Domain Swapping and Different Oligomeric States for the Complex Between Calmodulin and the Calmodulin-Binding Domain of Calcineurin A

BACKGROUND: Calmodulin (CaM) is a ubiquitously expressed calcium sensor that engages in regulatory interactions with a large number of cellular proteins. Previously, a unique mode of CaM target recognition has been observed in the crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A. METHODOLOGY/PRINCIPAL FINDINGS: We have solved a high-resolution crystal structure of a complex between CaM and the CaM-binding domain of calcineurin A in a novel crystal form, which shows a dimeric assembly of calmodulin, as observed before in the crystal state. We note that the conformation of CaM in this complex is very similar to that of unliganded CaM, and a detailed analysis revels that the CaM-binding motif in calcineurin A is of a novel '1-11' type. However, using small-angle X-ray scattering (SAXS), we show that the complex is fully monomeric in solution, and a structure of a canonically collapsed CaM-peptide complex can easily be fitted into the SAXS data. This result is also supported by size exclusion chromatography, where the addition of the ligand peptide decreases the apparent size of CaM. In addition, we studied the energetics of binding by isothermal titration calorimetry and found them to closely resemble those observed previously for ligand peptides from CaM-dependent kinases. CONCLUSIONS/SIGNIFICANCE: Our results implicate that CaM can also form a complex with the CaM-binding domain of calcineurin in a 1 ratio 1 stoichiometry, in addition to the previously observed 2 ratio 2 arrangement in the crystal state. At the structural level, going from 2 ratio 2 association to two 1 ratio 1 complexes will require domain swapping in CaM, accompanied by the characteristic bending of the central linker helix between the two lobes of CaM

Charge Isomers of Myelin Basic Protein: Structure and Interactions with Membranes, Nucleotide Analogues, and Calmodulin

As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers

Pollutant-Induced Modulation in Conformation and β-Lactamase Activity of Human Serum Albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA

Molecular genetics of Stickler and Marshall syndromes, and the role of collagen II and other candidate proteins in high myopia and impaired hearing

Abstract Stickler and Marshall syndromes are genetic disorders both inherited in an autosomal dominant manner. The genotype-phenotype correlation was performed in ten Stickler/Marshall syndrome patients with mutations in the COL11A1 gene. Four patients had a phenotype classified as Marshall syndrome based on early-onset severe hearing loss and characteristic facial dysmorphism. A splice site mutation in intron 50 of COL11A1 was found in these patients, while the remaining six patients had an overlapping Marshall-Stickler phenotype with a mutation elsewhere in the gene. These results indicate exon 50 as a hot spot for splice site mutations leading to a phenotype of Marshall syndrome rather than Stickler syndrome. Collagen II (COL2A1) precursor mRNA undergoes alternative splicing resulting in two different isoforms, IIA including exon 2 and IIB excluding exon 2. Recent evidence indicates that premature termination codon mutations in exon 2 cause Stickler syndrome with no or minimal extraocular manifestations. Two mutations were observed in this study: Cys64Stop, and a novel structural mutation, Cys57Tyr. Results from the COL2A1 mini-gene studies suggested that both mutations altered positive cis elements for splicing resulting in a lower IIA:IIB ratio. The results further emphasize the importance of exon 2 in the development and normal function of the eye. In addition, patients displaying eye phenotypes in the absence of extraocular manifestations should be analyzed first for exon 2 mutations. Linkage analysis identified a new locus for autosomal recessive nonsyndromic hearing loss (DFNB32) on chromosome 1p13.3-22.1 in a Tunisian family with congenital profound autosomal recessive deafness. The COL11A1 gene is located in this region and was analyzed as a candidate gene. No disease causing sequence variation was observed. The analysis of 85 English and 40 Finnish subjects with high myopia resulted in the identification 23 sequence variations in the SLRP genes LUM, FMOD, PRELP, and OPTC. The two intronic variations and seven amino acid changes, one synonymous and six non-synonymous, were not found in the 308 controls analyzed. Five changes were detected in opticin, and all but one were shown to co-segregate with high myopia in families with incomplete penetrance. The results suggested that sequence variations in the SLRP genes expressed in the eye are genetic risk factors underlying the pathogenesis of high myopia

Analysis of service transfer within the CONNECT transnational innovation network:case Nordic countries

Abstract The CONNECT service transfer analysis reported here is part of BECSI (Business ecosystems and platforms for innovations) project. The aim of the project is to enhance Finnish in- novation systems and innovation policy by analysing world-class innovation environments and ecosystem cases from different levels: Smart cities as innovation platforms, Health and Life Science ecosystems and emerging small business ecosystems. BECSI project is granted by TEKES – the Finnish Funding Agency for Innovation through 2013 innovation research thematic call “Ecosystems, spillovers and new policy approaches”. This report is part of BECSI project’s Work Package 4 (WP4) Health & Life Sciences Eco- system Cases task 4.1: San Diego health and life sciences ecosystem study. The purpose of the WP4 is to study theme-specific “local-global” ecosystems that engage both public and private actors in a wide Public-Private Partnership (PPP)-network and to analyse their growth drivers’ transferability into Finnish context. The research activities included screening case studies, transferability analyses, road-mapping and research exchange. CONNECT was founded in 1985 in San Diego, USA. The organisation has been very suc- cessful in catalysing new innovations in the region. However, there is insufficient knowledge about the transferability of the CONNECT to other business contexts at the international level. To fill this gap, this study first describes the service portfolio of CONNECT in San Diego and global CONNECT activities. Then, the study analyses the results of the CONNECT transfer activities at the Nordic level from Swedish and Norwegian perspectives. By doing so, it is possible to compare the current service portfolio of CONNECT with the service portfolios of the currently active Nordic CONNECT organisations. This is also a good way to see to which services developed by CONNECT have been implemented in the Nordic context and to what extent. As Sweden and Norway are both culturally and geographically close to Finland, it is, to certain extent, possible to reflect the transferability potential of CONNECT to Finland through the experiences in these countries. The results of this study show that Springboard is the only service that has been licensed from San Diego to Sweden and Norway. Other services have mainly been developed by local CONNECT organisations. Despite collaboration between different entities, CONNECT can be considered a relatively loose international network. Tighter collaboration would require dedi- cated leadership and sufficient funding. Finally, geographical distances and cultural differ- ences can also be considered barriers for international collaboration

Is there a recreational misuse potential for Pregabalin? : Analysis of anecdotal online reports in comparison with related Gabapentin and Clonazepam data

Original article can be found at: http://content.karger.com/ Copyright Karger.Background: Although pregabalin abuse potential is reportedly low, online data related to its recreational misuse potential have recently been identified. We aimed here at formally analysing pregabalin misuse web reports and comparing these with data pertaining to similar drugs, e.g. gabapentin and clonazepam, for which an abuse potential has already been identified. Methods: An 8-language analysis of the online information on pregabalin acquisition/misuse was carried out. An initial sample of 203 websites was considered and 108 relevant websites were monitored on a regular basis. To obtain material relating to gabapentin and clonazepam, an English-language search was carried out and 32 relevant websites were examined. Results: Misuse of pregabalin, which is commonly offered for sale online, was allegedly associated with sedative and/or psychedelic effects. This was associated with both higher dosages than clinically advised and idiosyncratic (i.e.: IV, rectal, intranasal) drug intake modalities. Although perceived as less powerful than pregabalin, gabapentin misuse was associated with similar sedative/psychedelic effects. Clonazepam allegedly induced either sedation or stimulation, depending on the dosage. Conclusions: Pregabalin experimenters might be profiled as individuals with a history of recreational polydrug misuse. Pregabalin misuse might be facilitated by easy online accessibility; potency; and peculiar, including anxiolytic, psychoactive effects. Physicians should carefully evaluate patients for history of drug abuse and observe them for signs of pregabalin/gabapentin/clonazepam misuse.Peer reviewe