Impact of Mutations on NPAC Structural Dynamics: Mechanistic Insights from MD Simulations.

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the ß-hydroxyacid dehydrogenases (ß-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other ß-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.

LSD1/KDM1A mutations associated to a newly described form of intellectual disability impair demethylase activity and binding to transcription factors.

Genetic diseases often lead to rare and severe syndromes and the identification of the genetic and protein alterations responsible for the pathogenesis is essential to understand both the physiological and pathological role of the gene product. Recently, de novo variants have been mapped on the gene encoding for the lysine-specific histone demethylase 1 (LSD1)/lysine(K)-specific histone demethylase 1A in three patients characterized by a new genetic disorder. We have analyzed the effects of these pathological mutations on the structure, stability and activity of LSD1 using both in vitro and cellular approaches. The three mutations (Glu403Lys, Asp580Gly and Tyr785His) affect active-site residues and lead to a partial impairment of catalytic activity. They also differentially perturb the ability of LSD1 to engage transcription factors that orchestrate key developmental programs. Moreover, cellular data indicate a decrease in the protein cellular half-life. Taken together, these results demonstrate the relevance of LSD1 in gene regulation and how even moderate alterations in its stability, catalytic activity and binding properties can strongly affect organism development. This depicts a perturbed interplay of catalytic and non-catalytic processes at the origin of the pathology.

Probing the interaction of the p53 C-terminal domain to the histone demethylase LSD1.

The p53 transcription factor plays a central role in the regulation of the expression of several genes, and itself is post-translationally regulated through its different domains. Of particular relevance for p53 function is its intrinsically disordered C-terminal domain (CTD), representing a hotspot for post-translational modifications and a docking site for transcriptional regulators. For example, the histone H3 lysine demethylase 1 (LSD1) interacts with p53 via the p53-CTD for mutual regulation. To biochemically and functionally characterize this complex, we evaluated the in vitro interactions of LSD1 with several p53-CTD peptides differing in length and modifications. Binding was demonstrated through thermal shift, enzymatic and fluorescence polarization assays, but no enzymatic activity could be detected on methylated p53-CTD peptides in vitro. These experiments were performed using the wild-type enzyme and LSD1 variants that are mutated on three active-site residues. We found that LSD1 demethylase activity is inhibited by p53-CTD. We also noted that the association between the two proteins is mediated by mostly non-specific electrostatic interactions involving conserved active-site residues of LSD1 and a highly charged segment of the p53-CTD. We conclude that p53-CTD inhibits LSD1 activity and that the direct association between the two proteins can contribute to their functional cross-talk.

The Structural-Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications.

Calsequestrin (CASQ) is a key intra-sarcoplasmic reticulum Ca2+-handling protein that plays a pivotal role in the contraction of cardiac and skeletal muscles. Its Ca2+-dependent polymerization dynamics shape the translation of electric excitation signals to the Ca2+-induced contraction of the actin-myosin architecture. Mutations in CASQ are linked to life-threatening pathological conditions, including tubular aggregate myopathy, malignant hyperthermia, and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The variability in the penetrance of these phenotypes and the lack of a clear understanding of the disease mechanisms associated with CASQ mutations pose a major challenge to the development of effective therapeutic strategies. In vitro studies have mainly focused on the polymerization and Ca2+-buffering properties of CASQ but have provided little insight into the complex interplay of structural and functional changes that underlie disease. In this review, the biochemical and structural natures of CASQ are explored in-depth, while emphasizing their direct and indirect consequences for muscle Ca2+ physiology. We propose a novel functional classification of CASQ pathological missense mutations based on the structural stability of the monomer, dimer, or linear polymer conformation. We also highlight emerging similarities between polymeric CASQ and polyelectrolyte systems, emphasizing the potential for the use of this paradigm to guide further research.

A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.

LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.

A pilot study with early adolescents: dealing with diet, tobacco and air pollution using practical experiences and biological markers.

BACKGROUND: Tobacco use and the Western diet are two of the most important and investigated topics in relation to adolescents' health. In addition, air pollution is a crucial subject for future generations. School is a key social environment that should promote healthy behaviors in children and adolescents. In this field many different programs have been conducted, with mixed results and effectiveness. Research data suggest that comprehensive and multicomponent approaches may have a greater effect on tobacco use and diet, especially when integrated into a community-wide approach. METHODS: The present work describes a multi-area pilot study called "La Scuola della Salute" (the School of Health) with a focus on the methodological aspects of the intervention. In our study we assessed different web-based and practical experiences related to adolescents' smoking and dietary behaviors and awareness of smoke-related air pollution. Furthermore, to make adolescents more conscious of smoking and dietary behaviors, we conducted experiential workshops that addressed smoking and environmental pollution, food education, and lifestyle. Teachers and school administrators were involved in the project. RESULTS: At baseline we investigated dietary habits, tobacco use, and individual and social characteristics by means of lifestyle questionnaires. In addition, we collected anthropometric parameters and performance indicators such as exhaled carbon monoxide and urinary fructose to assess smoking and nutrition habits. At the end of the intervention lifestyle questionnaire and biological markers were collected again: knowledge about these topics was significantly improved, and the urinary fructose was able to estimate the levels of obesity in the classes. CONCLUSIONS: The integrated approach, combined with the use of biological markers, could be an innovative approach to the promotion of healthy lifestyles among adolescents, but further research is needed.

The growing structural and functional complexity of the LSD1/KDM1A histone demethylase.

LSD1 was the first discovered histone demethylase. Using a flavin-dependent oxidative mechanism, LSD1 demethylates the N-terminal tail of histone H3 in the context of a variety of developmental processes. This functional complexity involves the association with nuclear factors and non-coding RNAs. A number of exciting studies are uncovering the bases of these specific and diverse molecular interactions, which occur both at catalytic and non-catalytic regions of the enzyme. Alternative splicing and post-translation modifications represent further layers for modulating this complex molecular network. By combining structural methods with the usage of chemically modified histones, it is becoming possible to visualize how LSD1 and associated co-repressors recognize the nucleosome. The enzyme clamps the nucleosomal particle through multivalent interactions mediated by the non-catalytic domains, which represent prospective sites for drug design.

Extended varenicline treatment in a severe cardiopathic cigarette smoker: a case report.

INTRODUCTION: Tobacco smoking is the leading cause of cardiovascular morbidity and mortality and quitting tobacco use should be fundamental for cardiovascular patients. Varenicline is a smoking cessation pharmacological therapy able to improve the possibilities to successfully achieve this result. In 2011 the US Food and Drug Administration issued a safety announcement that varenicline may be associated with an increased risk of certain cardiovascular adverse events in patients who have cardiovascular disease. Following studies found no significant increase in cardiovascular serious adverse events associated with varenicline. For the first time in the literature, we describe the case of a cardiopathic hard smoker who received varenicline for 9 months without any side effect. By describing this case we want to underline the safety of varenicline, to illustrate the setting and the method that we used to support him and to underline the importance of promoting smoking cessation in heart patients. CASE PRESENTATION: Varenicline was used to promote smoking cessation in a 52-year-old Caucasian man who smoked 40 cigarettes per day, despite two ischemic cardiovascular events. He asked for a consultation in a pharmacy's smoking cessation service and after the assessment phase varenicline was prescribed. Due to his difficulty to quit smoking and given his good tolerance of the drug, we extended the treatment with varenicline to 9 months in order to achieve and maintain a complete smoking abstinence; intensive behavioural counselling was combined with the pharmacological therapy. By using exhaled carbon monoxide measurement we assessed smoking abstinence up to 2 years. CONCLUSIONS: The use of varenicline for a period longer than 6 months has not been described in the literature, particularly in heart patients. The extended varenicline therapy was clinically monitored and allowed the patient to consolidate his abstinence; the intensive behavioural counselling helped him to overcome his strong psychological dependence. Promoting smoking cessation in people who have cardiovascular disease is crucial. Currently available medications, such as varenicline, increase the chances of success and the risk of possible side effects is outweighed by the lifetime benefits and we hope that clinicians use them more frequently and confidently.

Antismoking centers in Milan's communal pharmacies: analysis of the 2010-2011 campaign.

AIMS AND BACKGROUND: According to recent assessments from the Italian Istituto Superiore della Sanità, information and assistance to smokers are still far from satisfactory. We evaluated the impact of a new smoking cessation service located in pharmacies. Smokers' individual characteristics were also considered. MATERIAL AND METHODS: A 1-year pilot study was carried out from October 2010 to September 2011. Five pharmacies in Milan were selected. A psychologist with experience in smoking cessation was present in each pharmacy one afternoon per week, and pharmacists were trained by a team from the Antismoking Center of the Fondazione IRCCS Istituto Nazionale dei Tumori. Each pharmacy was equipped with informative material, carbon monoxide analyzers, and motivational and nicotine dependence questionnaires, in addition to a clinical briefcase. Counseling sessions were also arranged upon request. RESULTS: In the first 12 months of activity, 216 persons asked for a consultation. The sample, aged 15-79 years, reported the following median values: 30 pack/years, 14 ppm CO, and a Fagerström Test of Nicotine Dependence score of 5. More than one-third of the sample (40.3%) had one pathology and 25% had more than one. In some cases (15.7%), people just wanted information about what the service offered. For those who tried to quit, smoking cessation rates were 33.3% at 3 months, 28% at 6 months, and 24.6% at 1 year. Three kinds of pharmacologic therapies were suggested to smokers: nicotine replacement therapy (75.5%), varenicline (17.5%), and bupropion (7%). CONCLUSIONS: The results show that an accessible and free smoking cessation service is considered useful by smokers as demonstrated by the large number of requests compared with other smoking centers in Italy. Increased involvement of pharmacists in supporting smoking cessation makes this a promising initiative for the near future.