In vivo isolation of a pPJ3a-λ cosmid mediated by the Tn2307 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn2301.     

In vivo isolation of a pPJ3a-lambda cosmid mediated by the Tn2301 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn2301.     

Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.     

Purification and identification of G protein-coupled receptor protein complexes under native conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.     

NanoLC-MS/MS analysis provides new insights into the phosphorylation pattern of Cdc25B in vivo: full overlap with sites of phosphorylation by Chk1 and Cdk1/cycB kinases in vitro

NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.     

Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.