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We mathematically modeled the receptor-dependent mitogen-activated protein kinase (MAPK) signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway. A novel regulatory motif was, thus, generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu(156) to Ser(170), Val(196) to Ile(210), and Ser(312) to Asn(326) of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu(156)-Ser(170) and Val(196)-Ile(210) were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.

Activation of p53 is an important mechanism in apoptosis. However, whether the presence of p53 in mitochondria plays an important role in p53-mediated apoptosis is unclear. Here, we demonstrate that overexpression of NPM (nucleophosmin) significantly suppresses 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated apoptosis, in part, by blocking the mitochondrial localization of p53. Within 1 h following TPA treatment of skin epithelial (JB6) cells, p53 accumulated in mitochondria. Expression of NPM enhances p53 levels in the nucleus but reduces p53 levels in mitochondria, as detected by immunocytochemistry and Western blot analysis. The suppressive effect of NPM on p53 mitochondrial localization is also observed in TPA-treated primary epithelial cells and in JB6 cells treated with doxorubicin. NPM enhances the expression of p53 target gene p21 and bax. However, the increase in Bax level in the absence of p53 in mitochondria did not lead to an increase in TPA-induced apoptosis, suggesting that the presence of p53 in mitochondria is important. Suppression of NPM by NPM small interfering RNA leads to an increase of p53 levels in mitochondria and apoptosis. Furthermore, suppression of NPM in tumor cells with a high constitutive level of NPM results in p53 translocation to mitochondria and enhances TPA-mediated apoptosis. The results demonstrate the effect of NPM on p53 localization in mitochondria and apoptosis. Together, the data indicate that the presence of p53 in mitochondria plays an important role in stress-induced apoptosis and suggest that NPM may protect cells from apoptosis by reducing the mitochondrial level of p53.

Small ubiquitin-like modifier 1 (SUMO-1) modification of IkappaBalpha has been described to actively participate in NFkappaB regulation. Following proteosomal degradation of IkappaBalpha, an auto-regulatory loop consisting of transcriptional activation of IkappaBalpha gene and SUMO-1 modification of newly synthesized IkappaBalpha proceeds. The SUMOylated IkappaBalpha form is resistant to signal-induced degradation, consequently halting NFkappaB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IkappaBalpha with subsequent attenuation of NFkappaB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IkappaBalpha/SUMO-1. We demonstrate a dose-dependent increase in IkappaBalpha/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IkappaBalpha, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFkappaB activation and NFkappaB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IkappaBalpha, a major control point for NFkappaB activation via Ado signaling.

The physiological relevance of the activation of hepatocyte growth factor (Hgf) by the plasminogen (Plg) system of proteases and its contribution to tissue repair are largely undefined. Here, we investigated whether the defective liver repair in mice lacking Plg is due to impaired activation of Hgf. Loss of Plg in vivo suppressed Hgf activation and signaling through its Met tyrosine kinase receptor. Without Plg, hepatocytes were unresponsive to Hgf-induced proliferation and migration, with a more pronounced impairment in hepatocyte movement within the hepatic environment. Most notably, circumventing the defect in proteolytic activation of Hgf by the downstream expression of an activated Met receptor corrected the functional deficits and improved liver repair in Plg-deficient mice. These findings support a fibrinolysis-unrelated role for Plg in modulating cell proliferation and migration by activation of Hgf.

Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. The number of transcripts expressed progressively decreased during the evolution from K to BP cells. Bioinformatic analysis, validated by detailed biochemical analysis, revealed substantial contraction of both pro- and antiapoptotic networks during transformation. Nonetheless, L and BP cells were not resistant to apoptotic stimuli. At doses of cisplatin that led to 30-60% apoptosis of K and E cells, transformed L and BP cells underwent 80% necrotic cell death, which became the default response to genotoxic stress. Moreover, appreciable necrotic fractions were observed in the cervical carcinoma cell line, HeLa, in response to comparable doses of cisplatin. The shrinkage of biochemical networks, including the apoptotic network, may allow a cancer cell to economize on energy usage to facilitate enhanced proliferation but leaves it vulnerable to stress. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis.

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.

Fatty-acid synthase (FAS) is up-regulated in a broad range of cancers, including those of the breast, prostate, and ovaries. In tumor cells, the inhibition of FAS elicits cell cycle arrest and apoptosis, so it is considered a potential drug target for oncology. Results from this study show that inhibition of FAS, by either knockdown with small interfering RNA or inhibition with the small molecule drug orlistat, leads to activation of the receptor-mediated apoptotic cascade (caspase-8-mediated) and ultimately to cell death. However, knockdown of two enzymes upstream of FAS, acetyl-CoA carboxylase-α and ATP-citrate lyase, fails to activate caspase-8 or to elicit apoptosis in tumor cells, even though palmitate synthesis was suppressed. Using differential gene analysis, we traced the unique apoptotic effect of FAS inhibition to up-regulation of DDIT4 (DNA damage-inducible transcript 4), a stress-response gene that negatively regulates the mTOR pathway. These findings indicate that suppression of palmitate synthesis is not sufficient for eliciting tumor cell death and suggest that the unique effect of inhibition of FAS results from negative regulation of the mTOR pathway via DDIT4.

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. We found that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmids segregating as a unit.

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.