdel, J., Finckh, U., Menzer, G., Nitsch, R.M., Richard, G., 2002. CST3 genotype associated with exudative age related macular degeneration. Br. J. Ophthalmol. 86, 214e219.