Metabolism of AGEs--bacterial AGEs are degraded by metallo-proteases.

Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism.

A genetic investigation of the KEOPS complex in halophilic Archaea.

KEOPS is an important cellular complex conserved in Eukarya, with some subunits conserved in Archaea and Bacteria. This complex was recently found to play an essential role in formation of the tRNA modification threonylcarbamoyladenosine (t(6)A), and was previously associated with telomere length maintenance and transcription. KEOPS subunits are conserved in Archaea, especially in the Euryarchaea, where they had been studied in vitro. Here we attempted to delete the genes encoding the four conserved subunits of the KEOPS complex in the euryarchaeote Haloferax volcanii and study their phenotypes in vivo. The fused kae1-bud32 gene was shown to be essential as was cgi121, which is dispensable in yeast. In contrast, pcc1 (encoding the putative dimerizing unit of KEOPS) was not essential in H. volcanii. Deletion of pcc1 led to pleiotropic phenotypes, including decreased growth rate, reduced levels of t(6)A modification, and elevated levels of intra-cellular glycation products.

AGEs secreted by bacteria are involved in the inflammatory response.

Advanced Glycated End Products (AGEs) are formed by non-enzymatic protein glycation and are implicated in several physiological aspects including cell aging and diseases. Recent data indicate that bacteria--although short lived--produce, metabolize and accumulate AGEs. Here we show that Escherichia coli cells secret AGEs by the energy-dependent efflux pump systems. Moreover, we show that in the presence of these AGEs there is an upshift of pro-inflammatory cytokins by mammalian cells. Thus, we propose that secretion of AGEs by bacteria is a novel avenue of bacterial-induced inflammation which is potentially important in the pathophysiology of bacterial infections. Moreover, the sensing of AGEs by the host cells may constitute a warning system for the presence of bacteria.

Oncogenic synergism between ErbB1, nucleolin, and mutant Ras.

Alterations in the ErbB family of growth factor receptors, their signaling components, and mutational activation of Ras proteins are major contributors to malignant transformation. Recently, mutant Ras was shown to be capable of activating ErbB receptors in a ligand-independent manner. Furthermore, it was observed that nucleolin, a transcriptional regulator and ribosome biogenesis factor, can bind both K-Ras and the cytoplasmic tail of ErbB receptors to enhance ErbB receptor activation. However, the functional significance of these interactions to cancer pathogenesis has not been probed. Here, we show that endogenous nucleolin interacts simultaneously in vivo with endogenous Ras and ErbB1 (EGFR) in cancer cells. The C-terminal 212 amino acids of nucleolin were determined to be sufficient to interact with ErbB1 and all Ras protein isoforms (H-, N-, and K-Ras). Nucleolin partially colocalizes with Ras at the plasma membrane. Moreover, activated but not wild-type Ras facilitates nucleolin interaction with ErbB1 and stabilizes ErbB1 receptor levels. Most importantly, these three oncogenes synergistically facilitate anchorage-independent cell growth in vitro and tumor growth in vivo. Our findings suggest strategies to target nucleolin as a general approach to inhibiting ErbB- and Ras-driven cancers.

The ubiquitous conserved glycopeptidase Gcp prevents accumulation of toxic glycated proteins.

Amadori-modified proteins (AMPs) are the products of nonenzymatic glycation formed by reaction of reducing sugars with primary amine-containing amino acids and can develop into advanced glycated end products (AGEs), highly stable toxic compounds. AGEs are known to participate in many age-related human diseases, including cardiovascular, neurological, and liver diseases. The metabolism of these glycated proteins is not yet understood, and the mechanisms that reduce their accumulation are not known so far. Here, we show for Escherichia coli that a conserved glycopeptidase (Gcp, also called Kae1), which is encoded by nearly every sequenced genome in the three domains of life, prevents the accumulation of Amadori products and AGEs. Using mutants, we show that Gcp depletion results in accumulation of AMPs and eventually leads to the accumulation of AGEs. We demonstrate that Gcp binds to glycated proteins, including pyruvate dehydrogenase, previously shown to be a glycation-prone enzyme. Our experiments also show that the severe phenotype of Gcp depletion can be relieved under conditions of low intracellular glycation. As glycated proteins are ubiquitous, the involvement of Gcp in the metabolism of AMPs and AGEs is likely to have been conserved in evolution, suggesting a universal involvement of Gcp in cellular aging and explaining the essentiality of Gcp in many organisms.

CspC regulates rpoS transcript levels and complements hfq deletions.

The general stress response in Escherichia coli is activated by several stress agents, including entering the stationary growth phase. This response constitutes a complex regulatory network in which a large number of genes are induced and others are repressed. The stress response is regulated by the alternative sigma factor σ(S) encoded by the rpoS gene. The rpoS transcripts are substrates of the RNA binding protein, Hfq, which is essential for its translation. The rpoS mRNA is also a substrate of the cold shock protein C (CspC) which stabilizes the transcripts. Here we demonstrate, using pull-down assays, that CspC interacts with Hfq via mRNA molecules. We also show that CspC acts on the 5' UTR of the rpoS transcript, but its activity on rpoS is independent of Hfq. Moreover, we show that CspC suppresses the phenotypes of an hfq deletion. These results elucidate a new aspect in the post-transcriptional regulation of the stress response and will further our understanding of this complex network.