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Biomedical subjects

A Kihara

Publications and source records attributed to A Kihara.

At least 19 recordsLinked to original sources
PubMed1999

Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition.

The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4. 1.1.31) has been determined by x-ray diffraction methods at 2.8-A resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a "dimer-of-dimers" form with respect to subunit contact, resulting in an overall square arrangement. The contents of alpha-helices and beta-strands are 65% and 5%, respectively. All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the beta-barrel. The binding site for L-aspartate is located about 20 A away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding. The participation of Arg-587 is unexpected, because it is known to be catalytically essential. Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site. There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure. The importance of this loop in catalytic activity was also shown. Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate.

Allosteric Regulation↗
PubMed1998

Translocation, folding, and stability of the HflKC complex with signal anchor topogenic sequences.

HflK and HflC are plasma membrane proteins of Escherichia coli, each having a large C-terminal domain exposed to the periplasmic space and an N-terminally located transmembrane segment, which should act as a signal anchor sequence for their biogenesis. They form a complex, HflKC. We studied in vivo processes of biogenesis of this pair of membrane proteins. Translocation of the C-terminal domains across the membrane, as assessed by their accessibility to externally added protease, was completed within 1 min after the synthesis in wild-type cells as well as in the secB mutant cells or in the FtsY-depleted cells. In contrast, translocation of these domains was retarded markedly when sodium azide was added to inhibit SecA ATPase and blocked almost completely in secY- or secD-defective mutant cells. Thus, although targeting of these membrane proteins depends neither on the SecB chaperone nor on the SRP pathway, their translocation occurs exclusively via the Sec translocase complex. Translocated HflK molecules were then folded into a partially protease-resistant conformation, taking a few minutes, and this folding was induced upon association with HflC. Singly expressed HflK and HflC were unstable in vivo and periplasmic proteases DegP and Prc were involved in the degradation of the HflK subunit. We characterized several hflA alleles isolated in early studies; they alter the HflK or the HflC sequence and destabilize the HflKC complex.

Amino Acid Sequence↗
PubMed1998

Roles of the periplasmic domain of Escherichia coli FtsH (HflB) in protein interactions and activity modulation.

FtsH is a membrane-bound and ATP-dependent protease of Escherichia coli, known to degrade SecY, a membrane protein for protein translocation, and CII, a soluble transcription factor for lysis/lysogeny decision of phage lambda. FtsH forms a homo-oligomeric complex as well as a hetero-oligomeric complex with HflKC, a putative modulator of FtsH. Although FtsH has a small periplasmic region, HflKC is mostly exposed to the periplasmic space. We studied the roles of the periplasmic region of FtsH by engineering mutations in this protein. FtsHDelta236, lacking most of the periplasmic region, retained the in vivo ability to degrade SecY but not CII, resulting in high frequency lysogenization of lambda. Several insertion mutations in the periplasmic region of FtsH also differentially affected the proteolytic activities of FtsH. Interestingly, purified and detergent-solubilized FtsHDelta236 was as active as the wild-type enzyme in degrading SecY and CII, although its ATPase activity was lowered 5-fold. Affinity chromatography using histidine-tagged derivatives showed that the periplasmic domain-deleted FtsH no longer interacted with FtsH or HflKC. Although FtsHDelta236-His6-Myc lost the static FtsH-FtsH interaction, it retained the ability to change its conformation in an ATP-dependent manner at 37 degreesC, leading to a limited oligomerization. These results suggest that the periplasmic region of FtsH has crucial roles in the protein-protein interactions of this complex and in the modulation of its proteolytic functions against different substrates.

ATP-Dependent Proteases↗
PubMed1998

Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: an implication from the interference by a mutant form of a new substrate protein, YccA.

Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here, we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein. From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.

ATP-Dependent Proteases↗
PubMed1998

Polypeptide binding of Escherichia coli FtsH (HflB).

The Escherichia coli FtsH protein is a membrane-bound and ATP-dependent protease. In this study, we describe ATP-dependent conformational changes in FtsH as well as a polypeptide binding ability of this protein. A 33 kDa segment of FtsH became trypsin resistant in the presence of ATP. ATP and ATPgammaS prevented self-aggregation of detergent-solubilized FtsH-His6-Myc at 37 degrees C, again suggesting that the binding of ATP induces a conformational change in FtsH. Affinity chromatography showed that FtsH-His6-Myc can associate with denatured alkaline phosphatase (PhoA) but not with the native enzyme. Denatured PhoA also prevented the aggregation of FtsH, and these two proteins co-sedimented through a sucrose gradient. Binding between FtsH-His6-Myc and detergent-solubilized SecY was also demonstrated. Although FtsH-bound SecY was processed further for ATP-dependent proteolysis, FtsH-bound PhoA was not. Thus, FtsH association with denatured PhoA is uncoupled from proteolysis. Overproduction of FtsH significantly increased the cytoplasmic localization of the PhoA moiety of a MalF-PhoA hybrid protein, in which a charged residue had been introduced into a transmembrane segment. Thus, denatured PhoA binding of FtsH may also occur in vivo.

ATP-Binding Cassette Transporters↗
PubMed1998

Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo.

Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.

Animals↗
PubMed1997

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

ATP-Dependent Proteases↗
PubMed1996

Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli.

Escherichia coli FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase. We now report that subunit a of the membrane-embedded Fo part of H+-ATPase is another substrate of FtsH. Pulse-chase experiments showed that subunit a is unstable when it alone (without Fo subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants. Selective and ATP-dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro. These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane.

ATP-Dependent Proteases↗
PubMed1996

FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins.

The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein). We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH. We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini. Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation. These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.

ATP-Dependent Proteases↗
PubMed1996

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY.

Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

ATP-Dependent Proteases↗
PubMed1995

Intracellular stability of alpha fragments of beta-galactosidase: effects of amino-terminally fused polypeptides.

Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.

Amino Acid Sequence↗
PubMed1995

FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit.

When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY.

ATP-Dependent Proteases↗
PubMed1995

Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli.

A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

ATP-Binding Cassette Transporters↗
PubMed1995

Cytotoxic activity of chimeric toxins containing the epidermal growth factor-like domain of heregulins fused to PE38KDEL, a truncated recombinant form of Pseudomonas exotoxin.

The EGF-like domains of heregulin alpha, beta 1, beta 2, and beta 3 were fused to a truncated form of Pseudomonas exotoxin (PE38KDEL), which contains a modified carboxyl-terminal sequence, KDEL, that increases that toxin activity. The resulting chimeric toxins were produced in Escherichia coli, purified to near homogeneity, and shown to be cytotoxic to target cells with very high activity on HTB20, N-87 MCF-7, and HepG2 cells; high activity on A431 and MDA-MB468 cells; and low activity toward SK-OV3, L929, and KB cells. The fact that cytotoxicity did not correlate with the levels of erbB2 expression indicated that another receptor in the erb family might be involved. Accordingly, cytotoxicity assays were performed on NIH/3T3 cell lines transfected with EGFR, ErbB2, ErbB3, or ErbB4. The results indicate that the heregulin toxins target ErbB4 or possibly ErbB3 but not ErbB2.

3T3 Cells↗
PubMed1994

Small chimeric toxins containing only transforming growth factor alpha and domain III of Pseudomonas exotoxin with good antitumor activity in mice.

Chimeric toxins composed of transforming growth factor alpha (TGF alpha) fused to mutant forms of Pseudomonas exotoxin (PE) bind to the epidermal growth factor receptor and kill cells bearing epidermal growth factor receptors. Initially, the binding domain (Ia; amino acids 1-252) of PE was deleted and replaced with TGF alpha to make TGF alpha-PE40 in which TGF alpha is fused to domains II, Ib, and III of PE (amino acids 253-613). That drug is currently undergoing clinical study for the intravesical therapy of bladder cancer. To generate smaller molecules that would have increased tumor penetration, several deletion mutants were constructed. In one of these, TGF alpha was inserted near the carboxyl terminus of PE, and residues in domains II and Ib of PE (amino acids 253-279 and 365-380) were deleted so that the chimeric toxin did not need to be cleaved by an intracellular protease to be activated (Theuer et al., J. Biol. Chem., 267: 16872-16877, 1992). We have now constructed chimeric toxins which contain only domain III, yet still exhibit high cytotoxic activity on epidermal growth factor receptor-containing cells and produce substantial tumor regressions in mice bearing a human xenograft. The high cytotoxic activity of these severely truncated toxins provides new insights on the proposed functions of domains II and III of PE.

Adenosine Diphosphate Ribose↗