Human AE1 performs electroneutral exchange of Cl ؊ for HCO 3 ؊ across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.

The PI3K/AKT signaling pathway is aberrant in a wide variety of cancers. Downstream effectors of AKT are involved in survival, growth, and metabolic-related pathways. In contrast, contradictory data relating to AKT effects on cell motility and invasion, crucial pro-metastatic processes, have been reported pointing to a potential cell type and isoform type-specific AKT driven function. By implication, study of AKT signaling should optimally be conducted in the appropriate intracellular environment. Prognosis in soft-tissue sarcoma (STS), aggressive malignancies of mesenchymal origin, is poor reflecting our modest abilities to control metastasis, an effort hampered by lack of insight into molecular mechanisms driving STS progression and dissemination. We examined the impact of the cancer progression relevant AKT pathway on the mesenchymal tumor cell internal milieu. We demonstrate that AKT1 activation induces STS cell motility and invasiveness at least partially via a novel interaction with the intermediate filament vimentin. The binding of AKT (tail region) to vimentin (head region) results in vimentin Ser39 phosphorylation enhancing the ability of vimentin to induce motility and invasion while protecting vimentin from caspase induced proteolysis. Moreover, vimentin phosphorylation was shown to enhance tumor and metastasis growth in vivo. Insights into this mesenchymal-related molecular mechanism may facilitate development of critically lacking therapeutic options for these devastating malignancies.

BackgroundVimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting “epithelial to mesenchymal transition” phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses.Methods and FindingsWithaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens.ConclusionsIn light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and “epithelial to mesenchymal transition” clinical contexts is warranted.

We report here the first example using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. With this novel array, proteins are site-specifically immobilized on the glass surface and are able to retain their native activity. The advantage of the avidin/biotin linkage over his-tag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which makes this new protein array compatible with most biological assays.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that .70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A 2 receptor-1 (PLA 2 R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA 2 R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA 2 R by probing isolated truncated PLA 2 R extracellular domains with sera from patients with IMN that contain anti-PLA 2 R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteinerich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA 2 R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA 2 R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA 2 R. These results indicate that the immunodominant epitope in PLA 2 R is exclusively located in the CysR-FnII-CTLD1 region.

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NBCe1-A electrogenically cotransports Na

The unusually high MAO-B activity consistently observed in Parkinson's disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level.