Chorein,the protein responsible for chorea-acanthocytosis,interacts with β-adducin and β-actin

Chorea-acanthocytosis (ChAc) is an autosomal, recessive hereditary disease characterized by striatal neurodegeneration and acanthocytosis, and caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene. VPS13A encodes chorein whose physiological function at the molecular level is poorly understood. In this study, we show that chorein interacts with β-adducin and β-actin. We first compare protein expression in human erythrocyte membranes using proteomic analysis. Protein levels of β-adducin isoform 1 and β-actin are markedly decreased in erythrocyte membranes from a ChAc patient. Subsequent co-immunoprecipitation (co-IP) and reverse co-IP assays using extracts from chorein-overexpressing human embryonic kidney 293 (HEK293) cells, shows that β-adducin (isoforms 1 and 2) and β-actin interact with chorein. Immunocytochemical analysis using chorein-overexpressing HEK293 cells demonstrates co-localization of chorein with β-adducin and β-actin. In addition, immunoreactivity of β-adducin isoform 1 is significantly decreased in the striatum of gene-targeted ChAc-model mice. Adducin and actin are membrane cytoskeletal proteins, involved in synaptic function. Expression of β-adducin is restricted to the brain and hematopoietic tissues, corresponding to the main pathological lesions of ChAc, and thereby implicating β-adducin and β-actin in ChAc pathogenesis.     

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca²⁺‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg²⁺, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca²⁺ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca²⁺‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.     

De novo trisomy 20p characterized by array comparative genomic hybridization: Report of a novel case and review of the literature

We report on a boy with speech delay, mental retardation, motor clumsiness, hyperactivity, dysmorphic facial features, brachytelephalangy and short stature. Electrocardiogram, echocardiography, renal ultrasound, electroencephalogram, fundoscopic exam and auditory brainstem responses were all normal. Brain magnetic resonance imaging showed a left temporal arachnoid cyst and a small pineal gland cyst.     

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.     

Introduction of a negative charge at Arg82 in thaumatin abolished responses to human T1R2-T1R3 sweet receptors

Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50 nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors. Mutations at Arg82 had a more deteriorative effect on sweetness than mutations at Lys67. Particularly, a charge inversion at Arg82 (R82E) resulted in an abolishment of the response to sweet receptors even at a concentration as high as 1 mM. These results indicate that Arg82 plays a central role in determining the sweetness of thaumatin. A strict spatial charge location at residue 82 appears to be required for interaction with sweet receptors.     

Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells

Mammalian cells play a dominant role in the industrial production of biopharmaceutical proteins. However, the productivity of producer cells is often hindered by a bottleneck in the saturated secretory pathway, where a sophisticated mechanism of vesicle trafficking is mediated by numerous proteins and their complexes, among which are the cross‐kingdom conserved SNAREs [soluble NSF (N‐ethylmaleimide‐sensitive factor) receptor]. The SNAREs assemble into complexes by means of four interactive α‐helices and, thus, trigger the fusion of transport vesicles with the respective target membranes. We report that the transgenic expression of exocytic SNAREs, which control the fusion of secretory vesicles to the plasma membrane, differentially impacts the secretory capacity of HEK‐293, HeLa, and CHO‐K1 cells. While other exocytic SNAREs have no effect or a negative effect, SNAP‐23 [synaptosome‐associated protein of 23 kDa] and VAMP8 [vesicle‐associated membrane protein 8] specifically increase the production of recombinant proteins when they are ectopically and stably expressed in mammalian cells. The targeted and effective intervention in the secretory capacity of SNARE proteins is a novel engineering strategy, which could lead to the development of new therapies by increasing the production of biopharmaceutical proteins or by boosting the secretion of cell implants in cell therapy initiatives. Biotechnol. Bioeng. 2011; 108:611–620. © 2010 Wiley Periodicals, Inc.     

Human serum albumin interaction,in silico and anticancer evaluation of Pine-Gold nanoparticles

Unique characteristics displayed by phytoconstituent conjugated nanoparticles and their crucial interactions with proteins serve to develop nanoparticle-bio-interface platform. Gold nanoparticles of 16 nm in size were generated using aqueous extracts of pine bark and further conjugated to human serum albumin. The gold nanoparticles-protein complex was characterized by surface plasmon resonance, UV–vis and emission spectroscopy techniques. Further, it was characterized for surface morphology and elemental composition, crystallographic quality, nanoparticles size, shape, stability, structural determination and the identification of capping agent. Moreover, the interaction of gold nanoparticles with human serum albumin was investigated using conventional spectroscopy techniques. Fluorescence quenching and absorption studies demonstrated an effective binding of human serum albumin with oleamide capped gold nanoparticles. The molecular docking study showed a binding affinity of -6.1 kcal/mol whereas the molecular dynamics simulation indicated that the binding of oleamide to human serum albumin. A biological evaluation of pine bark extract-gold nanoparticles showed cytotoxicity with increased cell mortality in lung cancer cells and minimal toxicity on non-cancerous human embryonic kidney cells, respectively.     

An anti-apoptotic HEK293 cell line provides a robust and high titer platform for transient protein expression in bioreactors

ABSTRACT

HEK293 transient expression systems are used to quickly generate proteins for research and pre-clinical studies. With the aim of engineering a high-producing host that grows and transfects robustly in bioreactors, we deleted the pro-apoptotic genes Bax and Bak in an HEK293 cell line. The HEK293 Bax Bak double knock-out (HEK293 DKO) cell line exhibited resistance to apoptosis and shear stress. HEK293 DKO cells sourced from 2 L seed train bioreactors were most productive when a pH setpoint of 7.0, a narrow pH deadband of ±0.03, and a DO setpoint of 30% were used. HEK293 DKO seed train cells cultivated for up to 60 days in a 35 L bioreactor showed similar productivities to cells cultivated in shake flasks. To optimize HEK293 DKO transfection cultures, we first evaluated different pH and agitation parameters in ambr15 microbioreactors before scaling up to 10 L wavebag bioreactors. In ambr15 microbioreactors with a pH setpoint of 7.0, a wide pH deadband of ±0.3, and an agitation of 630 rpm, HEK293 DKO transient cultures yielded antibody titers up to 650 mg/L in 7 days. The optimal ambr15 conditions prompted us to operate the 10 L wavebag transfection without direct pH control to mimic the wide pH deadband ranges. The HEK293 DKO transfection process produces high titers at all scales tested. Combined, our optimized HEK293 DKO 35 L bioreactor seed train and 10 L high titer transient processes support efficient, large-scale recombinant protein production for research studies.     

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions

Members of the peroxidase-cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase-cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase-cyclooxygenase family, including the catalysis of dityrosine formation.