Polyglutamine expansion affects huntingtin conformation in multiple Huntington's disease models

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine-and temperaturedependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperatureand polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins

L’ingegneria proteica nello studio della struttura e funzione di (macro)molecole

The present Ph.D. project deals with the use of protein engineering techniques to express in recombinant form and characterize disulphide rich bioactive peptides, such as conotoxins. Moreover, taking advantage of their high target specificity and stability, the potential application of this class of macromolecules in “green” (environmental) and “white” (industrial) biotechnology fields have been explored. Protein engineering is a powerful tool to improve some proteins parameters, acting on their sequence and structure, or to insert a desired function. There are three basic approaches in protein engineering: the rational design is an "intelligent" design that aims to change the structure or function of a protein, starting from the analysis of its three-dimensional structure. Its nucleotide sequence can be modified and the protein finally “built” and characterized (Hellinga, 1997). The second approach is the directed evolution or DNA breeding, a technique that simulates biological evolution mechanisms (Stemmer, 1994). In fact it involves the generation of random mutations in the gene coding for a protein, or shuffling the genes encoding different domains, to generate a high number of new proteins that will be then selected for a desired function. The third one, named de novo design, allows to design and build artificial proteins which don’t exist in nature, with novel functions and properties (Dahiyat and Mayo, 1997). This strategy takes advantage of computational biology to draw novel proteins both using natural scaffolds as templates and starting completely from scratch (Dahiyat and Mayo, 1997). The idea of using stable protein structural motifs as scaffold for reproducing functional epitopes or stabilize bioactive conformations is currently one of the most successful approaches of protein engineering. The knottin family of proteins is particularly interesting from this point of view, since they share a common structural fold while having very different sequences and functions in animals and plants (Norton and Pallaghy, 1998). The multiple disulphide bridges that characterize this protein family give the greatest contribution to the structure stability, allowing a high variability of the remaining amino acid sequence regions. Among knottins, the highest tolerance to sequence variability is observed in conotoxins, small disulphide-rich neurotoxic peptides extracted from cone snails venom. Nature, during the evolution, has engineered this stable scaffold to express a great variety of neurotoxins with a remarkable high target specificity for different ion channels, both voltage-gated and ligand-gated (Olivera and Teichert, 2007). As a result, their scaffold represents an excellent starting point for design strategies aimed at developing new miniproteins with new properties as well as more stable/active conotoxins for therapeutics (Clark et al., 2005). From the natural source, the purification of conotoxins proved to be very difficult because of the large number of specimens needed to obtain a sufficient amount of venom to process. Thus, during this thesis work, a recombinant expression system has been developed to express conotoxins as GST-fusion proteins, overcoming both the problem of the small amount of a single conotoxin that can be extracted from the Conus venom, and their propensity to form insoluble aggregates because of the formation of intermolecular disulfide bridges. As a test case, the new protocol has been used to express conotoxin Vn2 from the Mediterranean Sea Conus ventricosus, a 33 amino acids long highly hydrophobic neurotoxic peptide, and its Asp2His mutant (Spiezia et al., 2012). This first part of the work involved cloning of both the conotoxins in pET-CM plasmid and their purification by affinity chromatography, exploiting the GST tag. Since the worm-hunting C. ventricosus has an array of toxins acting on invertebrate ion channels, GST-conotoxins have been tested for their neurotoxic activity on the larvae of the moth Galleria mellonella, taken as an insect model system (Spiezia et al., 2012). Further, using ω-conotoxin GVIA as template, novel metal-binding peptides were designed to generate novel biocatalysts. Through computational modeling, two disulphide bridges in the original conotoxin have been replaced with His residues and the redesigned peptides, named Cupricyclin-1 and Cupricyclin-2, have been synthesized and characterized for their copper binding ability and superoxide dismutase activity (Barba et al., 2012). The last part of the work concerned the generation of Cupricyclin-1 mutants with the aim to improve its superoxide dismutase activity and to change metal-binding specificity from copper to iron and manganese. The new expression system developed in the first part of the present project was successfully used to express these Cupricyclin mutants as GST-fusion proteins. Following GST tag removal using thrombin cleavage, the mutants have been purified and their metal binding properties analyzed by optical and fluorescence spectroscopy and mass spectrometry. Further studies will be necessary to elucidate the redox properties of these novel Cupricyclins and their potential as biosensors for the detection of heavy metals or as therapeutics in oxidative stress injuries, given the ability of Cupricyclin-1 to dismutate O2- radicals

Growth hormone treatment on atherosclerosis: results of a 5-year open, prospective, controlled study in male patients with severe growth hormone deficiency.

BACKGROUND: Severe GH deficiency (GHD) is associated with, increased cardiovascular risk and intima-media thickness (IMT) at major arteries. OBJECTIVE: The objective of the study was to investigate the 5-yr effects of GH replacement on common carotid IMT and insulin resistance syndrome (IRS) (at least two of the following: triglycerides levels > or = 1.7 mmol/liter, high-density lipoprotein-cholesterol levels < or = 1.0 mmol/liter, blood pressure above 130/85 mm Hg, fasting glucose 6.1-7 or 2 hr after glucose 7.7-11.1 mmol/liter). DESIGN: This was an interventional, open, prospective, controlled study. PATIENTS: Patients included 35 men with severe GHD and 35 age-matched healthy men as controls. INTERVENTION: All patients received standard replacement therapy; GH replacement was added in 22 patients (group A) and refused by 13 others (group B). MEASUREMENTS: Five-year changes in IMT and IRS prevalence were measured. RESULTS: At baseline, IMT was higher in the patients with (P < 0.001) and without IRS (P = 0.004) than in controls. Eighteen patients (51.4%) and two controls (5.7%; P < 0.0001) had IRS. At study end, use of lipid-lowering drugs (92.3, vs. 13.6 and 34.3%, P < 0.0001), glucose-lowering drugs (69.2 vs. 31.4 and 22.7%; P = 0.016), and antihypertensive drugs (61.5 vs. 20.0 and 4.5%; P < 0.0001) was higher in group B patients than controls and group A patients. IGF-I levels normalized in all group A patients and remained lower than -1 sd score in 77% of group B patients. IMT significantly decreased only in group A and significantly increased in controls and nonsignificantly in group B patients. IRS prevalence significantly reduced only in group A patients. CONCLUSIONS: Severely hypopituitary GHD men have more frequently increased IMT at common carotid arteries and IRS than controls. After 5 years, only in GH replaced patients, IMT and prevalence of IRS decreased

Short-term effects of growth hormone (GH) treatment or deprivation on cardiovascular risk parameters and intima-media thickness at carotid arteries in patients with severe GH deficiency

To explore early effects of GH treatment or deprivation on cardiovascular risk factors and carotid intima-media thickness (IMT), we designed this randomized, cross-over study in 34 adult patients with severe GH deficiency. At study entry, the patients were randomized into two groups (A and B); group A (n = 17) received appropriate replacement therapy including GH at standard doses for 6 months and then were withdrawn from GH for the subsequent 6 months; group B (n = 17) received appropriate replacement therapy excluding GH for 6 months with the addition of GH in the subsequent 6 months. After the first 6 months, we observed a significant increase in IGF-I levels and of high-density lipoprotein (HDL)-cholesterol together with a significant decrease in diastolic blood pressure, the total/HDL-cholesterol ratio, and C-reactive protein in the patients in group A, whereas vascular parameters did not significantly change. In the patients in group B, none of the parameters studied significantly changed. After 6 months of GH withdrawal in the patients in group A, a significant decrease in IGF-I levels, a significant increase in the total/HDL-cholesterol ratio and C-reactive protein, and a trend toward an impairment of carotid IMT and peak velocities were observed. In the patients in group B, the addition of GH to the standard replacement induced a significant increase in IGF-I levels together with a decrease in systolic and diastolic blood pressure, total cholesterol and total/HDL-cholesterol ratio, and C-reactive protein, and an increase in HDL-cholesterol levels with a trend toward an improvement of vascular parameters. At the end of the study, mean IMT was significantly lower than at baseline both in group A (from 0.88 +/- 0.28 to 0.85 +/- 0.27 mm, P = 0.0003) and in group B (from 0.83 +/- 0.21 to 0.80 +/- 0.20 mm, P = 0.003). In conclusion, 6 months of GH replacement has beneficial effects whereas 6 months of GH deprivation has detrimental effects on cardiovascular risk factors and atherosclerosis. These findings support the indication for GH replacement in severe GH deficiency adult patients

Cardiovascular risk factors and common carotid artery caliber and stiffness in patients with Cushing's disease during active disease and 1 year after disease remission

Cardiovascular accidents represent the most important cause of death in patients with Cushing's syndrome. This prospective study aims at evaluating carotid arteries by echo-Doppler ultrasonography and clinical and metabolic markers of atherosclerosis in 25 patients with Cushing's disease (CD) before and after 1 yr of remission. Thirty-two sex- and age-matched subjects (control-1) and 32 body mass index-matched subjects (control-2) served as controls. At diagnosis, CD patients had higher body mass index, waist to hip ratio (WHR), total, low-density lipoprotein-cholesterol and total/high-density lipoprotein (HDL) ratio, glucose and insulin, as well as lower HDL-cholesterol than control-1; they had higher WHR and total/HDL ratio and lower HDL-cholesterol than control-2. They also had higher intima-media thickness (IMT), and lower systolic lumen diameter and distensibility coefficient (DC) than either control group. Atherosclerotic plaques were detected in 31.2% of patients, 0 control-1, and 6.2% of control-2 subjects. One year after remission, WHR, LDL-cholesterol, and IMT significantly decreased, whereas systolic lumen diameter and DC significantly increased. However, all of the above parameters were still abnormal compared with control-1, but not control-2. A significant correlation was found between WHR, glucose and insulin levels, and right and left carotid IMT. WHR was the best predictor of left IMT and left DC in active, but not in cured, patients. The duration of hypercortisolism was the best predictor of right DC in active but not in cured patients. In conclusion, patients with CD have severe atherosclerotic damage. The persistence of a metabolic syndrome, vascular damage, and atherosclerotic plaques after cortisol level normalization makes these subjects still at high cardiovascular risk despite disease remission

Circulating insulin-like growth factor-I levels are correlated with the atherosclerotic profile in healthy subjects independently of age

To investigate the relationships between the GH-IGF-I axis and the atherosclerotic profile, we designed this open, observational, prospective study. Peak GH after GHRH+arginine (ARG) test, serum IGF-I and IGF binding protein-3 (IGFBP-3), lipid profile, homeostasis model assessment (HOMA) index and intima-media thickness (IMT) at common carotid arteries were measured in 174 healthy individuals (92 women, 82 men, aged 18-80 yr). Exclusion criteria for this study were: 1) body mass index (BMI) > or = 30 kg/m2; 2) personal history of cardiovascular diseases; 3) previous or current treatments of diabetes or hypertension; 4) previous corticosteroids treatment for longer than 2 weeks or estrogens for longer than 3 months; 5) smoking of more than 15 cigarettes/day and alcohol abuse. Subjects were divided according to age in decade groups from 70 yr. BMI increased with age, as did systolic and diastolic blood pressures, although they remained in the normal range. The GH peak after GHRH+ARG test was significantly higher in the subjects aged 1 (p = 0.006). At multi-step regression analysis, age was the best predictor of HDL-cholesterol levels and mean IMT, IGF-I level was the best predictor of total cholesterol and total/HDL-cholesterol ratio, the IGF-I/IGFBP-3 molar ratio was the best predictor of triglycerides levels. The z-scores of IGF-I and IGFBP-3 were the second best predictors of mean IMT after age. In conclusion, IGF-I and IGFBP-3 were negatively correlated with common cardiovascular risk factors, studied as total/HDL-cholesterol ratio, and/or early atherosclerosis, studied as IMT at common carotid arteries. The prevalence of atherosclerotic plaques, though not hemodinamically significant, was higher in the subjects having a z-score of IGF-I of < or = -2 to -1. Our results support a role of the IGF/IGFBP-3 axis in the pathogenesis of atherosclerosis

Titration of Cupricyclin-1 with CuSO₄ monitored by ¹H NMR.

<p>The molar ratio CuSO₄/Cupricyclin-1 is reported on the left side of each spectrum.</p