Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.     

An intestinal parasitic protist, Entamoeba histolytica, possesses a non-redundant nitrogen fixation-like system for iron-sulfur cluster assembly under anaerobic conditions

We have characterized the iron-sulfur (Fe-S) cluster formation in an anaerobic amitochondrial protozoan parasite, Entamoeba histolytica, in which Fe-S proteins play an important role in energy metabolism and electron transfer. A genomewide search showed that E. histolytica apparently possesses a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe-S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. Amino acid alignment and phylogenetic analyses revealed that both amebic NifS and NifU (EhNifS and EhNifU, respectively) showed a close kinship to orthologs from epsilon-proteobacteria, suggesting that both of these genes were likely transferred by lateral gene transfer from an ancestor of epsilon-proteobacteria to E. histolytica. The EhNifS protein expressed in E. coli was present as a homodimer, showing cysteine desulfurase activity with a very basic optimum pH compared with NifS from other organisms. Eh-NifU protein existed as a tetramer and contained one stable [2Fe-2S]2+ cluster per monomer, revealed by spectroscopic and iron analyses. Fractionation of the whole parasite lysate by anion exchange chromatography revealed three major cysteine desulfurase activities, one of which corresponded to the EhNifS protein, verified by immunoblot analysis using the specific EhNifS antibody; the other two peaks corresponded to methionine gamma-lyase and cysteine synthase. Finally, ectopic expression of the EhNifS and EhNifU genes successfully complemented, under anaerobic but not aerobic conditions, the growth defect of an Escherichia coli strain, in which both the isc and suf operons were deleted, suggesting that EhNifS and EhNifU are necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.     

Gem GTPase and tau: morphological changes induced by gem GTPase in cho cells are antagonized by tau

A series of observations have indicated that tau, one of the major microtubule-associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance. Tau is also the major component of paired helical filaments found in brains affected by Alzheimer's disease. To explore an as yet unidentified role of tau in vivo, approximately 11,000 mRNAs were profiled from tau-deficient mouse brains and compared with those from control brains at the same ages. The expression of Gem GTPase, a small GTP-binding protein of the ras superfamily, was significantly increased in the brains of tau-deficient mice at 8 weeks of age. Because Gem GTPase is a negative regulator of the Rho-Rho kinase pathway for cytoskeletal organization, this protein was transiently overexpressed in Chinese hamster ovary cells that do not express tau. Overexpression of Gem GTPase induced a marked elongation of Chinese hamster ovary cells, and simultaneous expression of tau eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-elongation activity of tau was attributed to its microtubule-binding domain, and homologous domains of microtubule-associated proteins 2 and 4 exhibited similar antagonistic activities. Taken together, the present results indicate that the level of Gem GTPase and its cell elongation activity are modulated by tau and suggest that tau may be involved in a Gem GTPase-mediated signal transduction pathway.     

Abnormal lipid metabolism in cystathionine beta-synthase-deficient mice, an animal model for hyperhomocysteinemia

Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine beta-synthase (CBS). Although HHCY is an important and independent risk factor for cardiovascular diseases that are commonly associated with hepatic steatosis, the mechanism by which homocysteine promotes the development of fatty liver is poorly understood. CBS-deficient (CBS(-/-)) mice were previously generated by targeted deletion of the Cbs gene and exhibit pathological features similar to HHCY patients, including endothelial dysfunction and hepatic steatosis. Here we show abnormal lipid metabolism in CBS(-/-) mice. Triglyceride and nonesterified fatty acid levels were markedly elevated in CBS(-/-) mouse liver and serum. The activity of thiolase, a key enzyme in beta-oxidation of fatty acids, was significantly impaired in CBS(-/-) mouse liver. Hepatic apolipoprotein B100 levels were decreased, whereas serum apolipoprotein B100 and very low density lipoprotein levels were elevated in CBS(-/-) mice. Serum levels of cholesterol/phospholipid in high density lipoprotein fractions but not of total cholesterol/phospholipid were decreased, and the activity of lecithin-cholesterol acyltransferase was severely impaired in CBS(-/-) mice. Abnormal high density lipoprotein particles with higher mobility in polyacrylamide gel electrophoresis were observed in serum obtained from CBS(-/-) mice. Moreover, serum cholesterol/triglyceride distribution in lipoprotein fractions was altered in CBS(-/-) mice. These results suggest that hepatic steatosis in CBS(-/-) mice is caused by or associated with abnormal lipid metabolism.     

X-ray-induced telomeric instability in Atm-deficient mouse cells

The gene responsible for ataxia telangiectasia (AT) encodes ATM protein, which plays a major role in the network of a signal transduction initiated by double strand DNA breaks. To determine how radiation-induced genomic instability is modulated by the dysfunction of ATM protein, we examined radiation-induced delayed chromosomal instability in individual cell lines established from wild-type Atm(+/+), heterozygote Atm(+/-), and knock-out Atm(-/-) mouse embryos. The results indicate that Atm(-/-) mouse cells are highly susceptible to the delayed induction of telomeric instability and end-to-end chromosome fusions by radiation in addition to the elevated spontaneous telomeric instability detected by telomere fluorescence in situ hybridization (FISH). The telomeric instability was characterized by abnormal telomere FISH signals, including loss of the signals and the extra-chromosomal signals that were associated and/or not associated with chromosome ends, suggesting that Atm deficiency makes telomeres vulnerable to breakage. Thus, the present study shows that Atm protein plays an essential role in maintaining telomere integrity and prevents chromosomes from end-to-end fusions, indicating that telomeres are a target for the induction of genomic instability by radiation.     

Potent receptor-mediated cytotoxicity of granulocyte colony-stimulating factor-Pseudomonas exotoxin, a fusion protein against myeloid leukemia cells

A chimeric toxin in which the cell-surface binding domain of Pseudomonas exotoxin A was replaced with mature human granulocyte colony-stimulating factor (G-CSF) was produced in Escherichia coli, purified and tested for its biological activity on the human G-CSF-responsive myeloid leukemia cell line, UT7/GR. This fusion protein, termed G-CSF-PE40, showed potent cytotoxicity in the cell line in a dose-dependent manner. G-CSF-PE40 displaced binding of biotinylated G-CSF to its receptor, and the cytotoxicity of G-CSF-PE40 was neutralized by an excess of wild-type G-CSF, indicating the receptor-mediated effects of this chimeric toxin. When G-CSF-PE40 was injected into normal mice, they showed transient neutropenia but no significant changes in the numbers of red blood cells or platelets. Furthermore, G-CSF-PE40 prolonged the survival of mice transplanted with syngeneic myeloid leukemia cells. These observations suggest that G-CSF-PE40 may be useful in targeted therapy of myeloid leukemia cells expressing G-CSF receptors.     

NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74-78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62-79). Further, it was found that a network of hydrogen bonds within the beta sheet and the 3(10) helix in the beta domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.     

Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.     

Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity

To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.