Expression of human BK ion channels in Sf9 cells, their purification using metal affinity chromatography, and functional reconstitution into planar lipid bilayers

This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250-300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.     

Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase

The current study examined the aromatic degradation and central metabolism in Corynebacterium glutamicum by proteomic and molecular methods. Comparative analysis of proteomes from cells grown on gentisate and on glucose revealed that 30% of the proteins of which their abundance changed were involved in aromatic degradation and central carbon metabolism. Similar results were obtained from cells grown on benzoate, 4-cresol, phenol, and resorcinol. Results from these experiments revealed that (i) enzymes involved in degradation of benzoate, 4-cresol, gentisate, phenol, and resorcinol were specifically synthesized and (ii) that the abundance of enzymes involved in central carbon metabolism of glycolysis/gluconeogenesis, pentose phosphate pathway, and TCA cycles were significantly changed on various aromatic compounds. Significantly, three novel proteins, NCgl0524, NCgl0525, and NCgl0527, were identified on 4-cresol. The genes encoding NCgl0525 and NCgl0527 were confirmed to be necessary for assimilation of 4-cresol with C. glutamicum. The abundance of fructose-1,6-bisphosphatase (Fbp) was universally increased on all the tested aromatic compounds. This Fbp gene was disrupted and the mutant WT(Deltafbp) lost the ability to grow on aromatic compounds. Genetic complementation by the Fbp gene restored this ability. We concluded that gluconeogenesis is a necessary process for C. glutamicum growing on various aromatic compounds.     

Targeting autophagic regulation of NFkappaB in HTLV-I transformed cells by geldanamycin: implications for therapeutic interventions

The IkappaB kinase (IKK)/NFkappaB signaling pathway plays an essential role in the development and survival of many types of cancers including adult T-cell leukemia (ATL) caused by the human T-cell leukemia virus type I (HTLV-I) infection. Accordingly, targeting NFkappaB provides an attractive strategy for cancer therapy. We recently found that specific inhibition of Hsp90 by geldanamycin (GA) results in autophagic degradation of IKK and NFkappaB-inducing kinase (NIK), an upstream kinase of IKK, and inactivation of NFkappaB in various cell lines. Here, we further report that GA inhibition of Hsp90 also led to IKK autophagic degradation and NFkappaB inhibition in both HTLV-transformed T cells and ATL-derived cell lines. Importantly, GA treatment led to efficient apoptosis of these malignant cells, whereas inhibition of autophagic degradation of IKK significantly ameliorated the cytotoxic effect of GA. These findings thus not only provide mechanistic insights into the tumor suppression function of autophagy and the anti-tumor activity of GA, but also suggest an immediate therapeutic strategy for ATL and other diseases associated with NFkappaB activation by targeting autophagic degradation of the central NFkappaB activating kinases.     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Identification of novel proteins associated with both alpha-synuclein and DJ-1

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between alpha-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of alpha-synuclein, and vice versa, in the setting of PD.     

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.     

Community response grids: using information technology to help communities respond to bioterror emergencies

Access to accurate and trusted information is vital in preparing for, responding to, and recovering from an emergency. To facilitate response in large-scale emergency situations, Community Response Grids (CRGs) integrate Internet and mobile technologies to enable residents to report information, professional emergency responders to disseminate instructions, and residents to assist one another. CRGs use technology to help residents and professional emergency responders to work together in community response to emergencies, including bioterrorism events. In a time of increased danger from bioterrorist threats, the application of advanced information and communication technologies to community response is vital in confronting such threats. This article describes CRGs, their underlying concepts, development efforts, their relevance to biosecurity and bioterrorism, and future research issues in the use of technology to facilitate community response.     

Identification and Validation of Proteinase 3 and Latent Matrix-Metalloproteinase 9 as Potential Biomarkers for Chronic Lung Transplant Rejection

Introduction

This study examined potential biomarkers for the diagnosis and early detection of chronic allograft rejection after lung transplantation.

Methods

Protein ratios in pooled samples of bronchoalveolar lavage fluid (BALF) from lung transplant recipients at different stages of pre- and postchronic rejection were determined by iTRAQ labeling and mass spectrometry. The potential biomarkers were validated using enzyme-linked immunosorbent assay (ELISA) assay.

Results

Two hundred sixty-five proteins were identified, about two thirds of which showed more than a twofold difference between a pooled control sample (individuals who did not develop chronic rejection in 100 months) and a pooled sample from those with chronic rejection. Proteinase 3 (PR-3) and matrix metalloproteinase 9 (MMP-9) were validated by ELISA assay of 124 individual samples. PR-3 and the latent form of MMP-9 (proMMP9) both demonstrated a specificity of 92% with sensitivities of 76% and 82%, respectively, for disease diagnosis; both were also predictors of developing chronic rejection up to 15 months before diagnosis. While immunoglobulin M (IgM) was upregulated in the pooled samples, individual sample analysis revealed that this arose from outlier values.

Conclusions

iTRAQ can be used to detect a large number of proteins in pooled samples for the discovery of potential biomarkers, but the findings must be validated with technology capable of distinguishing broadly based changes from outcomes as a result of a few extreme cases. The proteins identified in this study expanded the panel of potential biomarkers for the diagnosis and prediction of chronic rejection and provided additional insight into the mechanism of the disease.     

Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy

Pressure overload–induced hypertrophy is a key step leading to heart failure. The Ca²⁺-induced Ca²⁺ release (CICR) process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca²⁺ channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of “intermolecular failure.” Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca²⁺ release, visualized as “Ca²⁺ spikes,” became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca²⁺ transients in CHT. These data suggested that, within a certain limit, termed the “stability margin,” mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of “hidden” intermolecular failure in CHT has important clinical implications.     

Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action

Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.