Drosophila EGFR signalling is modulated by differential compartmentalization of Rhomboid intramembrane proteases

We explore the role of differential compartmentalization of Rhomboid (Rho) proteases that process the Drosophila EGF receptor ligands, in modulating the amount of secreted ligand and consequently the level of EGF receptor (EGFR) activation. The mSpitz ligand precursor is retained in the ER, and is trafficked by the chaperone Star to a late compartment of the secretory pathway, where Rho-1 resides. This work demonstrates that two other Rho proteins, Rho-2 and Rho-3, which are expressed in the germ line and in the developing eye, respectively, cleave the Spitz precursor and Star already in the ER, in addition to their activity in the late compartment. This property attenuates EGFR activation, primarily by compromising the amount of chaperone that can productively traffic the ligand precursor to the late compartment, where cleavage and subsequent secretion take place. These observations identify changes in intracellular compartment localization of Rho proteins as a basis for signal attenuation, in tissues where EGFR activation must be highly restricted in space and time.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD₅₀ B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD₅₀ of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.     

Diethyldithiocarbamate and Nitric Oxide Synergize with Oxidants and with Membrane-Damaging Agents to Injure Mammalian Cells

The effect of diethyldithiocarbamate (DDC) and sodium nitroprusside (SNP) on the killing of endothe-lial cells and on the release of arachidonate by mixtures of oxidants and membrane-damaging agents was studied in a tissue culture model employing bovine aortic endothelial cells labeled either with ⁵¹Chromium or ³arachidonic acid. While exposure to low, subtoxic concentrations of oxidants (reagent H₂O₂, glucose-oxidase generated peroxide, xanthine xanthine oxidase, AAPH-generated peroxyl radical, menadione-generated oxidants) did not result either in cell death or in the loss of membrane-associated arachidonic acid, the addition of subtoxic amounts of a variety of membrane-damaging agents (streptolysin S, PLA₂, histone, taurocholate, wheatgerm agglutinin) resulted in a synergistic cell death. However, no significant amounts of arachidonate were released unless proteinases were also present. The addition to these reaction mixtures of subtoxic amounts of DDC (an SOD inhibitor and a copper chelator) not only very markedly enhanced cell death but also resulted in the release of large amounts of arachidonate (in the complete absence of added proteinases). Furthermore, the inclusion in DDC-containing reaction mixtures of subtoxic amounts of SNP, a generator of NO, further enhanced, in a synergistic manner, both cell killing and the release of arachidonate. Cell killing and the release of arachidonate induced by the DDC and SNP- containing mixtures of agonists were strongly inhibited by catalase, glutathione, N-acetyl cysteine, vitamin A, and by a nonpenetrating PLA_z inhibitor as well as by tetracyclines. A partial inhibition of cell killing was also obtained by 1,10-phenanthroline and by antimycin. It is suggested that DDC might amplify cell damage by forming intracellular, loosely-bound complexes with copper and probably also by depleting antioxidant thiols. It is also suggested that “cocktails” containing oxidants, membrane-damaging agents, DDC, and SNP might be beneficial for killing of tumor cells in vivo and for the assessment of the toxicity of xenobiotics in vitro.     

NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.     

Rapid diagnosis of infection etiology in febrile pediatric oncology patients using infrared spectroscopy of leukocytes

Rapid diagnosis of the etiology of infection is highly important for an effective treatment of the infected patients. Bacterial and viral infections are serious diseases that can cause death in many cases. The human immune system deals with many viral and bacterial infections that cause no symptoms and pass quietly without treatment. However, oncology patients undergoing chemotherapy have a very weak immune system caused by leukopenia, and even minor pathogen infection threatens their lives. For this reason, physicians tend to prescribe immediately several types of antibiotics for febrile pediatric oncology patients (FPOPs). Uncontrolled use of antibiotics is one of the major contributors to the development of resistant bacteria. Therefore, for oncology patients, a rapid and objective diagnosis of the etiology of the infection is extremely critical. Current identification methods are time‐consuming (>24 h). In this study, the potential of midinfrared spectroscopy in tandem with machine learning algorithms is evaluated for rapid and objective diagnosis of the etiology of infections in FPOPs using simple peripheral blood samples. Our results show that infrared spectroscopy enables the diagnosis of the etiology of infection as bacterial or viral within 70 minutes after the collection of the blood sample with 93% sensitivity and 88% specificity.     

The MEK/ERK cascade: from signaling specificity to diverse functions

The ERK signaling cascade is a central MAPK pathway that plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, learning, survival and, under some conditions, also apoptosis. The ability of this cascade to regulate so many distinct, and even opposing, cellular processes, raises the question of signaling specificity determination by this cascade. Here we describe mechanisms that cooperate to direct MEK-ERK signals to their appropriate downstream destinations. These include duration and strength of the signals, interaction with specific scaffolds, changes in subcellular localization, crosstalk with other signaling pathways, and presence of multiple components with distinct functions in each tier of the cascade. Since many of the mechanisms do not function properly in cancer cells, understanding them may shed light not only on the regulation of normal cell proliferation, but also on mechanisms of oncogenic transformation.