Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs

VKORC1 Pharmacogenetics and Pharmacoproteomics in Patients on Warfarin Anticoagulant Therapy: Transthyretin Precursor as a Potential Biomarker

Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke.We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation.This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism

Endoplasmic Reticulum Stress Pathway-Mediated Apoptosis in Macrophages Contributes to the Survival of Mycobacterium tuberculosis

BACKGROUND: Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response. CONCLUSION/SIGNIFICANCE: These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria

Cluster Analysis of Symptoms Among Patients with Upper Extremity Musculoskeletal Disorders

Introduction Some musculoskeletal disorders of the upper extremity are not readily classified. The study objective was to determine if there were symptom patterns in self-identified repetitive strain injury (RSI) patients. Methods Members (n = 700) of the Dutch RSI Patients Association filled out a detailed symptom questionnaire. Factor analysis followed by cluster analysis grouped correlated symptoms. Results Eight clusters, based largely on symptom severity and quality were formulated. All but one cluster showed diffuse symptoms; the exception was characterized by bilateral symptoms of stiffness and aching pain in the shoulder/neck. Conclusions Case definitions which localize upper extremity musculoskeletal disorders to a specific anatomical area may be incomplete. Future clustering studies should rely on both signs and symptoms. Data could be collected from health care providers prospectively to determine the possible prognostic value of the identified clusters with respect to natural history, chronicity, and return to work

The interactions of disability and impairment

Theoretical work on disability is going through an expansive period, built on the growing recognition of disability studies as a discipline and out of the political and analytical push to bring disability into a prominent position within accounts of the intersecting social categories that shape people's lives. A current debate within critical disability studies is whether that study should include impairment and embodiment within its focus. This article argues it should and does so by drawing from symbolic interactionism and embodiment literatures in order to explore how differences in what bodies can do-defined as impairments-come to play a role in how people make sense of themselves through social interaction. We argue that these everyday interactions and the stories we tell within them and about them are important spaces and narratives through which impairment and disability are produced. Interactions and stories are significant both in how they are shaped by wider social norms, collective stories and institutional processes, and also how they at times can provide points of resistance and challenges to such norms, stories and institutions. Therefore, the significance of impairment and interaction is the role they play in both informing self-identity and also broader dynamics of power and inequality

Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes

Unfolded protein response in cancer: the Physician's perspective

The unfolded protein response (UPR) is a cascade of intracellular stress signaling events in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER). Cancer cells are often exposed to hypoxia, nutrient starvation, oxidative stress and other metabolic dysregulation that cause ER stress and activation of the UPR. Depending on the duration and degree of ER stress, the UPR can provide either survival signals by activating adaptive and antiapoptotic pathways, or death signals by inducing cell death programs. Sustained induction or repression of UPR pharmacologically may thus have beneficial and therapeutic effects against cancer. In this review, we discuss the basic mechanisms of UPR and highlight the importance of UPR in cancer biology. We also update the UPR-targeted cancer therapeutics currently in clinical trials

Redox regulation of mitochondrial fission, protein misfolding, synaptic damage, and neuronal cell death: potential implications for Alzheimer’s and Parkinson’s diseases

Normal mitochondrial dynamics consist of fission and fusion events giving rise to new mitochondria, a process termed mitochondrial biogenesis. However, several neurodegenerative disorders manifest aberrant mitochondrial dynamics, resulting in morphological abnormalities often associated with deficits in mitochondrial mobility and cell bioenergetics. Rarely, dysfunctional mitochondrial occur in a familial pattern due to genetic mutations, but much more commonly patients manifest sporadic forms of mitochondrial disability presumably related to a complex set of interactions of multiple genes (or their products) with environmental factors (G × E). Recent studies have shown that generation of excessive nitric oxide (NO), in part due to generation of oligomers of amyloid-β (Aβ) protein or overactivity of the NMDA-subtype of glutamate receptor, can augment mitochondrial fission, leading to frank fragmentation of the mitochondria. S-Nitrosylation, a covalent redox reaction of NO with specific protein thiol groups, represents one mechanism contributing to NO-induced mitochondrial fragmentation, bioenergetic failure, synaptic damage, and eventually neuronal apoptosis. Here, we summarize our evidence in Alzheimer’s disease (AD) patients and animal models showing that NO contributes to mitochondrial fragmentation via S-nitrosylation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission. These findings may provide a new target for drug development in AD. Additionally, we review emerging evidence that redox reactions triggered by excessive levels of NO can contribute to protein misfolding, the hallmark of a number of neurodegenerative disorders, including AD and Parkinson’s disease. For example, S-nitrosylation of parkin disrupts its E3 ubiquitin ligase activity, and thereby affects Lewy body formation and neuronal cell death