Proteome analysis of NIH3T3 cells transformed by activated Galpha12: regulation of leukemia-associated protein SET

Galpha(12), the alpha-subunit of the G12 family of heterotrimeric G proteins is involved in the regulation of cell proliferation and neoplastic transformation. GTPase-deficient, constitutively activated mutant of Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) has been previously shown to induce oncogenic transformation of NIH3T3 cells promoting serum- and anchorage-independent growth. Reduced growth-factor dependent, autonomous cell growth forms a critical defining point at which a normal cell turns into an oncogenic one. To identify the underlying mechanism involved in such growth-factor/serum independent growth of Galpha(12)QL-transformed NIH3T3, we carried out a two-dimensional differential proteome analysis of Galpha(12)QL-transformed NIH3T3 cells and cells expressing vector control. This analysis revealed a total of 22 protein-spots whose expression was altered by more than 3-folds. Two of these spots were identified by MALDI-MS analysis as proliferating cell nuclear antigen (PCNA) and myeloid-leukemia-associated SET protein. The increased expressions of these proteins in Galpha(12)QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated Galpha(12) readily increased the expression of SET protein by 24 h. As SET has been previously reported to be an inhibitor of phosphatase PP2A, the nuclear phosphatase activity was monitored in cells expressing activated Galpha(12). Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in Galpha(12)QL cells compared to vector control cells. Thus, our results from differential proteome analysis presented here report for the first time a role for SET in Galpha(12)-mediated signaling pathways and a role for Galpha(12) in the regulation of the leukemia-associated SET-protein expression.     

Helix orientation of the functional domains in apolipoprotein e in discoidal high density lipoprotein particles

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.     

Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.     

Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing approximately 3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal alpha-helix (residues 223-243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20-450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target

The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.     

Evolutionary dynamics and emergence of panzootic H5N1 influenza viruses

The highly pathogenic avian influenza (HPAI) H5N1 virus lineage has undergone extensive genetic reassortment with viruses from different sources to produce numerous H5N1 genotypes, and also developed into multiple genetically distinct sublineages in China. From there, the virus has spread to over 60 countries. The ecological success of this virus in diverse species of both poultry and wild birds with frequent introduction to humans suggests that it is a likely source of the next human pandemic. Therefore, the evolutionary and ecological characteristics of its emergence from wild birds into poultry are of considerable interest. Here, we apply the latest analytical techniques to infer the early evolutionary dynamics of H5N1 virus in the population from which it emerged (wild birds and domestic poultry). By estimating the time of most recent common ancestors of each gene segment, we show that the H5N1 prototype virus was likely introduced from wild birds into poultry as a non-reassortant low pathogenic avian influenza H5N1 virus and was not generated by reassortment in poultry. In contrast, more recent H5N1 genotypes were generated locally in aquatic poultry after the prototype virus (A/goose/Guangdong/1/96) introduction occurred, i.e., they were not a result of additional emergence from wild birds. We show that the H5N1 virus was introduced into Indonesia and Vietnam 3-6 months prior to detection of the first outbreaks in those countries. Population dynamics analyses revealed a rapid increase in the genetic diversity of A/goose/Guangdong/1/96 lineage viruses from mid-1999 to early 2000. Our results suggest that the transmission of reassortant viruses through the mixed poultry population in farms and markets in China has selected HPAI H5N1 viruses that are well adapted to multiple hosts and reduced the interspecies transmission barrier of those viruses.     

Contributions of the carboxyl-terminal helical segment to the self-association and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms

To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.     

Selective cholinergic depletion in medial septum leads to impaired long term potentiation and glutamatergic synaptic currents in the hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning.     

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual’s blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.