Mayra de A. Marques

Structural basis for the dissociation of α-synuclein fibrils triggered by pressure perturbation of the hydrophobic core.

Parkinson’s disease is a neurological disease in which aggregated forms of the α-synuclein (α-syn) protein are found. We used high hydrostatic pressure (HHP) coupled with NMR spectroscopy to study the dissociation of α-syn fibril into monomers and evaluate their structural and dynamic properties. Different dynamic properties in the non-amyloid-β component (NAC), which constitutes the Greek-key hydrophobic core, and in the acidic C-terminal region of the protein were identified by HHP NMR spectroscopy. In addition, solid-state NMR revealed subtle differences in the HHP-disturbed fibril core, providing clues to how these species contribute to seeding α-syn aggregation. These findings show how pressure can populate so far undetected α-syn species, and they lay out a roadmap for fibril dissociation via pathways not previously observed using other approaches. Pressure perturbs the cavity-prone hydrophobic core of the fibrils by pushing water inward, thereby inducing the dissociation into monomers. Our study offers the molecular details of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. Understanding the molecular forces behind the formation of pathogenic fibrils uncovered by pressure perturbation will aid in the development of new therapeutics against Parkinson’s disease.

Insights into the Intramolecular Coupling between the N- and C‑Domains of Troponin C Derived from High-Pressure, Fluorescence, Nuclear Magnetic Resonance, and Small-Angle X‑ray Scattering Studies

Troponin C (TnC), the Ca(2+)-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu, A., Ballard, L., Sorenson, M. M., Foguel, D., and Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here, we analyzed the stability of full-length F29W TnC using structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a fluorescent mutant, in which Phe 29, located in the N-domain, was replaced with Trp. From these experiments, we calculated the thermodynamic parameters (ΔV and ΔG°(atm)) that govern the folding of the intact F29W TnC in the absence or presence of Ca(2+). We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca(2+). However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca(2+)-bound state (i.e., when Ca(2+) was also bound to sites III and IV of the C-domain). An accompanying decrease in the thermodynamic stability of the N-domain generated a reduction in ΔΔG°(atm) in absolute terms, and Ca(2+) binding affects the Ca(2+) affinity of the N-domain in full-length TnC. Cross-talk between the C- and N-domains may be mediated by the central helix, which has a smaller volume and likely greater rigidity and stability following binding of Ca(2+) to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.

Cardiac Troponin and Tropomyosin: Structural and Cellular Perspectives to Unveil the Hypertrophic Cardiomyopathy Phenotype.

Inherited myopathies affect both skeletal and cardiac muscle and are commonly associated with genetic dysfunctions, leading to the production of anomalous proteins. In cardiomyopathies, mutations frequently occur in sarcomeric genes, but the cause-effect scenario between genetic alterations and pathological processes remains elusive. Hypertrophic cardiomyopathy (HCM) was the first cardiac disease associated with a genetic background. Since the discovery of the first mutation in the β-myosin heavy chain, more than 1400 new mutations in 11 sarcomeric genes have been reported, awarding HCM the title of the “disease of the sarcomere.” The most common macroscopic phenotypes are left ventricle and interventricular septal thickening, but because the clinical profile of this disease is quite heterogeneous, these phenotypes are not suitable for an accurate diagnosis. The development of genomic approaches for clinical investigation allows for diagnostic progress and understanding at the molecular level. Meanwhile, the lack of accurate in vivo models to better comprehend the cellular events triggered by this pathology has become a challenge. Notwithstanding, the imbalance of Ca2+ concentrations, altered signaling pathways, induction of apoptotic factors, and heart remodeling leading to abnormal anatomy have already been reported. Of note, a misbalance of signaling biomolecules, such as kinases and tumor suppressors (e.g., Akt and p53), seems to participate in apoptotic and fibrotic events. In HCM, structural and cellular information about defective sarcomeric proteins and their altered interactome is emerging but still represents a bottleneck for developing new concepts in basic research and for future therapeutic interventions. This review focuses on the structural and cellular alterations triggered by HCM-causing mutations in troponin and tropomyosin proteins and how structural biology can aid in the discovery of new platforms for therapeutics. We highlight the importance of a better understanding of allosteric communications within these thin-filament proteins to decipher the HCM pathological state.

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca 2+ -binding affinity in a C-domain mutant of cardiac troponin C

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

FRI0558 Predictors of mortality and re-fracture at 1 and 3years after hip fracture

Background Osteoporosis is a major health problem, particularly in the elderly, because of fragility fractures and their consequences. Hip fractures (HF) are the most ominous in terms of morbi-mortality. Objectives The aim of our work was to establish the current mortality and re-fracture rate at 1 and 3 years after HF, as well as their predictors. Methods The study included all patients aged >40 years, admitted to Coimbra University Hospital between May and October 2013 with the diagnosis of HF. Demographic and clinical data related to the fracture episode was collected from medical files. Patients or the caregiver were contacted to assess potential risk factors at baseline and major post-fracture events at 1 and 3 years after the index HF. The mortality and re-fracture rate 1 and 3 years after fracture were calculated. Possible predictor variables were tested by cox regression analysis: age, gender, physiotherapy, number of re-fractures, BMI, parent hip fracture, current smoking, corticotherapy, rheumatoid arthritis, secondary osteoporosis, alcohol, history of falls, anti-osteoporotic treatment, Katz index of independence in activities of daily living and Charlson comorbidity index. All FRAX® variables were defined as established in this algorithm. Results A total of 130 patients satisfied the inclusion criteria, with a mean age of 82±8,7 years, 69% being female). Mortality rates were of 30% and 41% at 1 and 3 years after HF respectively (Fig.1). Age, physiotherapy, parent fractured hip, corticotherapy, alcohol consumption (>3/day) and Charlson índex were statistically significant predictors of mortality at 3 years in multivariable analysis (Tab.1). Re-fracture rates at 1 and 3 years after the index fracture was 3,8% and 11% respectively (Fig.1). We were unable to identify any statistically significant predictors of re-fracture.Table 1. Mortality and refracture predictors Mortality Re-fracture p-value Exp (b) p-value Exp (b) Gender 0,106 2,052 0,265 3,089 Age 0,002 1,075 0,276 0,953 Katz index 0,116 1,154 0,752 0,918 Physiotherapy 0,020 2,167 0,499 0,638 BMI 0,812 0,991 0,142 0,891 Parent hip fracture 0,015 0,355 0,196 0,322 Current smoking 0,453 0,615 0,394 0,417 Corticotherapy 0,013 0,404 0,639 0,637 Rheumatoid arthritis 0,071 2,848 0,798 1,410 Secondary osteoporosis 0,172 0,566 0,154 0,321 Alcohol intake 0,037 0,370 0,980 348544,658 Charlson index 0,000 1,384 0,835 0,941 Number of re-fractures 0,660 0,781 Anti-osteoporotic treatment 0,430 0,474 Conclusions We concluded that HF have a great impact on the older population, leading to high morbidity and mortality. In our study, age, physiotherapy, parent hip fracture, corticotherapy, alcohol consumption and Charlson índex are related with increasing mortality in patients who suffered a fagility HF. Disclosure of Interest None declared

AB0841 Perfectionism in Chronic Pain: Are There Differences between fibromyalgia, Rheumatoid Arthritis and Healthy Controls?

Background Perfectionism has been associated with a plethora of medical conditions and psychopathological symptoms/disorders.1 However, the potential impact of perfectionism in different chronic pain conditions is scarcely explored. Objectives Compare patients with fibromyalgia (FM), rheumatoid arthritis (RA) and pain-free controls regarding mean scores of perfectionism dimensions and cognitions; and test if perfectionism dimensions are significantly associated with the disease group. Methods Participants were recruited among consecutive attendees with FM, RA and pain-free controls at rheumatology and gynecology practices in Coimbra. Patients with FM and RA satisfied currently accepted classification criteria.2,3 The absence of chronic pain in healthy controls (HC) was established by a score of zero in the London Fibromyalgia Epidemiological Study Screening4. All participants completed the Portuguese versions of the Frost and of the Hewitt and Flett Multidimensional Perfectionism Scales (MPS) and the Multidimensional Perfectionism Cognitions Inventory. To achieve our aims, descriptive analyses, one-way independent ANOVA (followed by post hoc tests) and linear multiple regression (LMR) were performed. Results The total sample comprised 302 participants [103 FM (34.11%), 98 RA (32.45%) and 101 HC (33.44%)]. Differences between samples were found regarding age (F=30.598; p<.001) and education years (F=25.883; p<.001). Results revealed significant differences between the samples regarding: parental criticism (PC), organization (O), Frost-MPS total score, perfectionism cognitions related with concern over mistakes (CoM) and pursuit of perfection (PP). Post hoc comparisons indicated that FM patients exhibited higher levels than RA patients and HC in all of these variables (except for PP that did not significantly differ between FM and HC). No significant differences were found between RA and HC, except for PC and O, with RA patients scoring higher. Regarding LMR, the model was significant (F(8,245)=5,203; p<.001), with PC (β=-.372; t=-5.014; p<.001) and O (β=-.161; t=-2.567; p=.011) significantly predicting the type of sample. Conclusions FM patients seem to be noticeably more perfectionistic than RA patients and HC, which in turn, do not differ significantly, in the majority of the perfectionism traits and cognitions. PC and O appear to be the most discriminative perfectionism dimensions. Perfectionism seems a variable of utmost importance, especially in FM, and may deserve consideration as a target for interventions. References Sirois F & Molnar DS (Eds). Perfectionism, health and well-Being. Springer International Publishing, 2016. Wolfe F et al. The American College of Rheumatology. Criteria for the classification of Fibromyalgia. Report of the multicenter criteria committee. Arthritis Rheum 1990; 33: 160–172. Arnett FC et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum, 1988; 315–324. White KP et al. Testing an instrument to screen for fibromyalgia syndrome in general population studies: The London Fibromyalgia Epidemiology Study Screening Questionnaire. J Rheumatol. 1999; 26: 880–884. Disclosure of Interest None declared

Supramolecular interactions in 3-ferrocenyl-methoxy-benzothiophenes, non-steroidal drug precursors

Supramolecular interactions in 3-ferrocenyl-methoxybenzothiophenes, non steroidal drug precursors Joao L.A. Ferreira da Silva, Andre P Ferreira, Matilde Marques, Fatima Minas da Piedade Instituto Superior Tecnico, Centro de Quimica Estrutural, Avenida Rovisco Pais, Lisboa, Lisboa, 1049-001, Portugal, Departamento de Quimica e Bioquimica, FCUL, Campo Grande, 1749-016 Lisboa, Portugal, E-mail:joao.luis@ist.utl.pt