John R. Strahler

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30–90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.

High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

Comment on "Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry"

We used authentication tests developed for ancient DNA to evaluate claims by Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon samples pass these tests, absence of amino acid composition data, lack of evidence for peptide deamidation, and association of α1(I) collagen sequences with amphibians rather than birds suggest that T. rex does not.

Temporal quantitative proteomics by iTRAQ 2D-LC-MS/MS and corresponding mRNA expression analysis identify post-transcriptional modulation of actin-cytoskeleton regulators during TGF-β-Lnduced epithelial-mesenchymal transition

To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-beta-induced EMT. Further analysis of cofilin1 and beta-actin revealed an increase in their mRNA stability in response to TGF-beta, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-beta-induced EMT.

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre‐rRNA processing

The precise functions of most of the ∼200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly—processing of 27SA3 pre‐rRNA. Six of seven assembly factors required for this step (A3 factors) are mutually interdependent for association with preribosomes. These A3 factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A3 factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A3 factors establishes and maintains stability of functional preribosomes containing 27S pre‐rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre‐rRNA.

Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

Involvement of Op18 in cell proliferation

Op18 is a highly conserved major cytosolic phosphoprotein that is expressed at high levels in acute leukemia and in neuroblastoma. In this study we present evidence pointing to a role for Op18 in cellular proliferation. Blocking of Op18 mRNA translation using antisense oligonucleotides delayed entrance of mitotically stimulated normal peripheral blood lymphocytes into the S phase. Moreover treatment of HL-60 promyelocytic leukemia cells with DMSO or PMA which induced terminal differentiation resulted in a decrease in the level of Op18 RNA and protein. Inhibition of lymphoid proliferation with cyclosporin also resulted in reduced Op18 levels.