ErbB2 degradation mediated by the co-chaperone protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.     

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.     

Synthesis and SAR study of 4,5-diaryl-1H-imidazole-2(3H)-thione derivatives,as potent 15-lipoxygenase inhibitors

A series of 4,5-diaryl-1H-imidazole-2(3H)-thione was synthesized and their inhibitory potency against soybean 15-lipoxygenase and free radical scavenging activities were determined. Compound 11 showed the best IC₅₀ for 15-LOX inhibition (IC₅₀ = 4.7 μM) and free radical scavenging activity (IC₅₀ = 14 μM). Methylation of SH at C₂ position of imidazole has dramatically decreased the 15-LOX inhibition and radical scavenging activity as it can be observed in the inactive compound 14 (IC₅₀ >250 μM). Structure activity similarity (SAS) showed that the most important chemical modification in this series was methylation of SH group and Docking studies revealed a proper orientation for SH group towards Fe core of the 15-LOX active site. Therefore it was concluded that iron chelating could be a possible mechanism for enzyme inhibition in this series of compounds.     

Manual 768 or 384 well microplate gel ‘dry’ electrophoresis for PCR checking and SNP genotyping

Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid–protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient ‘dry’ gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5–10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.     

Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus

The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of 2-DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.     

Prion protein attenuates excitotoxicity by inhibiting NMDA receptors

It is well established that misfolded forms of cellular prion protein (PrP [PrP(C)]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrP(C) remains incompletely understood. To determine the physiological role of PrP(C), we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrP(C). The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrP(C) mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.     

The next step of neurogenesis in the context of Alzheimer’s disease

Molecular Biology Reports - Among different pathological mechanisms, neuronal loss and neurogenesis impairment in the hippocampus play important roles in cognitive decline in Alzheimer’s...     

Mesenchymal stem cell transfusion for desensitization of positive lymphocyte cross‐match before kidney transplantation: outcome of 3 cases

Objectives

Donor specific antibodies (DSA) and a positive cross‐match are contraindications for kidney transplantation. Trials of allograft transplantation across the HLA barrier have employed desensitization strategies, including the use of plasmapheresis, intravenous immunoglobulins, anti‐B‐cell monoclonal antibodies and splenectomy, associated with high‐intensity immunosuppressive regimens. Our case 1 report suffered from repeatedly positive lymphocyte cross match after 1st renal transplantation. Graft nephrectomy could not correct the state of sensitization. Splenectomy was done in a trial to get rid of the antibody producing clone. Furthermore plasmapheresis with low dose IVIG could not as well revert the state of sensitization for the patient.

Material and methods

About 50 millions donor specific MSCs were injected to the patient.

Results

MSCs transfusion proved to be the only procedure which could achieve successful desensitization before performing the second transplantation owing to their immunosuppressive properties.

Conclusion

This case indicates that DS‐MSCs is a potential option for anti‐HLA desensitization. In cases 2 and 3 IV DS‐MSCs transfusion was selected from the start as a successful line of treatment for pre renal transplantation desensitization to save other unnecessary lines of treatment that were tried in case 1.

     

Genetic polymorphisms of vascular endothelial growth factor and risk for retinopathy of prematurity in South of Iran

Retinopathy of prematurity (ROP) is a multifactorial disease, that cause visual impairment in premature children. The exact pathogenesis and etiology of ROP is unknown and genetic susceptibility is considered as risk factor. Vascular endothelial growth factor (VEGF) plays a major role in retinal neovascularization and subsequently retinal detachment. VEGF polymorphism is associated with proliferative ROP in some studies. We examined the possible association of the VEGF gene polymorphisms with ROP in preterm infants in south of Iran. A total of 111 preterm infants were examined by ophthalmologist and after that were genotyped. Genotyping of the VEGF +405 (rs2010963) and VEGF +936 (rs3025039) was done by the polymerase chain reaction and restriction fragment length polymorphism methods. The frequency of VEGF alleles, genotypes and haplotype distribution were compared between groups. The patients were divided in three groups: 66 to the normal group (normal fundoscopy), and 45 to the ROP group; 30 infants were not treated with Lasertherapy (Regressive group) and 15 treated with Lasertherapy. The frequency of VEGF +405 and VEGF +936 G/C genotypes as well as allele frequencies was not different between groups. No significant difference was found between ROP with treatment and ROP without lasertherapy. Our report indicate that there is no association between the carrier states of gene polymorphisms VEGF +405, VEGF +936 and progression or spontaneous regression of ROP in preterm infants in Iranian population. However, it should be considered that angiogenesis is a complex process and genetic factors in addition to environmental factors are contributed in this pathway.     

Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice

Background

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).

Methods

Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.

Results

In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.

Conclusion

Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.