Soluble Variants of Human Recombinant Glutaminyl Cyclase

Soluble variants QPC

Left ventricular wall thickness and severity of cardiac disease in women and men with transthyretin amyloidosis

Aims: Cardiac amyloidosis (CA) is due to a deposition of amyloid fibrils in the heart causing an increase in wall thickness. A left ventricular (LV) wall thickness ≥12 mm plus at least one red flag should raise the suspicion of CA. As normal values of LV wall thickness are lower in women, the adoption or the same cut-off values for men and women could lead to underdiagnosis or delayed diagnosis in women. We investigated the relationship between LV wall thickness and the severity of cardiac involvement in women and men with transthyretin (ATTR) CA. Methods and results: We evaluated 330 consecutive patients diagnosed with ATTR-CA at three centres (Pisa, n = 232; Brescia, n = 69; Trieste, n = 29). Interventricular septum (IVS) and posterior wall (PW) thickness values were lower in women (n = 53, 16%) than men, but most differences were abolished when indexing by body surface area (BSA), height, or height, suggesting similar disease severity when accounting for the smaller body size of women. PW thickness indexed for height was even higher in women. We also searched for correlations between IVS and PW thickness and other indicators of the severity of cardiac disease. IVS values indexed by height displayed tighter associations with N-terminal pro-B-type natriuretic peptide values than non-indexed IVS values. Similarly, indexed values displayed closer relationships with relative wall thickness, E/e' ratio, and tricuspid annular plane systolic excursion. Conclusions: Indexed LV wall thickness values, particularly by height, reflect more accurately the severity of cardiac involvement than non-indexed values

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development

Magnetic resonance imaging of infarct-induced canonical wingless/integrated (Wnt)/β-catenin/T-cell factor pathway activation, in vivo.

Aims Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/β-catenin/TCF pathway activation, an important regulator of post-ischemic cardiac remodeling. Methods and Results We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the β-catenin-responsive TCF/Lef promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n=11). By 1.5-Tesla MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, postmortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n=6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of β-catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischemic remodeling process, as demonstrated by the significant preservation of cardiac function, the 42±1% increase of peri-infarct arteriolar density and 43±3% reduction in infarct scar size compared with untreated animals. Conclusions The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/β-catenin/TCF activation state in response to cardiac injury and therapeutic interventions

Improving Upper Extremity Bradykinesia in Parkinson’s Disease: A Randomized Clinical Trial on the Use of Gravity-Supporting Exoskeletons

Hand movements are particularly impaired in patients with Parkinson’s Disease (PD), contributing to functional disability and difficulties in activities of daily living. Growing evidence has shown that robot-assisted therapy may be considered an effective and reliable method for the delivery of the highly repetitive training that is needed to trigger neuroplasticity, as intensive, repetitive and task-oriented training could be an ideal strategy to facilitate the relearning of motor function and to minimize motor deficit. The purpose of this study is to evaluate the improvement of hand function with semi-autonomous exercises using an upper extremity exoskeleton in patients with PD. A multicenter, parallel-group, randomized clinical trial was then carried out at the IRCCS Centro Neurolesi Bonino-Pulejo (Messina, Italy). Thirty subjects with a diagnosis of PD and a Hoehn–Yahr score between 2 and 3 were enrolled in the study. Patients were 1:1 randomized into either the experimental group (ERT), receiving 45 min training daily, 6 days weekly, for 8 weeks with Armeo®Spring (Volketswil, Switzerland) (a gravity-supporting device), or the control group (CPT), which was subjected to the same amount of conventional physical therapy. Motor abilities were assessed before and after the end of the training. The main outcomes measures were the Nine-hole peg test and the motor section of the UPDRS. All patients belonging to ERT and 9 out of 15 patients belonging to the CPT completed the trial. ERT showed a greater improvement in the primary outcome measure (nine-hole peg test) than CPT. Moreover, a statistically significant improvement was found in ERT concerning upper limb mobility, and disease burden as compared to CPT. Using an upper extremity exoskeleton (i.e., the Armeo®Spring) for semi-autonomous training in an inpatient setting is a new perspective to train patients with PD to improve their dexterity, executive function and, potentially, quality of life

Soluble Variants of Human Recombinant Glutaminyl Cyclase

<div><p>Recombinant human Glutaminyl Cyclase expressed in <i>E. coli</i> is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of ¹H-¹⁵N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease.</p></div

NMR spectra of wild type, 2xmut and 6xmut.

<p>2D ¹H-¹⁵N-HSQC spectra of: <b>A,</b> 30 µM wild type hQPCT at 700 MHz; <b>B,</b> of 90 µM 2xmut hQPCT at 950 MHz; <b>C,</b> 70 µM 6xmut hQPCT at 950 MHz. Spectra were recorded at 298 K, in 150 mM NaCl and 50 mM Tris pH 8 buffer.</p

3D structure of hQPCT.

<p>Ribbon representation of the X-ray structure of hQPCT (PDB id 2AFM). The zinc ion is shown by a yellow sphere, the zinc ligands are shown as orange sticks and the two Cys residues responsible for the disulphide bridge formation as green sticks. The loop connecting β1 with β2 is highlighted in red, while those forming the crown-like structure around the zinc are in orange.</p

Purification of wild type hQPCT.

<p><b>A,</b> Imidazole gradient (green line) (a) 50 mM, (b) 50–500 mM, (c) 500 mM in FPLC Akta (GE Healthcare). Blue line: UV measure (mAU). <b>B,</b> SDS-PAGE of purified protein fractions. Lane 1: insoluble fraction, lane 2: protein marker, lane 3: total fraction, lane 4: flow-through, lane 5: wash unbound, lanes 6–7: fractions 50 mM imidazole, lanes 8–15: fractions 50–500 mM imidazole.</p