Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Axin1 expression facilitates cell death induced by aurora kinase inhibition through PARP activation

Axin, a negative regulator of Wnt signaling, participates in apoptosis, and Axin1 localizes to centrosomes and mitotic spindles, which requires Aurora kinase activity. In this study, Aurora inhibition of Axin1-expressing cells (L-Axin) produced polyploid cells, which died within 48 h posttreatment, whereas Axin2-expressing cells (L-Axin2) survived the same period. These cell death events showed apoptotic signs, such as chromatin condensation and increased sub-G1 populations, as well as cell membrane rupture. Further analysis showed that Aurora kinase inhibitor (AKI) treatment of L-Axin cells induced poly(ADP-ribose) polymerase (PARP) activation, which increased the poly(ADP-ribosyl)ation of cellular proteins and reduced cellular ATP content. PARP inhibition reduced a proportion of dead cells, suggesting PARP involvement in AKI-induced cell death. Also, AKI treatment of L-Axin cells induced mitochondrial apoptosis-inducing factor (AIF) release, but not mitochondrial cytochrome c release or caspase-3 activation. Knockdown of AIF attenuated AKI-induced cell death in L-Axin cells. Thus, our results suggest that Axin1 expression renders L929 cells sensitive to Aurora inhibition-induced cell death in a PARP- and AIF-dependent manner.     

Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.     

Ganoderma lucidum extract stimulates glucose uptake in L6 rat skeletal muscle cells

The effect of Ganoderma lucidum extract on glucose uptake was studied in L6 rat skeletal muscle cells. G. lucidum extract increased glucose uptake about 2-fold compared to control. The extract stimulated the activity of phosphatidylinositol (PI) 3-kinase which is a major regulatory molecule in the glucose uptake pathway. About 7-fold increased activity of a PI 3-kinase was observed after treatment with G. lucidum extract, whereas PI 3-kinase inhibitor, LY294002, blocked the G. lucidum extract-stimulated PI 3-kinase activity in L6 skeletal muscle cells. Protein kinase B, a downstream mediator of PI 3-kinase, was also activated by G. lucidum extract. We then assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in the glucose uptake pathway. G. lucidum extract increased the phosphorylation level of both AMPK alpha1 and alpha2. Activity of p38 MAPK, a downstream mediator of AMPK, was also increased by G. lucidum extract. Taken together, these results suggest that G. lucidum extract may stimulate glucose uptake, through both PI 3-kinase and AMPK in L6 skeletal muscle cells thereby contributing to glucose homeostasis.     

Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Axin localizes to mitotic spindles and centrosomes in mitotic cells

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.     

Proteomic analysis of parthenogenetic and in vitro fertilized porcine embryos

Proteomic data from embryos are essential for the completion of whole proteome catalog due to embryo‐specific expression of certain proteins. In this study, using reverse phase LC‐MS/MS combined with 1‐D SDS‐PAGE, we identified 1625 mammalian and 735 Sus scrofa proteins from porcine zygotes that included both cytosolic and membranous proteins. We also found that the global protein profiles of parthenogenetically activated (PA) and in vitro fertilized (IVF) zygotes were similar but differences in expression of individual proteins were also evident. These differences were not due to culture conditions, polyspermy or non‐activation of oocytes, as the same culture method was used in both groups, the frequency of polyspermy was 24.3±3.0% and the rates of oocyte activation did not differ (p>0.05) between PA and IVF embryos. Consistent with proteomic data, fluorescent Hoechst 33 342 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed that PA embryos were of poor quality as they contained less cells per blastocyst and were more predisposed to apoptosis (p<0.05), although their in vitro development rates were similar. To our knowledge, this is the first report on global peptide sequencing and quantification of protein in PA and IVF embryos by LC‐MS/MS that may be useful as a reference map for future studies.     

Isolation and characterization of <Emphasis Type="Italic">NgRLK1</Emphasis>, a receptor-like kinase of <Emphasis Type="Italic">Nicotiana glutinosa</Emphasis> that interacts with the elicitin of <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (~10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn²⁺ than Mg²⁺.