Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426 ), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.     

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells

Complement is the canonical innate immune system involved in host defense and tissue repair with the clearance of cell debris. In contrast to the robust armory mounted against microbial nonself-pathogens, complement is selectively activated on altered self (i.e. apoptotic and necrotic cells) to instruct the safe demise by poorly characterized mechanisms. Our data shed new light on the role of complement C1q in sensing nucleic acids (NA) rapidly exposed on apoptotic Jurkat T cell membranes and in driving C3 opsonization but without the lytic membrane attack complex. DNA/RNase-treated apoptotic cells failed to activate complement. We found that several other apoptotic cell models, including senescent keratinocytes, ionophore-treated sperm cells, and CMK-derived platelets, stained for cleaved caspase 3 were rapidly losing the key complement regulator CD46. CD46 from nuclear and membrane stores was found to cluster into blebs and shed into microparticles together with NA, phosphatidylserine, C1q, and factor H. Classical and alternative pathways of complement were involved in the recognition of H2O2-treated necrotic cells. Membrane attack complex was detected on necrotic cells possibly as a result of CD46 and CD59 shedding into soluble forms. Our data highlight a novel and universal paradigm whereby the complement innate immune system is using two synergistic strategies with the recognition of altered self-NA and missing self-CD46 signals to instruct and tailor the efficient removal of apoptotic and necrotic cells in immunoprivileged sites.     

Calcium plays a central role in the sensitization of TRPV3 channel to repetitive stimulations

Transient receptor potential channels are involved in sensing chemical and physical changes inside and outside of cells. TRPV3 is highly expressed in skin keratinocytes, where it forms a nonselective cation channel activated by hot temperatures in the innocuous and noxious range. The channel has also been implicated in flavor sensation in oral and nasal cavities as well as being a molecular target of some allergens and skin sensitizers. TRPV3 is unique in that its activity is sensitized upon repetitive stimulations. Here we investigated the role of calcium ions in the sensitization of TRPV3 to repetitive stimulations. We show that the sensitization is accompanied by a decrease of Ca(2+)-dependent channel inhibition mediated by calmodulin acting at an N-terminal site (amino acids 108-130) and by an acidic residue (Asp(641)) at the pore loop of TRPV3. These sites also contribute to the voltage dependence of TRPV3. During sensitization, the channel displayed a gradual shift of the voltage dependence to more negative potentials as well as uncoupling from voltage sensing. The initial response to ligand stimulation was increased and sensitization to repetitive stimulations was decreased by increasing the intracellular Ca(2+)-buffering strength, inhibiting calmodulin, or disrupting the calmodulin-binding site. Mutation of Asp(641) to Asn abolished the high affinity extracellular Ca(2+)-mediated inhibition and greatly facilitated the activation of TRPV3. We conclude that Ca(2+) inhibits TRPV3 from both the extracellular and intracellular sides. The inhibition is sequentially reduced, appearing as sensitization to repetitive stimulations.     

Glucose 6-phosphate plays a central role in the activation of glycogen synthase by glucose in hepatocytes

The state of activation of glycogen synthase enhanced by glucose, other sugars and gluconeogenic precursors shows a strong positive correlation with the intracellular concentrations of glucose 6-P when ATP concentrations remain constant. The concentrations of glucose 6-P achieved upon incubation of hepatocytes with glucose plus mannoheptulose, an inhibitor of glucokinase and hexokinase, were lower than those found when the incubation was carried out with glucose alone. Under these conditions, in keeping with the decrease in glucose 6-P, the activation of glycogen synthase by glucose was also impaired. On the other hand the inactivation of glycogen phosphorylase was not altered in the presence of mannoheptulose.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Peptidylarginine deiminase (PAD) is a mouse cortical granule protein that plays a role in preimplantation embryonic development

Background  

While mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa cortical granule protein (p75) present in a subpopulation of mouse cortical granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.     

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.     

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM d-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 μM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123±10 μg/10⁶ cells (n=5) to 3.9±1.9 μg/10⁶ cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.     

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3^fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3^−/− cells. Notably, Ada3^−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3^fl/fl and Ada3^−/− cells confirmed higher residual DNA damage in Ada3^−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability.     

Alteration/deficiency in activation-3 (Ada3) plays a critical role in maintaining genomic stability

Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3^fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3^?/? cells. Notably, Ada3^?/? cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G? or G? phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3^fl/fl and Ada3^?/? cells confirmed higher residual DNA damage in Ada3^?/? cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability.     

Antistasin, an inhibitor of coagulation and metastasis, binds to sulfatide (Gal(3-SO4) beta 1-1Cer) and has a sequence homology with other proteins that bind sulfated glycoconjugates

Antistasin, a 15-kDa salivary protein from the Mexican leech Haementeria officinalis, inhibits both blood coagulation and the metastasis of tumors (Tuszynski, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. Chem. 262, 9718-9723). Antistasin binds to heparin-agarose, suggesting the protein interacts with sulfated glycoconjugates. The specificity of the interaction between antistasin and heparin was tested by measuring the binding of antistasin to various lipids and by comparing the ability of several charged glycoconjugates to inhibit binding. Of the lipids tested, antistasin binds with high affinity only to sulfatide (Gal(3-SO4)beta 1-1Cer) and does not bind to comparable levels of phospholipids, neutral glycosphingolipids, gangliosides, or cholesterol-3-SO4. The binding of antistasin to sulfatide is inhibited by dextran sulfate, fucoidan, and heparin, with I50 values of 1.5, 9.2, and 16 micrograms/ml, respectively. Comparable levels of chondroitin sulfates A, B, C, keratan sulfate, or hyaluronic acid do not inhibit binding. Comparisons of the amino acid sequences of antistasin and other sulfatide or heparin-binding proteins revealed a region of homology, based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg, which may be a sulfated glycoconjugate binding domain. In addition, homologies were found with the alternate complement pathway protein properdin and coat proteins from malaria circumsporozoites and Herpes simplex I.     

Cytoplasmic ribosomal protein S3 (rpS3) plays a pivotal role in mitochondrial DNA damage surveillance

Ribosomal protein S3 (rpS3) is known to play critical roles in ribosome biogenesis and DNA repair. When cellular ROS levels increase, the mitochondrial genes are highly vulnerable to DNA damage. Increased ROS induces rpS3 accumulation in the mitochondria for DNA repair while significantly decreasing the cellular protein synthesis. For the entrance into the mitochondria, the accumulation of rpS3 was regulated by interaction with HSP90, HSP70, and TOM70. Pretreatment with geldanamycin, which binds to the ATP pocket of HSP90, significantly decreased the interaction of rpS3 with HSP90 and stimulated the accumulation of rpS3 in the mitochondria. Furthermore, cellular ROS was decreased and mtDNA damage was rescued when levels of rpS3 were increased in the mitochondria. Therefore, we concluded that when mitochondrial DNA damages accumulate due to increased levels of ROS, rpS3 accumulates in the mitochondria to repair damaged DNA due to the decreased interaction between rpS3 and HSP90 in the cytosol.     

Akt plays a central role in the anti-apoptotic effect of estrogen in endothelial cells

Estrogen has been reported to inhibit apoptosis in vascular endothelial cells. However, its precise mechanism still remains to be elucidated. Here we determined the role of Akt in the anti-apoptotic effect of estrogen. 17Beta-estradiol prevented the apoptosis induced by TNF-alpha in bovine aortic endothelial cells, as evaluated by double staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Introducing a dominant negative mutant of Akt by using a cell-penetrating peptide of Tat protein inhibited the anti-apoptotic effect of estrogen in a concentration-dependent manner, and resulted in the complete inhibition of the anti-apoptotic effect of 17beta-estradiol at 1nM and higher concentrations. The dominant negative mutant without the cell-penetrating peptide and Tat peptide-conjugated protein A had no effect. The intracellular protein transduction was confirmed by immunoblot analysis. Our observations thus provide first direct evidence that Akt plays a central role in the anti-apoptotic effect of estrogen in vascular endothelial cells.