George Tsaprailis

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Refining the model for selective cleavage at acidic residues in arginine-containing protonated peptides

Abstract Simple glycine-based peptides containing acidic [aspartic (D) and glutamic acid (E)] and basic residues [arginine (R)] were dissociated by surface-induced dissociation (SID) both in a tandem double quadrupole (Q1Q2) and a hybrid sector/time-of-flight (TOF) mass spectrometer, as well as by low-energy collision-induced dissociation in an ion trap mass spectrometer. The synthetic peptides investigated were G D GGG D G, G D GGG D GR, RG D GGG D G, RG D GGG D GR, GGG D GR, GGG E GR, and G D GGG E GR. The mass spectral results obtained support and extend our previous findings that selective cleavages at the C–(O)–N bond adjacent to the acidic residues (C-side) predominate in the spectra when the number of ionizing protons equals or is less than the number of arginine residues. They also support our conclusion that these cleavages are induced by the side-chain acidic hydrogens of D or E residues. Stochastic molecular modeling procedures have been employed in this work to probe the gas-phase conformations for these protonated peptides. These searches have revealed possible conformers of singly protonated GGG D GR, and RG D GGG D G peptide ions where the protonated arginine is solvated by nearby carboxylic and carbonyl oxygens along with simultaneous intramolecular H bonding between the D side-chain acidic hydrogen(s) and the adjacent C-side peptide bonds. Electrospray ionization/surface-induced dissociation fragmentation efficiency curves (percent fragmentation versus SID laboratory collision energy) are also presented for some of these peptides. The relative position of these curves both with the Q1Q2 and sector/TOF instruments along with less pronounced selective cleavages for the E-containing peptides support our previous conclusion that selective cleavage at E residues require longer time frames for dissociation than for D-containing peptides. The total sum of these findings underscores the idea that gas-phase secondary structure (i.e. conformation) can have an influence in peptide fragmentation.

Media ion composition controls regulatory and virulence response of Salmonella in spaceflight.

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.

Cystatin C increases in cardiac injury: a role in extracellular matrix protein modulation.

AIMS Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology. METHODS AND RESULTS Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III. CONCLUSION Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.

The multifunctional Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin’s spring elements

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titins elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titins spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titins molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.

Improved MALDI-TOF imaging yields increased protein signals at high molecular mass.

Matrix assisted laser desorption ionization (MALDI) mass spectrum images are created from an array of mass spectra collected over a tissue surface. We have increased the mass range of proteins that can be detected in tissue sections from kidneys, heart, lung and brain of different rodent species by a modification of the sandwich technique, which involves co-crystallizing matrix with analyte. A tissue section is placed upon a drop of sinapinic acid matrix dissolved in 90% ethanol and 0.5% Triton X-100. Once the matrix has dried, a seed layer of sinapinic crystals is added as a dispersion in xylene. Additional layers of sinapinic acid are added as solutions in 90% ethanol followed by 50% acetonitrile. Numerous peaks with signal to noise ratio of four or greater are observed between 25 kDa to 50 kDa. This represents ∼10 times as many peaks as are detected using traditional matrix spotting and spraying.

Proteomic Identification of Insulin-like Growth Factor-binding Protein-6 Induced by Sublethal H2O2 Stress from Human Diploid Fibroblasts

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen α1(VI), collagen α2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, l-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.

La Autoantigen Mediates Oxidant Induced De Novo Nrf2 Protein Translation

Nrf2 gene encodes a transcription factor that regulates the expression of a cluster of antioxidant and detoxification genes. Recent works from our laboratory indicate that oxidative stress causes rapid de novo synthesis of Nrf2 protein. We have found that 5′ Untranslated Region (5′UTR) of Nrf2 allows the mRNA to undergo an Internal Ribosomal Entry Site (IRES) mediated protein translation. Using liquid chromatography tandem MS, we have discovered that La/SSB protein bound to Nrf2 5′UTR in response to oxidative stress. In vitro RNA binding and in vivo ribonucleoprotein immunoprecipitation showed H2O2 dose and time dependent increases of La/SSB binding to Nrf2 5′UTR. La/SSB protein translocated from the nuclei to cytoplasm and distributed in the perinuclear space in cells treated with H2O2. Isolation of ribosomal fractions indicated that oxidants caused an association of La/SSB with ribosomes. Physical interaction of La/SSB with representative proteins from the small or large subunits of ribosomes was found to increase in cells responding to H2O2 treatment. Knocking down La/SSB gene with siRNA prevented Nrf2 protein elevation or Nrf2 5′UTR activation by oxidants. In contrast, overexpression of La/SSB gene was able to enhance Nrf2 5′UTR activation and Nrf2 protein increase. Our data suggest that oxidants cause nuclear export of La/SSB protein and subsequent association of La/SSB with Nrf2 5′UTR and ribosomes. These events contribute to de novo Nrf2 protein translation because of oxidative stress.

Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries.

New Site(s) of Methylglyoxal-Modified Human Serum Albumin, Identified by Multiple Reaction Monitoring, Alter Warfarin Binding and Prostaglandin Metabolism

Methylglyoxal (MG) is a biologically reactive byproduct of glucose metabolism, levels of which increase in diabetes. MG modification of protein generates neutral hydroimidazolone adducts on arginine residues which can alter functional active sites. We investigated the site-specificity of MG adduction to human serum albumin (HSA) using multiple reaction monitoring (MRM) of 13 MG-modified tryptic peptides, each containing an internal arginine. Seven new sites for MG modification (R257>R209>R222>R81>R485>R472>R10) are described. Analysis of MG-treated HSA showed substantial R257 and R410 modification, with MG-modified R257 (at 100μM MG) in drug site I causing significant inhibition of prostaglandin catalysis. The MG hydroimidazolone (MG-H1) adduct was modeled at R257, and molecular dynamics simulations and affinity docking revealed a decrease of 12.8-16.5kcal/mol (S and R isomers, respectively) for warfarin binding in drug site I. Taken together, these results suggest that R257 is a likely site for MG modification in vivo, which may have functional consequences for prostaglandin metabolism and drug bioavailability.