Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.     

Late endosome motility depends on lipids via the small GTPase Rab7

We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.     

Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans

Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant.     

Identification of novel isoforms of the BH3 domain protein Bim which directly activate Bax to trigger apoptosis

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspase-dependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X(L)) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Bax-mediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting

Ghrelin, a circulating growth-hormone releasing peptide derived from stomach, stimulates food intake through neuropeptide Y (NPY) neurons of the arcuate nucleus in the hypothalamus (ARC). We examined the effect of ghrelin microinjected into the ARC and the influence of intracerebroventricular (i.c.v.) pretreatment with a GHRH or NPY receptor antagonist on ghrelin-induced food intake in free-feeding male rats. Ghrelin (0.1-1 microg) stimulated food intake in a dose-dependent manner, and this effect was reduced by 55-60% by the Y(5) NPY receptor antagonist (10 microg i.c.v.), but not by the GHRH receptor antagonist MZ-4-71 (10 microg i.c.v.). We also evaluated the effects of passive ghrelin immunoneutralization by the microinjection of anti-ghrelin immunoglobulins (IgGs) intracerebroventricularly or directly into the ARC on food intake in free-feeding and fasted male rats. i.c.v. administration of anti-ghrelin IgGs decreased cumulative food intake over 24 h, whereas microinfusion of anti-ghrelin IgGs into the ARC induced only a short-lived (2 and 6 h) effect. Collectively, these data would indicate that centrally derived ghrelin has a major role in the control of food intake in rats and, in this context, blood-born ghrelin would be effective only in relation to its ability to reach the ARC, which is devoid of blood-brain barrier.     

2'-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell line

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.