Structure-Based Design of Non-Natural Amino Acid Inhibitors of Amyloid Fibrillation

Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures.We thank M.I. Ivanova, J. Corn, T. Kortemme, D. Anderson, M.R. Sawaya, M. Phillips, S. Sambashivan, J. Park, M. Landau, Q. Zhang, R. Clubb, F. Guo, T. Yeates, J. Nowick, J. Zheng, and M.J. Thompson for discussions, HHMI, NIH, NSF, the GATES foundation, and the Joint Center for Translational Medicine for support, R. Peterson for help with NMR experiments, E. Mandelkow for providing tau constructs, R. Riek for providing amyloid beta, J. Stroud for amyloid beta preparation. Support for JK was from the Damon Runyon Cancer Research Foundation, for HWC by the Ruth L. Kirschstein National Research Service Award, for JM from the programme for junior-professors by the ministry of science, Baden-Württemberg, and for SAS by a UCLA-IGERT bioinformatics traineeship

The Extended Cleavage Specificity of Human Thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times

Atrial fibrillation in pure rheumatic mitral valvular disease is expression of an atrial histological change

BACKGROUND: Some of theories try to explain the insurgence of atrial fibrillation (AF) in patients with acute articular rheumatism (AAR). These theories remind the close relation between AF and left atrium, or with valvular vitium degree, or monophasic action potential and histological cardiac structure. In 15 years of work in the academic Department of Heart and Big Vessels in Rome, the Authors studied 243 patients with mitral valvular disease post AAR before and after surgical manoeuvres. MATERIALS AND METHODS: Patients were divided in order to monitor atrium and ventricle morphological and functional modifications of the valve according to cardiac rhythm. Patients classification was based on surgical therapy adopted, kind of mitral disease and cardiac rhythm. An histological examination was performed, only in patients treated with valvular replacement. During the operation an histological examination in an atrial tissue fragment was performed. 243 patients with mitral valvular disease post AAR with indication in valvular adjustment were studied. The whole population was treated with mitral transcutaneous valvuloplasty (Group B--130 patients) or with mitral valve replacement surgery (Group A--113 patients). These two groups were divided: in Gr.A in Gr.A1 and Gr.A2, and Gr.B in Gr.B1 and Gr.B2, according to cardiac rhythm (sinus rhythm iSR, AF). These subgroups were also divided in Gr.A1SR, Gr.A1AF; Gr.A2SR, Gr.A2AF; Gr.A3SR, Gr.A3AF, according to mitralic disease's kind (stenosis, stenosis/regurgitation, regurgitation). A complex screening were exerted to all patients using echocardio-doppler technology. Morphological parameters of atrium and ventricle, and functional parameters of mitral valve, aorta and tricuspid were evaluated. In Gr.A group patients during the operation were execute a bioptic sampling from left atrium and a consecutive histological valuation. RESULTS: In Gr.A1 mitral valve area (MtVA) arises smaller (p0.05). Left atrium volume arises elder in patients in AF than in patients in SR (p<0.01), either in patients of subgroups Gr.A1, Gr.A2 or in patients of the whole Gr.B before and after valvuloplasty. In the whole population Gr.B, either Gr.BRS or Gr.BFA, left and right atrial volumes decrease eloquently (p<0.01) after valvoplasty. There's no linear relationship (Pearson r<0.5) between the different subgroups of Gr.A (Gr.A1, Gr.A2, Gr.A3) and those of Gr.B according to mitral valve area (MtVA), volume and left atrial area. Left atrial biopsy shows in patients of SR a normal atrial tissue in the 48% of cases and lightly altered in remaining 52%. On the contrary in patients of AF there are strong anomalies in the 100% of cases. CONCLUSIONS: According to histological view, atrial volumes variations and valvular area variations before and after surgical treatment, and according also to their comparisons in different groups, authors could assume that insurgence of AF and its chronicization could be an expression of a strong atrial myocardial histological alteration. Furthermore while starting moment of AF genesis is characterized by histological alterations of atrial myocardium (expression of rheumatic chronic disease), its chronicization hands to anatomic-volumetric progressive deterioration of the atrial dysfunction

Catheter ablation of atrial tachycardia after interatrial defect repair with patch apposition

A 54-year-old woman with history of septal atrial mixoma surgically treated and drug-refractory supraventricular tachyarrhythmia underwent catheter ablation of macro-reentry areas near the pericardial patch placed to repair an interatrial defect. The use of ablative therapy has been successful to cure this arrhythmia

Functional changes cardiovascular: normobaric activity and microgravity in young healthy human subjects Eur Rev Med Pharmacol Sci 2012; 16 (3): 310-315

Abstract. – Background: The cardiovascular system works to maintain homeostasis through a series of adaptive responses to physiological requirements. Different self-regulatory mechanism prevent the effects induced by hydrostatic pressure changes on oncotic pressure caused by postural changes. Gravity exerts a strong influence on the postural changes with implications on the cardiovascular system. In orbit, gravity (+Gz) is responsible of mass redistribution of circulating blood flow. The aim of this study was the evaluation of the adaptive responses of cardiovascular system to postural changes with and without the use of the Lower Body Negative Pressure (LBNP). We considered that pressure changes that occur in human body in orbit can be simulated experimentally with use of Tilt-Test (Clino/ortho; Clino/head-down; head-down/ortho). This investigation could be useful for studying the influence on astronauts of long flights. Subjects and Methods: We studied in 12 months, 30 young healthy volunteers (20 males, 10 female) during postural change tests. In the first evaluation they were submitted to tilt-test for 40 minutes, remaining in head-up +60° (this state corresponds to a kind of gravitational stress +Gz) and in head-down to -30° (-Gz) for 20 minutes. During the second assessment (after 5 ± 1 days) all volunteers wear a device that simulate a state of LBNP at -20 mmHg. Afterwards, they were processed to 20 minutes in Head Down -8º and after 2 hours of rest to 20 minutes at -15°. Volunteers were monitored measuring blood pressure, heart rate and by Transthoracic Echocardiogram (TTE) Results: Collected data were elaborated by a statistical analysis. We observed during orthostatic position for 40 min (+60°) without LBNP, lower diameters and volumes of left and right ventricular (p < 0.05) and an increase in heart rate in comparison with the baseline conditions in clinostatism. Despite the reduction of preload volume, the mean value of cardiac output does not vary significantly. In Trendelemburg (-15°) data show a non-significant variation (p > 0.05) of left and right ventricular diameters and volumes, while cardiac output and systolic blood pressure varies significantly (p < 0.05) compared to clinostatic and orthostatic position. With LBNP in head down to -8° and -15°, systolic and diastolic arterial pressure, ventricular volumes and cardiac output were unchanged if compared to values obtained in clinostatism with and without LBNP. If compared to -30° in Trendelemburg without LBNP, data reached statistical significance (p < 0.05) Conclusions: The cardiovascular system and the autonomic nervous system, respond to postural changes and to volemia alterations, maintaining the physiological cardiac output, in order to preserve the metabolic requirements of body