H2a-specific proteolysis as a unique probe in the analysis of the histone octamer

We have utilized the H2a-specific protease as a unique probe to investigate the nature of the interactions between the protein subunits which form the core histone octamer. Upon incubation in high ionic strength media this protease, normally found tightly associated with isolated calf thymus chromatin, releases the 15 COOH-terminal amino acids of histone H2a by specifically cleaving the H2a polypeptide between Val114 and Leu115, yielding cleaved H2a (cH2a) and a free pentadecapeptide (Eickbush, T. H., Watson, D. K., and Moudrianakis, E. N. (1976) Cell 9, 785-792). We find that removal of this pentadecapeptide results in a marked dissociation of the octamer into its H2a:H2b dimer and H3:H4 tetramer subunits. Reconstitution experiments indicate that cH2a is capable of forming a dimer with H2b, but this cH2a:H2b dimer has a substantially lower affinity for the H3:H4 tetramer than native H2a:H2b dimer. Kinetic studies of H2a cleavage in high ionic strength solutions demonstrate that H2a molecules in the octamer are relatively resistant to proteolytic attack compared to H2a molecules in the dimer. The extent of this resistance, in response to various experimental parameters, is directly correlated to the strength of interaction between the H2a:H2b dimer and H3:H4 tetramer subunits. These reconstitution and kinetic experiments suggest that the histone domains proximal to the H2a cleavage site have an important function in maintaining the association of the histone octamer subunits.     

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.e., a twofold higher apparent Ca(2+) affinity, but twofold lower maximal turnover rate) can explain these compensatory changes, we simulated Ca(2+) dynamics in mouse ventricular myocytes. The model shows that the relative Ca(2+) transport capacity of SERCA2a and SERCA2b depends on the SERCA concentration. The simulations point to a dominant effect of SERCA2b's higher Ca(2+) affinity over its lower maximal turnover rate. The results suggest that increased systolic and decreased diastolic Ca(2+) levels in unstimulated conditions could contribute to the downregulation of SERCA in SERCA2(b/b) mice. In stress conditions, Ca(2+) handling is less efficient by SERCA2b than by SERCA2a, which might contribute to the observed hypertrophy in SERCA2(b/b) mice. Altogether, SERCA2a might be a better compromise between performance in basal conditions and performance during β-adrenergic stress.     

Expression of preproinsulin-2 gene shapes the immune response to preproinsulin in normal mice

Deciphering mechanisms involved in failure of self tolerance to preproinsulin-2 is a key issue in type 1 diabetes. We used nonautoimmune 129SV/Pas mice lacking preproinsulin-2 to study the immune response to preproinsulin-2. In these mice, a T cell response was detected after immunization with several preproinsulin-2 peptides and confirmed by generating hybridomas. Activation of some of these hybridomas by wild-type (wt) islet cells or recombinant murine proinsulin-2 demonstrated that two epitopes can be generated from the naturally expressed protein. Although T cells from wt mice responded to preproinsulin-2 peptides, we could not detect a response to the naturally processed epitopes in these mice. Moreover, after immunization with recombinant whole proinsulin-2, a T cell response was detected in preproinsulin-2-deficient but not in wt mice. This suggests that islet preproinsulin-2-autoreactive T cells are functionally eliminated in wt mice. We used a transplantation model to evaluate the relevance of reactivity to preproinsulin-2 in vivo. Wild-type preproinsulin-2-expressing islets transplanted in preproinsulin-2-deficient mice elicited a mononuclear cell infiltration and insulin Abs. Graft infiltration was further increased by immunization with preproinsulin-2 peptides. Preproinsulin-2 expression thus shapes the immune response and prevents self reactivity to the islet. Moreover, islet preproinsulin-2 primes an immune response to preproinsulin-2 in deficient mice.     

Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze ?1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.     

Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.     

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.     

In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts

Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL-P2, HDL-P2 or A-P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10-20 micrograms/ml) by LDL-P2 than by HDL-P2 or A-P2. Moreover, P2 delivery to cells by LDL-P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL-P2 or A-P2 is a linear process. Finally, reduction of the LDL-receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL-P2. These results suggest a special role of the LDL-receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication.

Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a.     

Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I

Synaptotagmin I (syt), a transmembrane protein localized to secretory vesicles, functions as a Ca2+ sensor that facilitates SNARE-mediated membrane fusion. The cytoplasmic domain of syt harbors two C2-domains designated C2A and C2B. Upon binding Ca2+, C2A and C2B partially penetrate into membranes that contain anionic phospholipids. However, it is unknown whether these tandem C2-domains engage membranes at the same time, in a sequential manner, or in a mutually exclusive manner. We have used site-directed fluorescent probes to monitor the penetration of syt's C2-domains into phosphatidylserine-harboring lipid bilayers. We report that, in response to Ca2+, C2A and C2B copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more efficient when synthetic rather than natural phospholipids were used to prepare bilayers. The membrane penetration activity of the intact cytoplasmic domain of syt (C2A-C2B) exhibits significant resistance to changes in ionic strength. In contrast, the ability of isolated C2B to bind membranes in response to Ca2+ can be disrupted by subtle changes in ionic strength. Tethering C2B to a mutant version of C2A that does not bind Ca2+ or membranes significantly increases the stability of Ca2+.C2B.membrane complexes, confirming that C2A affects the membrane-binding properties of the adjacent C2B domain.     

Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha in catalyzed nucleotide exchange

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2.GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The alpha-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2betagamma) completely devoid of the alpha-subunit to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude, with a smaller increase in V(max). These data indicate that eIF2alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the alpha-subunit to control the rate of nucleotide exchange through reversible phosphorylation.     

DOC-2/DAB2 is the binding partner of myosin VI

Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells. Here we found that DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain. DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain. The C-terminal 122 amino acid residues of DOC-2/DAB2, containing the Grb2 binding site, is identified to be critical for the binding to myosin VI. Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule. The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site. The present findings suggest that myosin VI plays a role in transporting DOC-2/DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways.     

Interaction among Btn1p, Btn2p, and Ist2p reveals potential interplay among the vacuole, amino acid levels, and ion homeostasis in the yeast Saccharomyces cerevisiae

Btn2p, a novel cytosolic coiled-coil protein in Saccharomyces cerevisiae, was previously shown to interact with and to be necessary for the correct localization of Rhb1p, a regulator of arginine uptake, and Yif1p, a Golgi protein. We now report the biochemical and physical interactions of Btn2p with Ist2p, a plasma membrane protein that is thought to have a function in salt tolerance. A deletion in Btn2p (btn2Delta strains) results in a failure to correctly localize Ist2p, and strains lacking Btn2p and Ist2p (btn2Delta ist2Delta strains) are unable to grow in the presence of 0.5 or 1.0 M NaCl. Btn2p was originally identified as being up-regulated in a btn1Delta strain, which lacks the vacuolar-lysosomal membrane protein, Btn1p, and serves as a model for Batten disease. This up-regulation of Btn2p was shown to contribute to the maintenance of a stable vacuolar pH in the btn1Delta strain. Btn1p was subsequently shown to be required for the optimal transport of arginine into the vacuole. Interestingly, btn1Delta ist2Delta strains are also unable to grow in the presence of 0.5 or 1.0 M NaCl, and ist2Delta suppresses the vacuolar arginine transport defect in btn1Delta strains. Although further investigation is required, we speculate that altered vacuolar arginine transport in btn1Delta strains represents a mechanism for maintaining or balancing cellular ion homeostasis. Btn2p interacts with at least three proteins that are seemingly involved in different biological functions in different subcellular locations. Due to these multiple interactions, we conclude that Btn2p may play a regulatory role across the cell in response to alterations in the intracellular environment that may be caused by changes in amino acid levels or pH, a disruption in protein trafficking, or imbalances in ion homeostasis resulting from either genetic or environmental manipulation.     

Mechanisms of cellular resistance to hydrogen peroxide, hyperoxia, and 4-hydroxy-2-nonenal toxicity: the significance of increased catalase activity in H2O2-resistant fibroblasts.

An H2O2-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line which demonstrates a 20-fold increase in catalase activity was utilized in the study of mechanisms responsible for cellular resistance to hydrogen peroxide, oxygen, and 4-hydroxy-2-nonenal toxicity. HA1 and OC14 cells were treated with 9 mM aminotriazole which resulted in a 60 to 80% reduction in catalase activity. Pretreatment with aminotriazole resulted in significant sensitization to the toxicity of 1-h exposures to exogenously applied H2O2, which was proportional to the reduction in catalase activity. Treatment with aminotriazole produced significant sensitization to the toxicity of 95% O2 after 45 h of O2 exposure but no sensitization to the toxicity of a 1-h exposure to 50 microM 4-hydroxy-2-nonenal. Inhibition of catalase activity by aminotriazole had no effect on the metabolism of 4-hydroxy-2-nonenal by either cell line tested. These results support the conclusion that in H2O2-resistant cells, catalase activity is a major determinant of cellular resistance to H2O2 toxicity, whereas catalase activity has a limited role in cellular resistance to an acute exposure to 95% O2 and is unrelated to cellular resistance to 4-hydroxy-2-nonenal.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.     

Suppression of rat Frizzled-2 attenuates hypoxia/reoxygenation-induced Ca2+ accumulation in rat H9c2 cells

Growing evidence suggests that Ca(2+) overload is one of the major contributors of myocardial ischemia/reperfusion-induced injury. Since Frizzled-2 receptor, a seven transmembrane protein, transduces downstream signaling by specialized binding of Wnt5a to increase intracellular Ca(2+) release, this work aimed to investigate the effect of Frizzled-2 on Ca(2+) accumulation in H9c2 cells, which were subjected to hypoxia/reoxygenation to mimic myocardial ischemia/reperfusion. After exposing H9c2 cells to hypoxia/reoxygenation, we observed higher expression of Frizzled-2 and Wnt5a as compared to control group cells. Hypoxia/reoxygenation-induced intracellular Ca(2+) accumulation approached that of cells transfected with frizzled-2 plasmid. In cells treated with RNAi specifically designed against frizzled-2, intracellular Ca(2+) in both hypoxia/reoxygenation-treated cells and plasmid-treated cells were decreased. Rats that underwent ischemia/reperfusion injury exhibited increased intracellular Ca(2+) with high expression levels of Frizzled-2 and Wnt5a as compared to the sham group. Our data indicates that upon binding to Wnt5a, increased Frizzled-2 expression after hypoxia/reoxygenation treatment activated intracellular calcium release in H9c2 cells. Our findings provide a new perspective in understanding calcium overload in myocardial ischemia/reperfusion.