Detergent-based but gel-free method allows identification of several hundred membrane proteins in single LC-MS runs

Detergents are indispensable solubilizing agents in the purification and analysis of membrane proteins. For mass spectrometric identification of proteins, it is essential that detergents are removed prior to analysis, necessitating an in-gel digestion step. Here, we report a procedure that allows use of detergents and in-solution digestion of proteins. Crude membrane preparations from mouse brain were solubilized with Triton X-100, CHAPS, or SDS, and the detergents were depleted from the membrane proteins using a desalting column equilibrated with 8 M urea. Following digestion with endoproteinase Lys-C, the resulting peptides were analyzed by LC-MS/MS on Linear ion trap-Orbitrap instrument. Applying stringent identification criteria, in single-LC-MS-runs, 1059 +/- 108 proteins, including 797 +/- 43 membrane proteins, were mapped from mouse brain. The identified proteins represented a broad spectrum of neurotransmitter receptors and other ion channels. The general applicability of the method is demonstrated by profiling of membrane proteins from four other mouse organs. Single-run analyses of eye, liver, spleen, and skeletal muscle allowed identification of 522 +/- 9, 610 +/- 7, 777 +/- 8, and 307 +/- 7 membrane proteins. Our results demonstrate that membrane proteins can be analyzed as efficiently as soluble proteins.     

Phosphorylation of L1-type cell-adhesion molecules--ankyrins away!

Neural cell-adhesion molecules (CAMs) are powerful initiators of neurite outgrowth during neural differentiation. In addition, their interactions with cytoskeletal components are often conserved and highly regulated. How these two aspects of neural CAM function relate to each other is not well understood. However, a recent publication from the Felsenfeld laboratory ( http://www.mssm.edu/labs/felsenfeld) fills a gap in our knowledge of how the interaction of L1-type CAMs with the membrane skeleton adaptor protein ankyrin is severed by phosphorylation and suggests a feedback mechanism whereby the neurite-stimulating activity of L1-CAM is inversely connected to its cytoskeleton binding.     

The anti-apoptotic function of human ��A-crystallin is directly related to its chaperone activity

αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.     

RAD51 as a potential biomarker and therapeutic target for pancreatic cancer

Chemotherapy is a very important therapeutic strategy for cancer treatment. The failure of conventional and molecularly targeted chemotherapeutic regimes for the treatment of pancreatic cancer highlights a desperate need for novel therapeutic interventions. Chemotherapy often fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Overexpression of RAD51 protein, a key player in DNA repair/recombination has been observed in many cancer cells and its hyperexpression is implicated in drug resistance. Recent studies suggest that RAD51 overexpression contributes to the development, progression and drug resistance of pancreatic cancer cells. Here we provide a brief overview of the available pieces of evidence in support of the role of RAD51 in pancreatic tumorigenesis and drug resistance, and hypothesize that RAD51 could serve as a potential biomarker for diagnosis of pancreatic cancer. We discuss the possible involvement of RAD51 in the drug resistance associated with epithelial to mesenchymal transition and with cancer stem cells. Finally, we speculate that targeting RAD51 in pancreatic cancer cells may be a novel approach for the treatment of pancreatic cancer.     

Aggregation properties of a short peptide that mediates amyloid fibril formation in model proteins unrelated to disease

Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The hexapeptide stretch ²¹DIDLHL²⁶ has been shown to be important in the self-assembly of the Src homology 3 (SH3) domain of p85α subunit of bovine phosphatidylinositol-3-kinase (PI3-SH3). The SH3 domain of chicken brain α-spectrin, which is otherwise non-amyloidogenic, is rendered amyloidogenic if ²²EVTMKK²⁷ is replaced by DIDLHL. In this article, we describe the aggregation behaviour of DIDLHL-COOH and DIDLHL-CONH₂. Our results indicate that DIDLHL-COOH and DIDLHL-CONH₂ aggregate to form spherical structures at pH 5 and 6. At pH 5, in the presence of mica, DIDLHL-CONH₂ forms short fibrous structures. The presence of NaCl along with mica results in fibrillar structures. At pH 6, DIDLHL-CONH₂ forms largely spherical aggregates. Both the peptides are unstructured in solution but adopt β-conformation on drying. The aggregates formed by DIDLHL-COOH and DIDLHL-CONH₂ are formed during drying process and their structures are modulated by the presence of mica and salt. Our study suggests that a peptide need not have intrinsic amyloidogenic propensity to facilitate the self-assembly of the full-length protein. The propensity of peptides to form self-assembled structures that are non-amyloidogenic could be important in potentiating the self-assembly of full-length proteins into amyloid fibrils.     

Breaking an Epigenetic Chromatin Switch: Curious Features of Hysteresis in Saccharomyces cerevisiae Telomeric Silencing

In addition to gene network switches, local epigenetic modifications to DNA and histones play an important role in all-or-none cellular decision-making. Here, we study the dynamical design of a well-characterized epigenetic chromatin switch: the yeast SIR system, in order to understand the origin of the stability of epigenetic states. We study hysteresis in this system by perturbing it with a histone deacetylase inhibitor. We find that SIR silencing has many characteristics of a non-linear bistable system, as observed in conventional genetic switches, which are based on activities of a few promoters affecting each other through the abundance of their gene products. Quite remarkably, our experiments in yeast telomeric silencing show a very distinctive pattern when it comes to the transition from bistability to monostability. In particular, the loss of the stable silenced state, upon increasing the inhibitor concentration, does not seem to show the expected saddle node behavior, instead looking like a supercritical pitchfork bifurcation. In other words, the ‘off’ state merges with the ‘on’ state at a threshold concentration leading to a single state, as opposed to the two states remaining distinct up to the threshold and exhibiting a discontinuous jump from the ‘off’ to the ‘on’ state. We argue that this is an inevitable consequence of silenced and active regions coexisting with dynamic domain boundaries. The experimental observations in our study therefore have broad implications for the understanding of chromatin silencing in yeast and beyond.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.