Molecular models of NS3 protease variants of the Hepatitis C virus

Background  

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.     

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.     

Study of response of Swiss Webster mice to light subunit of mushroom tyrosinase

The light subunit of mushroom, Agaricus bisporus, tyrosinase (LSMT), has been identified as an extrinsic component of the enzyme. Its function is unknown, but it can cross an epithelial cell layer, which suggests that it can be absorbed by the intestine. A similar capability has been demonstrated for the HA-33 component of the progenitor toxin from Clostridium botulinum, which is the closest structural homolog of LSMT. Unlike HA-33, LSMT appears to be non-immunogenic as shown by preliminary tests in Swiss Webster mice. We investigated the immunogenicity and histopathology of LSMT in mice to determine its safety in vivo. LSMT did not evoke generation of antibodies after prolonged periods of intraperitoneal administration. Histopathological observations confirmed the absence of responses in organs after twelve weekly administrations of LSMT. We found that LSMT is not toxic and is less immunogenic than the C. botulinum HA-33 protein, which supports further research and development for pharmaceutical application.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

A study of the evolution of inverted-topology repeats from LeuT-fold transporters using AlignMe

X-ray crystal structures have revealed that numerous secondary transporter proteins originally categorized into different sequence families share similar structures, namely, the LeuT fold. The core of this fold consists of two units of five transmembrane helices, whose conformations have been proposed to exchange to form the two alternate states required for transport. That these two units are related implies that LeuT-like transporters evolved from gene-duplication and fusion events. Thus, the origins of this structural repeat may be relevant to the evolution of transport function. However, the lack of significant sequence similarity requires sensitive sequence search methods for analyzing their evolution. To this end, we developed a software application called AlignMe, which can use various types of input information, such as residue hydrophobicity, to perform pairwise alignments of sequences and/or of hydropathy profiles of (membrane) proteins. We used AlignMe to analyze the evolutionary relationships between repeats of the LeuT fold. In addition, we identified proteins from the so-called DedA family that potentially share a common ancestor with these repeats. DedA domains have been implicated in, e.g., selenite uptake; they are found widely distributed across all kingdoms of life; two or more DedA domains are typically found per genome, and some may adopt dual topologies. These results suggest that DedA proteins existed in ancient organisms and may function as dimers, as required for a would-be ancestor of the LeuT fold. In conclusion, we provide novel insights into the evolution of this important structural motif and thus potentially into the alternating-access mechanism of transport itself.     

Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of β,γ-meATP, α,β-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.