Specific protein-membrane contacts are required for prepore and pore assembly by a cholesterol-dependent cytolysin

Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. This observation suggested that the D4 loops, which include loops L1-L3 and the undecapeptide, of ILY were no longer required for its cell binding. However, we show here that membrane insertion of the D4 loops is required for the cytolysis by ILY. Receptor binding triggers changes in the structure of ILY that are necessary for oligomerization, but membrane insertion of the D4 loops is critical for oligomer assembly and pore formation. Defects that prevent membrane insertion of the undecapeptide also block assembly of the prepore oligomer, while defects in the membrane insertion of the L1-L3 loops prevent the conversion of the prepore oligomer to the pore complex. These studies reveal that pore formation by ILY, and probably other CDCs, is affected by an intricate and coupled sequence of interactions between domain 4 and the membrane.     

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

Activity Blockade and GABAA Receptor Blockade Produce Synaptic Scaling through Chloride Accumulation in Embryonic Spinal Motoneurons and Interneurons

Synaptic scaling represents a process whereby the distribution of a cell''s synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABA_A receptors (GABA_ARs). We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABA_AR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABA_AR. The finding that GABA_AR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABA_AR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development.