Membrane perturbations of erythrocyte ghosts by spectrin release

The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.     

Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE(-/-)) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE(-/-) macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H(+)-ATPase activity in both cell types, the elevated lysosomal pH in CatE(-/-) macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

Increased attachment and confluence of skin epidermal cells in culture induced by ascorbic acid: detection by permeation of trypan blue across cultured cell layers

In culture epidermal cells from the skin of newborn rats became attached to Millipore filters coated with type IV collagen much better than to filters coated with type I collagen. Ascorbic acid markedly increased the attachment and viability of epidermal cells seeded on type I collagen, but had no significant effect on cells seeded on type IV collagen. It was also found to enhance the synthesis of type IV collagen by the cells, which, we concluded, enabled the cells to become well attached to type I collagen. This conclusion was supported by studies on the penetration of trypan blue through the cell layers. There was a lag in penetration through cell layers cultured both with and without ascorbic acid on Millipore filters coated with either type I or IV collagen, indicating that the cells were confluent over the whole surface of the filters. The lag was much longer in the cultures with ascorbic acid, indicating greater confluence and tighter attachment of cells due to production of type IV collagen. The penetration was found to be due to destruction of the confluent cell layers by its cytotoxic effect. The time lag before penetration of trypan blue is a good index of the confluence and attachment of cultured cells to collagen layers.     

Crystal structure of MqnD (TTHA1568), a menaquinone biosynthetic enzyme from Thermus thermophilus HB8

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670–1673]. Here, we report the 1.55 Å crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with α/β structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.     

Chinese hamster monomeric carbonyl reductases of the short-chain dehydrogenase/reductase superfamily

Chinese hamster monomeric carbonyl reductases (CHCRs) belong to the short-chain dehydrogenase/reductase (SDR) superfamily, which is a family of enzymes that metabolize many endogenous and xenobiotic compounds. We previously cloned three carbonyl reductase cDNAs-Chcr1, Chcr2, and Chcr3. By performing spectrophotometric analyses, we indicated that the enzymes CHCR1, CHCR2, and CHCR3 had similar specificities toward steroids; only CHCR3 did not show any reactivity with prostaglandins (PGs). In the present study, we investigated the characteristics of CHCRs in detail, that is, the differences in their expression patterns, physicochemical properties, and enzymatic activities. CHCR1 exhibited sex-dependent expression patterns. CHCRs showed multiple surface potentials in the zeta potential analysis and CHCR3 exhibited an isatin reductase activity with a high K(m) value. By the present HPLC-analysis, all the three enzymes exhibited PGF(2alpha) dehydrogenase activity and could oxidize PGF(2alpha) to PGE(2) and 15-keto-PGF(2alpha), i.e., the three enzymes exhibited 9- and 15-hydroxy PG dehydrogenase activities. Moreover, 15-keto-PGE(2) was detected in a comparatively higher amount in the dehydrogenase reaction products of CHCR2 than in those of CHCR1 and CHCR3, suggesting that CHCR2 can oxidize PGE(2) and/or 15-keto-PGF(2alpha) to 15-keto-PGE(2); however, these two PGs did not seem to be efficient substrates of CHCR1. Despite the differences in the dehydrogenase activities between CHCR1 and CHCR2, PGE(2) reductase activities of the two enzymes were similar, and PGF(2alpha) was predominantly produced from PGE(2) as a result of the PG 9-keto reductase activity. On the other hand, CHCR3 exhibited a reduced PGE(2) reductase activity. In conclusion, although the CHCRs share a high degree of sequence identity (>70%), they clearly differed in their enzymatic characteristics.     

A Non-canonical DNA Structure Enables Homologous Recombination in Various Genetic Systems

Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.Homologous recombination (HR)² is essential for gametogenesis during meiosis and plays an important role in the generation of genetic diversity, a process that is critical for natural selection. A general HR intermediate is the heteroduplex joint, which is formed between a single-stranded (ss) DNA tail derived from a double-stranded break and a homologous double-stranded (ds) DNA by homologous pairing (HP) (1, 2) and subsequent strand exchange (3, 4). HP is a switch from parental dsDNA base pairs to recombinant base pairs involving the ssDNA and the complementary strand of the dsDNA, which form the core of the recombination intermediate, and strand exchange is the unidirectional replacement of a dsDNA strand by the incoming ssDNA. The RecA/Rad51 family of proteins, which include bacterial RecA, archaeal RadA/Rad51, eukaryotic Rad51, and meiosis-specific Dmc1, are essential for HR in their respective organisms, and these proteins can promote ATP-dependent HP and ATP hydrolysis-dependent strand exchange in vitro (see Refs. 5–10, for reviews). In HP, ATP-bound RecA first binds to ssDNA, and this ssDNA·RecA complex then interacts with dsDNA without homologous recognition. Within the RecA·ssDNA·dsDNA complex, a homologous region is identified (11). The base pair switch in HP is formally carried out by base rotation or base flipping (rotation around the base-sugar bond), either of which requires the expansion of the spaces between neighboring bases or base pairs (see Refs. 7 and 12).Electron microscopic studies have shown that RecA/Rad51 proteins form a well conserved right-handed helical filament around ssDNA or dsDNA (13–15). In the absence of ATP, these proteins assemble as a shorter inactive filament (helical pitch, ≈65–85 Å) (16, 17). In the presence of ATP (or ATPγS, a non-hydrolyzable ATP analogue), the filament adopts an extended active conformation with a helical pitch of ≈95 Å, and the contour length of ssDNA and dsDNA within the active filament is elongated to the same extent (13–15, 17, 18). This equalized elongation has been inferred to widen the spacing between bases of ssDNA and dsDNA equally in the nucleoprotein filament to facilitate the homologous alignment of both DNA substrates to achieve base pair switching (13). Previously, we analyzed the three-dimensional structure of the RecA·ssDNA complex in the presence of ATPγS by NMR, which showed that the axial rise per ssDNA base was extended to nearly 5 Å (19), and that the interconversion of sugar puckers induced horizontal base rotation (20). On the basis of these results, and as there was no other structural information at that time, we proposed a base rotation mechanism to explain the base pair switch in HP by assuming that the ssDNA and dsDNA were extended equally and uniformly (20). Recently, the crystal structure of the RecA·ssDNA complex has revealed a non-uniformly extended structure for the ssDNA (21). The crystal structure contains “a three-nucleotide segment” (triplet) region and “a long untwisted inter-nucleotide” (inter-triplet) region (see Fig. 4). However, it remains unclear which structure contributes to HP and how it does so.Open in a separate windowFIGURE 4.Comparison of the solution and crystal structures of ssDNA bound to RecA. A, superimposition of the solution structure of RecA-d(TACG) (19) (in magenta) and four DNA residues in the RecA₅-(dT)₁₅ crystal structure (21) (in cyan). The crystal structure is similar to the solution structure. D1, D2, and D3 indicate distances between adjacent bases (see also 18, 20). Actually, the dsDNA was shown to be untwisted (unwound) within the homology-independent RecA-ssDNA-dsDNA intermediate of HP described above (22, 23). HP mediated by RecA or Rad51 was shown to be extensively stimulated by negative supercoiling of the dsDNA substrate in vitro (24, 25). This is probably because negative supercoils in the dsDNA substrate would compensate for the positive supercoils generated by the untwisting for HP. Closed circular dsDNA isolated from living cells, including DNA from bacteria, nuclei, or mitochondria of eukaryotic cells, is similarly supercoiled. However, in vivo, the supercoils of cellular dsDNA are relaxed by nucleosome assembly in eukaryotic nuclei and by the binding of HU (26) and/or other DNA-binding proteins in bacteria. Thus, dsDNA in vivo is an unfavorable substrate for HP mediated by RecA/Rad51 family proteins.In mitochondria, which do not have RecA/Rad51 family proteins, negative supercoils are relaxed by the binding of TFAM (in mammals) or Abf2 (in yeast) (27, 28). In this in vivo dsDNA state, Mhr1, an ATP-independent HP protein required for mitochondrial HR in the budding yeast Saccharomyces cerevisiae (29) catalyzes HP without the net untwisting of dsDNA, i.e. Mhr1 catalyzes HP with relaxed closed circular dsDNA with similar efficiency as with dsDNA lacking topological constraints (linear dsDNA and closed circular dsDNA in the presence of a topoisomerase) (30). Furthermore, in contrast to what is observed for RecA/Rad51, Mhr1-catalyzed HP is prevented by negative dsDNA supercoiling. The absence of net untwisting of dsDNA appears at first glance to mean HP without the extension of the parental dsDNA. However, HP requires the expansion of inter-base pair spaces for base pair switching as described above, and thus we proposed that right-handed wrapping of dsDNA around Mhr1 with an extended and untwisted configuration allows base rotation for HP (30). However, it remained to be experimentally determined whether Mhr1 and RecA/Rad51 share a common or different mechanism for HP.In addition to Mhr1, several proteins that promote HP in vitro in the absence of nucleotide cofactors have been identified. These include the human (hs) Xrcc3·Rad51c/Rad51L2 complex (human Rad51 paralogues; 31), hsRad52 (32), Escherichia coli (ec) phage λ β-protein (33), ecRecT (a homologue of λ β-protein 34), ecRecO (35), and Ustilago maydis Brh2 (36). Some of these proteins are termed recombination mediators, but we refer to them as ATP-independent HP proteins for the purpose of this study (supplemental Fig. S1). In contrast to RecA/Rad51 family members, ATP-independent HP proteins, except for those in the Xrcc3·Rad51C complex, do not exhibit any amino acid sequence homology with RecA/Rad51 proteins or other ATP-independent HP proteins. In addition, ATP-independent HP proteins exhibit significantly different quaternary structures (31, 32, 36–40). The N-terminal domain of hsRad52 forms an undecameric ring around which ssDNA and/or dsDNA wrap(s) (32, 41), and the interaction of closed circular dsDNA with hsRad52 generates negative supercoils (41), whereas binding to RecA generates positive supercoils in this substrate. On the other hand, like RecA, RecT was shown to untwist dsDNA during HP (42). The binding of dsDNA to Mhr1 causes neither untwisting nor twisting (30). Thus, the properties of HP proteins vary considerably except for their HP activities.In this study, we questioned whether the extended structure of ssDNA as seen in the RecA·ssDNA complex is conserved among HP proteins, or whether each HP protein uses a different principle to promote HP. If the extended structure is a common determinant of HP, the different HP proteins are likely to use a common mechanism to promote HP, but their variation may reflect requirements for optimizing HP in different cellular environments. Thus, we focused on the structure of the HP protein-bound ssDNA, an HP intermediate. We determined the three-dimensional structures of ssDNA bound to Mhr1 and of three other evolutionarily distinct HP proteins, ecRecT (ATP-independent, from the λ-like cryptic prophage Rac of E. coli, involved in plasmid HR (43)), Thermus thermophilus (tt) RecO (ATP-independent HP protein in bacteria), and hsRad51 (ATP-dependent, human nuclear homologue of RecA) and compared them with ssDNA bound to ecRecA (E. coli, the prototype of the RecA family; supplemental Fig. S1). This is the first demonstration that diverse HP proteins, both ATP-dependent (RecA/Rad51) and ATP-independent (Mhr1, RecO, RecT), use the non-canonical extended DNA structure as a common intermediate for HP, and this suggests that they use a common mechanism for HP.     

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca²⁺. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca²⁺-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca²⁺-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca²⁺ and valinomycin on the inner and outer mitochondrial membranes are discussed.Mitochondria are well known as the organelle for energy conversion in all eukaryotes. This energy conversion, i.e. ATP synthesis, is performed by using the electrochemical gradient of H⁺ across the inner mitochondrial membrane. To enable effective energy conversion, the mitochondrial inner membrane is highly resistant to the permeation of solutes and ions. However, under certain conditions, such as in the presence of Ca²⁺ and inorganic phosphate, the permeability of this inner membrane is known to be markedly increased. This phenomenon is referred to as the permeability transition (PT)¹ and is believed to result from the formation of a proteinaceous pore, referred to as the PT pore, which makes the inner membrane permeable to various solutes and ions smaller than 1.5 kDa (1–3). The physiological importance of the PT has long been uncertain; however, recent studies have revealed that the changes in the permeability of the inner mitochondrial membrane due to the induction of PT cause the release of cytochrome c into the cytosol and that the released cytochrome c then triggers subsequent steps of programmed cell death, which is known as apoptosis (4–6). Thus, the PT is considered to be one of the major regulatory steps of apoptosis. However, the questions as to how the PT is induced and how cytochrome c is released accompanied by the induction of PT have remained unanswered.To characterize the features of the mitochondrial PT and to understand the mechanism underlying the release of cytochrome c from mitochondria, investigators have studied the effects of various agents on this organelle. As a result, the PT and the release of cytochrome c were found to be induced not only by Ca²⁺ but also by other agents (7–9). We also found that copper-o-phenanthroline (10), metal ions (11), and cyanine dyes (12, 13) induced this PT and the release of cytochrome c from mitochondria. Furthermore we reported that valinomycin, known as a potassium-selective ionophore, also induces the release of cytochrome c from mitochondria but without the induction of PT (14). This finding indicated that cytochrome c could be released from mitochondria in two different manners: one with the induction of PT and the other without it. To understand how cytochrome c is released from mitochondria, it is very important to know what protein species are released from mitochondria concomitant with the release of cytochrome c. To address these questions, in the present study we used a mass spectrometry (LC-MS/MS system)-based proteome analysis approach, which allowed us to identify the protein species present in a limited amount of protein samples. Using proteomics techniques, we examined the protein species released from mitochondria treated with valinomycin or with Ca²⁺, and we discuss our findings on the status of inner and outer mitochondrial membranes treated with these agents.     

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-ΔC) was solved in an oligomeric state. The molecular structure of BMRF1-ΔC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-ΔC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-ΔC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-ΔC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-ΔC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-ΔC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.