The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.     

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.     

Numb is an endocytic protein

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.     

The luteinising hormone-releasing hormone analogue triptorelin with or without the aromatase inhibitor formestane in premenopausal breast cancer: effects on bone metabolism markers

BACKGROUND: the combination of a luteinising hormone-releasing hormone (LH-RH) analogue and an aromatase inhibitor (AI) induces greater oestrogen suppression than the analogue alone in premenopausal breast cancer. However, very few data on the biological effects of such a combination are currently available. AIM OF THE STUDY: the short-term effects of treatment with the LH-RH analogue triptorelin alone or in association with the AI formestane on bone metabolism were investigated in premenopausal breast cancer. Circulating levels of the bone formation markers carboxy-terminal and amino-terminal propeptides of type I procollagen (PICP and PINP) and the bone resorption marker cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were assessed. In addition, serum levels of insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and interleukin 6 (IL-6) were evaluated. PATIENTS AND METHODS: twenty-one patients with advanced breast cancer were randomly given triptorelin monthly alone (n=10, arm A) or in combination with formestane fortnightly (n=11, arm B). Blood samples were collected over a 3-month period. RESULTS: serum PICP and PINP levels increased significantly over time (P=0.0065 and 0.0197 in arm A and B, respectively); no change in ICTP levels was observed. A rise in IGF-I and IGFBP-3 levels was seen in each treatment group, but only the increase in IGF-I was significant (P=0.0138, always). The on-treatment levels of the bone turnover markers and IGF-system components were inversely correlated with serum oestrogens. Neither treatment modalities significantly affected serum IL-6 levels over time. No difference in the behaviour of any of the assessed biomarkers was observed between patients with or without skeletal metastases. CONCLUSION: it is worth noting that complete oestrogen depletion, at least in our case series, seems to increase only osteoblastic activity markers. The observed modifications appear to be related to oestrogen depletion per se rather than the degree of oestrogen suppression or the different therapeutic regimen administered.     

Impermeability of the GIRK2 weaver channel to divalent cations

Asingle amino acid mutation (G156S) in the putative pore-forming regionof the G protein-sensitive, inwardly rectifying K⁺ channelsubunit, GIRK2, renders the conductance constitutively active andnonselective for monovalent cations. The mutant channel subunit(GIRK2wv) causes the pleiotropic weaver disease inmice, which is characterized by the selective vulnerability ofcerebellar granule cells and Purkinje cells, as well as dopaminergicneurons in the mesencephalon, to cell death. It has beenproposed that divalent cation permeability through constitutivelyactive GIRK2wv channels contributes to a rise in internalcalcium in the GIRK2wv-expressing neurons, eventually leadingto cell death. We carried out comparative studies of recombinantGIRK2wv channels expressed in Xenopus oocytes and COS-7cells to determine the magnitude and relative permeability of theGIRK2wv conductance to Ca²⁺. Data from thesestudies demonstrate that the properties of the expressed current differin the two systems and that when recombinant GIRK2wv isexpressed in mammalian cells it is impermeable to Ca²⁺.

     

L-Propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia–reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H₂O₂ UV-photolysis. This revised version was published online in July 2006 with corrections to the Cover Date.