Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.     

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na⁺/glucose cotransporter. The Na⁺/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na⁺/Cl⁻/γ-aminobutyric acid, Na⁺/iodide and H⁺/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.     

Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

Mast cells derived from Bruton’s tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon FcRI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.     

A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities

We have previously identified and characterized a heparin-binding cell surface protein (heparin/heparan sulfate-interacting protein, or HIP) present on epithelial and endothelial cells. A synthetic peptide mimicking a heparin-binding domain of HIP is now shown to bind a small subset of heparin molecules with high affinity and, therefore, presumably recognizes a specific structural motif in the heparin molecule. Further analyses revealed that the heparin molecules exhibiting a high affinity for the HIP peptide also show an extremely high affinity for antithrombin III (AT-III), a cofactor required for heparin’s anticoagulant activity. The HIP peptide was shown to compete with AT-III for binding to heparin and to neutralize the anticoagulant activity of heparin in blood plasma assays. Furthermore, the heparin subfraction that binds to the HIP peptide with high affinity exhibits an extremely high anticoagulant activity. We conclude that although the HIP peptide shows no sequence similarity with AT-III, the two proteins recognize the same or similar structural motifs in heparin.     

A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.     

Fe-catalyzed cleavage of the α subunit of Na/K-ATPase: Evidence for conformation-sensitive interactions between cytoplasmic domains

Incubation of Na/K-ATPase with ascorbate plus H₂O₂ produces specific cleavage of the α subunit. Five fragments with intact C termini and complementary fragments with intact N termini were observed. The β subunit is not cleaved. Cleavages depend on the presence of contaminant or added Fe²⁺ ions, as inferred by suppression of cleavages with nonspecific metal complexants (histidine, EDTA, phenanthroline) or the Fe³⁺-specific complexant desferrioxamine, or acceleration of cleavages by addition of low concentrations of Fe²⁺ but not of other heavy metal ions. Na/K-ATPase is inactivated in addition to cleavage, and both effects are insensitive to OH radical scavengers. Cleavages are sensitive to conformation. In low ionic strength media (E₂) or media containing Rb ions [E₂(Rb)], cleavage is much faster than in high ionic strength media (E₁) or media containing Na ions (E₁Na). N-terminal fragments and two C-terminal fragments (N-terminals E²¹⁴ and V⁷¹²) have been identified by amino acid sequencing. Approximate positions of other cleavages were determined with specific antibodies. The results suggest that Fe²⁺ (or Fe³⁺) ions bind with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe²⁺ (or Fe³⁺) ion. Thus, cleavage patterns can provide information on spatial organization of the polypeptide chain. We propose that highly conserved regions of the α subunit, within the minor and major cytoplasmic loops, interact in the E₂ or E₂(Rb) conformations but move apart in the E₁ or E₁Na conformations. We discuss implications of domain interactions for the energy transduction mechanism. Fe-catalyzed cleavages may be applicable to other P-type pumps or membrane proteins.     

The atomic model of the human protective protein/cathepsin A suggests a structural basis for galactosialidosis

Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with β-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of β-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the β-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.     

13C/31P NMR studies of glucose transport in human skeletal muscle

The muscle intracellular (IC) free glucose concentration and the rate of muscle glycogen synthesis were measured by using in vivo ¹³C and ³¹P NMR spectroscopy in normal volunteers under hyperinsulinemic (≈300 pM) clamp conditions at the following three plasma glucose levels: euglycemia (≈6 mM), mild (≈10 mM), and high (≈16 mM) hyperglycemia. In keeping with biopsy studies, muscle IC free glucose concentration at euglycemia (−0.03 ± 0.03 mmol/kg of muscle, mean ± SEM, n = 10) was not statistically different from zero. A small but statistically significant amount of IC free glucose was observed during mild and high hyperglycemia: 0.15 ± 0.08 (n = 5) and 0.43 ± 0.20 mmol/kg of muscle (n = 5), respectively. Muscle glycogen synthesis rate, in mmol per kg of muscle per min, was 111 ± 11 at euglycemia (n = 10), 263 ± 29 during mild hyperglycemia (n = 5), and 338 ± 42 during high hyperglycemia (n = 5), these three rates being significantly different from each other. As previous in vitro and in vivo studies, these rates suggest a K_m (concentration at which unidirectional glucose transport reaches half-maximal rate) of the muscle glucose transport system in the 15–25 mM range under hyperinsulinemic conditions. The low concentrations of muscle IC free glucose observed under hyperinsulinemic conditions were interpreted, with this estimate and in the framework of metabolic control theory, as glucose transport being the predominant step controlling muscle glucose flux not only at euglycemia but also during hyperglycemia.     

Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK₁ cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK₁ cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.     

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na⁺- and K⁺-dependent pH homeostasis and in Na⁺ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H⁺, Na⁺/H⁺, and K⁺/H⁺ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H⁺ and Na⁺/H⁺ antiports is presented. K⁺ and Li⁺ inhibit Na⁺ uptake, but there is little cross-inhibition between Na⁺ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.     

A model of long-term memory storage in the cerebellar cortex: A possible role for plasticity at parallel fiber synapses onto stellate/basket interneurons

By evoking changes in climbing fiber activity, movement errors are thought to modify synapses from parallel fibers onto Purkinje cells (pf*Pkj) so as to improve subsequent motor performance. Theoretical arguments suggest there is an intrinsic tradeoff, however, between motor adaptation and long-term storage. Assuming a baseline rate of motor errors is always present, then repeated performance of any learned movement will generate a series of climbing fiber-mediated corrections. By reshuffling the synaptic weights responsible for any given movement, such corrections will degrade the memories for other learned movements stored in overlapping sets of synapses. The present paper shows that long-term storage can be accomplished by a second site of plasticity at synapses from parallel fibers onto stellate/basket interneurons (pf*St/Bk). Plasticity at pf*St/Bk synapses can be insulated from ongoing fluctuations in climbing fiber activity by assuming that changes in pf*St/Bk synapses occur only after changes in pf*Pkj synapses have built up to a threshold level. Although climbing fiber-dependent plasticity at pf*Pkj synapses allows for the exploration of novel motor strategies in response to changing environmental conditions, plasticity at pf*St/Bk synapses transfers successful strategies to stable long-term storage. To quantify this hypothesis, both sites of plasticity are incorporated into a dynamical model of the cerebellar cortex and its interactions with the inferior olive. When used to simulate idealized motor conditioning trials, the model predicts that plasticity develops first at pf*Pkj synapses, but with additional training is transferred to pf*St/Bk synapses for long-term storage.     

A Xenopus oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit

Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca²⁺ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α₁ cRNAs (α_1C and α_1E); (ii) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α₁ Ca²⁺ channel currents; and (iii) coinjection of mammalian α₁ cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca²⁺ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α₁ alone” channels are in fact tightly associated α₁β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α₁β]β_n complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α₁β, are [α₁β]β_n complexes that associate with α₂δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α_1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α₁ subunits.     

Importin/karyopherin protein family members required for mRNA export from the nucleus

The yeast Saccharomyces cerevisiae contains three proteins (Kap104p, Pse1p, and Kap123p) that share similarity to the 95-kDa β subunit of the nuclear transport factor importin (also termed karyopherin and encoded by KAP95/RSL1 in yeast). Proteins that contain nuclear localization sequences are recognized in the cytoplasm and delivered to the nucleus by the heterodimeric importin complex. A second importin-related protein, transportin, delivers a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) to the nucleoplasm. We now show that in contrast to loss of importin β (Kap95p/Rsl1p) and transportin (Kap104p), conditional loss of Pse1p in a strain lacking Kap123p results in a specific block of mRNA export from the nucleus. Overexpression of Sxm1p, a protein related to Cse1p in yeast and to the human cellular apoptosis susceptibility protein, relieves the defects of cells lacking Pse1p and Kap123p. Thus, a major role of Pse1p, Kap123p, and Sxm1p may be nuclear export rather than import, suggesting a symmetrical relationship between these processes.     

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor–ligand interactions

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-β very low density lipoprotein (βVLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-βVLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111–1210. From these studies we conclude that the binding of apoE-βVLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.