Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.     

Domain swapping in N-truncated human cystatin C

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.     

Identification and characterization of antibodies elicited by human cystatin C fragment

Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60‐70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti‐hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA.     

Inhibitory effects of human cystatin C on plum pox potyvirus proteases

The effect of different protease inhibitors on the proteolytic processing of the plum pox potyvirus (PPV) polyprotein has been analyzed. Human cystatin C, an inhibitor of cysteine proteases, interfered with the outoprocessing of the viral papain-like cysteine protease HCPro. Unexpectedly, it also had an inhibitory effect on the autocatalytic cleavage of the Nla protease which, although it has a Cys residue in its active center, has been described as structurally related to serine proteases. Other protease inhibitors tested had no effect on any of the cleavage events analyzed.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far-UV CD, Fourier-transform infrared (FTIR) spectroscopy, and heme-binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native-like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild-type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.     

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.     

Copeptin,B-type natriuretic peptide and cystatin C are associated with incident symptomatic PAD

Purpose: The aim of this study is to evaluate plasma biomarkers as predictors for peripheral arterial disease (PAD).

Materials and methods: Prospective longitudinal cohort study of middle-aged individuals from the cardiovascular cohort of the Malmö Diet and Cancer study (MDCS) (n?=?5550; 1991–94). Cystatin C, copeptin, N-terminal pro-B-type natriuretic peptide (N-BNP), midregional proatrial natriuretic peptide (MR-proANP), mid-regional proadrenomedullin (MR-proADM), and conventional risk factors were measured at baseline. The diagnosis of symptomatic PAD was validated in 97% of the cases.

Results: Cumulative incidence of PAD during median follow up of 23.4?years was 4.4% (men 5.9%, women 3.3%). Adjusted for age, sex, smoking, body mass index, hypertension, diabetes mellitus and total cholesterol, copeptin (hazard ratio [HR] 1.46; 95% confidence interval [CI] 1.19–1.80), N-BNP (HR 1.28; 95% CI 1.11–1.48), and cystatin C (HR 1.19; 95% CI 1.10–1.29) were independently associated with incident PAD. Subjects with the three biomarkers copeptin, N-BNP, and cystatin C in the highest quartiles, ran a high risk of incident PAD (HR 3.29; 95% CI 1.76–6.17) compared to those with no biomarker in the highest quartile.

Conclusion: Copeptin, N-BNP, and cystatin C were associated with incident symptomatic PAD, implying that these biomarkers are sensitive indicators of early subclinical PAD.

• Clinical significance

• First prospective longitudinal cohort study evaluating Cystatin C, copeptin, N-terminal pro-B-type natriuretic peptide (N-BNP), midregional proatrial natriuretic peptide (MR-proANP), and mid-regional proadrenomedullin (MR-proADM) as predictors for peripheral arterial disease (PAD).

• Copeptin, N-BNP, and Cystatin C where independently associated with incident symptomatic PAD after adjustment for conventional risk factors.

• Copeptin, N-BNP, and Cystatin C seem to be sensitive indicators of early subclinical PAD.

     

Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis

Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE‐deficient mice, demonstrating that CysC deficiency (CysC^?/?) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC^?/? mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7‐oxysterol‐mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol‐mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE‐deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.     

血清半胱氨酸蛋白酶抑制剂C在肾移植患者肾功能监测中的临床应用

目的针对肾移植术后稳定期患者,进行血清半胱氨酸蛋白酶抑制剂C(Cystatin C)和血清肌酐(SCr)的灵敏度的比较。方法在HITACHI-7600-020型自动生化分析仪上采用微粒子颗粒增强透射免疫比浊分析法等方法测定肾移植术后稳定期患者的Cystatin C、SCr等指标。结果对数据用SPSS 11.5软件包进行χ2检验,P<0.01,故可以认为2种检测指标间的差异有统计学意义,即Cystatin C与SCr相比有较好的敏感度。结论血清Cystatin C在稳定期肾移植患者中敏感性与SCr差异存在统计学意义,且优于SCr,能更敏感,更及时的反映GFR的变化,可作为稳定期肾移植患者判断急、慢性排斥和免疫抑制剂的毒性的最重要的观察指标之一。     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Design and synthesis of a peptide derived from positions 195–244 of human cdc25C phosphatase

We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14‐3‐3 partner in vitro by fluorescence spectroscopy. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.     

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10^?7 cm² s^?1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (?5.78 kcal mol^?1) and positive ΔS (11.01 cal mol^?1 deg^?1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (?9.19 kcal mol^?1) value implies a spontaneous CBC-papain interaction.     

Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B activity

Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effect of polyols and salts on the acid-induced state of human placental cystatin

Polyols (glycerol and sorbitol) and salts (magnesium sulfate, sodium sulfate, and magnesium chloride) have been used to study the refolding of the acid-induced state of human placental cystatin (HPC), which is a low molecular weight (12,500 daltons) thiol proteinase inhibitor, in terms of CD spectroscopy, binding of hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), and intrinsic fluorescence measurements. The helical content of acid-denatured HPC increased with increase in glycerol concentration (0–80%). At 80% glycerol concentration, the secondary structural features observed in the far UV-CD region are similar to those of the native state (pH 6.0). The intrinsic fluorescence and near UV-CD studies showed that this 80% glycerol-induced state has a significant amount of tertiary structure with decreased ANS binding compared to the acid-denatured state. It was found that glycerol is more effective in stabilizing the acid-denatured state of HPC as compared to sorbitol. Among salts the stability effect was more for MgCl₂ (used up to concentration of 3 M) compared to MgSO₄ and Na₂SO₄ (used up to the concentration of 1.5 M due to restricted solubility of HPC at higher sulfate salt concentrations) as determined by CD studies and fluorescence measurements, which showed secondary and tertiary structural resemblance of this MgCl₂-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl₂ (3 M)-induced state showed decreased ANS fluorescence as compared to the acid-denatured state but more than that of the native state. The results taken together suggest that the acid-denatured state of HPC in the presence of 80% glycerol or 3 M MgCl₂ has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 768–777.     

Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy

Here we report the enzymologic characterization of recombinant human pitrilysin metallopeptidase 1 (Pitrm1) and derivative mutants including the arginine‐to‐glutamine substitution mutant Pitrm1 R183Q, which has been implicated in inherited amyloidogenic neuropathy. Recombinant Pitrm1 R183Q was readily expressed in and purified from Escherichia coli, but was less active than the recombinant wild‐type enzyme against recombinant amyloid beta‐peptide (Aβ 1‐40). A novel fluorogenic substrate derived from the reported Aβ 1‐40 core peptide cleavage sequence, Mca‐KLVFFAEDK‐(Dnp)‐OH, was synthesized and applied to real‐time kinetic study of Pitrm1 and derivative mutants including Pitrm1 R183Q. The Pitrm1 R183Q mutant exhibited significantly decreased rate of fluorogenic peptide hydrolysis, yet retained similar binding affinity by comparison with the wild‐type enzyme. Targeted mutagenic analysis revealed a functional requirement for uncharged or electropositive residues in place of Pitrm1 R183. Residue R183 is positioned within an N‐terminal strand‐loop‐strand motif that is conserved among M16C, but not M16A or M16B family metallopeptidases. Truncation analysis revealed that this strand‐loop‐strand motif inclusive of residue R183 is essential Pitrm1 function. A requirement for charged residues within 4.5 Å of residue R183 was demonstrated, and Pitrm1 R183Q was found to exhibit increased sensitivity to heat inactivation. Our findings indicate that charge sharing in the vicinity of Pitrm1 R183 is critical to enzyme activity, providing potential insight into a molecular basis of Pitrm1 dysfunction.