Mitsunobu dehydration of N-Boc neomycin B.
Reaction of hexa-N-Boc neomycin B with TPP and DIAD in toluene results in the formation of an epoxide in ring IV, not an aziridine or azetidine as previously reported.
Epoxy Compounds↗
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Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells.
Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.
Animals↗
Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes.
Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity.
Animals↗
Active transport of L-proline in the protozoan parasite Trypanosoma brucei brucei.
The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
Adenosine Triphosphate↗
CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis.
The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.
Bacillus subtilis↗
Upregulation of cytosolic chaperonin CCT subunits during recovery from chemical stress that causes accumulation of unfolded proteins.
The chaperonin containing TCP-1 (CCT) is a molecular chaperone consisting of eight subunit species and assists in the folding of actin, tubulin and some other cytosolic proteins. We examined the stress response of CCT subunit proteins in mammalian cultured cells using chemical stressors that cause accumulation of unfolded proteins. Levels of CCT subunit proteins in HeLa cells were coordinately and transiently upregulated under continuous chemical stress with sodium arsenite. CCT subunit levels in several mammalian cell lines were also upregulated during recovery from chemical stress caused by sodium arsenite or a proline analogue, L-azetidine-2-carboxylic acid. Several unidentified proteins that were newly synthesized and associated with CCT were found to increase concomitantly with CCT subunits themselves and known substrates during recovery from the stress. These results suggest that CCT plays important roles in the recovery of cells from protein damage by assisting in the folding of proteins that are actively synthesized and/or renatured during this period.
Animals↗
Role of the yeast acetyltransferase Mpr1 in oxidative stress: regulation of oxygen reactive species caused by a toxic proline catabolism intermediate.
The MPR1 gene, which is found in the Sigma1278b strain but is not present in the sequenced laboratory strain S288C, of the budding yeast Saccharomyces cerevisiae encodes a previously uncharacterized N-acetyltransferase that detoxifies the proline analogue azetidine-2-carboxylate (AZC). However, it is unlikely that AZC is a natural substrate of Mpr1 because AZC is found only in some plant species. In our search for the physiological function of Mpr1, we found that mpr1-disrupted cells were hypersensitive to oxidative stresses and contained increased levels of reactive oxygen species (ROS). In contrast, overexpression of MPR1 leads to an increase in cell viability and a decrease in ROS level after oxidative treatments. These results indicate that Mpr1 can reduce intracellular oxidation levels. Because put2-disrupted yeast cells lacking Delta(1)-pyrroline-5-carboxylate (P5C) dehydrogenase have increased ROS, we examined the role of Mpr1 in put2-disrupted strains. When grown on media containing urea and proline as the nitrogen source, put2-disrupted cells did not grow as well as WT cells and accumulated intracellular levels of P5C that were first detected in yeast cells and ROS. On the other hand, put2-disrupted cells that overexpressed MPR1 had considerably lower ROS levels. In vitro studies with bacterially expressed Mpr1 demonstrated that Mpr1 can acetylate P5C, or, more likely, its equilibrium compound glutamate-gamma-semialdehyde, at neutral pH. These results suggest that the proline catabolism intermediate P5C is toxic to yeast cells because of the formation of ROS, and Mpr1 regulates the ROS level under P5C-induced oxidative stress.
Acetylation↗
An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants.
Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the Arabidopsis genome identified AtbZIP60, which was induced by tunicamycin. AtbZIP60 encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60 delta C) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane. AtbZIP60 was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60 delta C clearly activated any of three BiP and two calnexin promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60 delta C also appeared to induce the expression of AtbZIP60 through an ER stress-response element-like sequence in the promoter of AtbZIP60. These characteristics of AtbZIP60 imply a signal transduction pathway of the ER stress response unique to plants.
Amino Acid Sequence↗
Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomycescerevisiae.
Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3-2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3-1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.
Azetidinecarboxylic Acid↗
A nonconserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins.
A toxic l-proline analogue, l-azetidine-2-carboxylic acid (AZC), causes misfolding of the proteins into which it is incorporated competitively with l-proline, thereby inhibiting the growth of the cells. AZC enters budding yeast Saccharomyces cerevisiae cells primarily through the general amino acid permease Gap1, not through the proline-specific permease Put4. We isolated an AZC-hypersensitive mutant that cannot grow even at low concentrations of AZC because of the accumulation of intracellular AZC. By screening through a yeast genomic library, the mutant was found to carry an allele of RSP5 encoding an E3 ubiquitin ligase. A single amino acid change replacing Ala (GCA) at position 401 with Glu (GAA) showed that Ala-401 in the third WW domain (a protein interaction module) is not conserved in the domain. The addition of NH4+ to yeast cells growing on l-proline induced rapid ubiquitination, endocytosis, and vacuolar degradation of the plasma membrane protein Gap1. However, immunoblot and permease assays indicated that Gap1 in the rsp5 mutant remained stable and active on the plasma membrane probably with no ubiquitination, leading to AZC accumulation and hypersensitivity. The rsp5 mutants also showed hypersensitivity to various stresses (toxic amino acid analogues, high temperature in a rich medium, and oxidative treatments) and defects in spore growth. These results suggest that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogen-regulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.
Alanine↗
Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides.
It was previously shown that virus-specific polypeptides made in HEp-2 cells infected with herpes simplex 1 form three groups designated alpha, beta, and gamma whose synthesis is coordinately regulated and sequentially ordered. This report shows that one or more functional alpha polypeptides are necessary to turn on the synthesis of beta and gamma groups, and conversely, one or more polypeptides in the latter groups turn off the synthesis of alpha polypeptides. Specifically, infected cells maintained in medium containing either canavanine, an analogue of arginine, or azetidine-2-carboxylic acid an analogue of proline and hydroxyproline, synthesized alpha polypeptide at rates comparable to maximal rates in untreated infected cells but did not undergo the normal transition to beta and gamma polypeptide synthesis. The transition to gamma polypeptide synthesis and shut-off of synthesis of earlier polypeptide groups proceeded normally if addition of canavanine was delayed until at least 4-5 hr after infection. Addition of canavanine after the onset of beta and gamma polypeptide synthesis, i.e., between 2 and 3.5 hr after infection, resulted in sustained, simultaneous synthesis of all three polypeptide groups, a phenomenon not seen in untreated infected cells. Canavanine-treated infected cells, synthesizing alpha polypeptides, recovered the capacity to make beta and gamma polypeptides after removal of the analogue, but only after a 1-to 2-hr delay compared with infected untreated cells. The data indicate that the on and off controls inherent in the cascade regulation of viral polypeptide synthesis are mediated by one or more polypeptides in each group at transcriptional or post-transcriptional levels.
Azetidinecarboxylic Acid↗
Experimental control of the differentiation of Leydig cells in the rat fetal testis.
In the developing fetal testis, in vitro as well as in vivo, two kinds of endocrine cells differentiate successively: Sertoli cells, which produce the Müllerian inhibitor (or anti-Müllerian hormone) and aggregate with germ cells into seminiferous cords; and Leydig cells, which release androgens. Serum added to the synthetic culture medium prevents the morphogenesis of the seminiferous cords but not the cytodifferentiation of the endocrine cells. L-Azetidine 2-carboxylic acid (LACA), a proline competitor, introduced into the medium also prevents differentiation of seminiferous cords. In the present experiments, the effects of LACA on the endocrine cells were studied. It did not suppress production of the Müllerian inhibitor, but it opposed differentiation of Leydig cells. Histochemically detectable 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was virtually absent and the release of testosterone, delta 4-androstenedione, 17-hydroxyprogesterone, or progesterone into the medium became undetectable. Moreover, dibutyryl cAMP added to the medium during the final day in vitro had very little effect on the parameters of steroidogenesis. An excess of proline added to the LACA-containing medium permitted normal morphogenesis of seminiferous cords, normal steroidogenesis, and normal response to cAMP. LACA did not prevent the appearance of 3 beta-HSD activity in the adrenals, nor did it reduce the expression of laminin and fibronectin (data not shown) in the mesonephric structures as much as in the testes. The differentiation of the testis and especially of the Leydig cells appears to have special requirements for proline.
17-alpha-Hydroxyprogesterone↗
Chaperone function of a Grp 94-related protein for folding and transport of the pancreatic bile salt-dependent lipase.
In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion. This route was inhibited by drugs that affect protein synthesis and intracellular transport. In the presence of monensin, BSDL was solely detected in microsome membrane fractions. The association of BSDL with intracellular membranes involved a protein complex, formed by at least two proteins of 94 and 56 kDa. In cells experiencing the metabolic stress due to azetidine-2-carboxylic acid, BSDL was additionally associated with a protein of 46 kDa. Affinity blotting showed that BSDL bound directly to the 94-kDa protein (p94). It was suggested that p94 could be a molecular chaperone, further identified as related to the 94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL (i.e. BSDL bound to the Grp 94-related p94) was O- and N-glycosylated and consequently appeared released from membranes in the trans-Golgi compartment. Therefore and for the first time, it is suggested that a multiprotein complex including the chaperone Grp 94-related p94 protein may play an essential role in the folding and transport of BSDL. One hypothesis is that the association of BSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein.
Animals↗
AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1.
Interleukin-1 (IL-1) -alpha and -beta are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo.
Amino Acid Sequence↗
Activation of the unfolded protein response pathway induces human asparagine synthetase gene expression.
The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.
Aspartate-Ammonia Ligase↗
Protein-tyrosine phosphatase 1B potentiates IRE1 signaling during endoplasmic reticulum stress.
Protein-tyrosine phosphatase 1B (PTP-1B) is the prototypic tyrosine phosphatase whose function in insulin signaling and metabolism is well established. Although the role of PTP-1B in dephosphorylating various cell surface receptor tyrosine kinases is clear, the mechanisms by which it modulates receptor function from the endoplasmic reticulum (ER) remains an enigma. Here, we provide evidence that PTP-1B has an essential function in regulating the unfolded protein response in the ER compartment. The absence of PTP-1B caused impaired ER stress-induced IRE1 signaling. More specifically, JNK activation, XBP-1 splicing, and EDEM (ER degradation-enhancing alpha-mannosidase-like protein) gene induction, as well as ER stress-induced apoptosis, were attenuated in PTP-1B knock-out mouse embryonic fibroblasts in response to two ER stressors, tunicamycin and azetidine-2 carboxylic acid. We demonstrate that PTP-1B is not just a passive resident of the ER but on the contrary has an essential role in potentiating IRE1-mediated ER stress signaling pathways.
Animals↗
The sarco/endoplasmic reticulum calcium-ATPase 2b is an endoplasmic reticulum stress-inducible protein.
The sarco/endoplasmic reticulum calcium-ATPase (SERCA) translocates Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum. This Ca(2+) storage is important for cellular processes such as calcium signaling and endoplasmic reticulum (ER)-associated posttranslational protein modifications. We investigated the expression of the SERCA2 and SERCA3 isozymes in PC12 cells exposed to agents interfering with different aspects of the posttranslational protein processing within the ER, thereby activating the ER stress-induced unfolded protein response (UPR). All agents increased the SERCA2b mRNA level 3-4-fold, in parallel with increasing mRNA levels for the ER stress marker proteins BiP/GRP78 and CHOP/GADD153. In contrast, SERCA3 mRNA levels did not change. SERCA2b mRNA stability was not changed, indicating that the mechanism of its up-regulation was transcriptional, in accordance with the presence of ER stress response elements in the promoter region of the SERCA2 gene. SERCA2b was also increased at the protein level upon ER stress treatments. Induction of ER stress by tunicamycin, dithiothreitol, or l-azetidine 2-carboxylic acid did not result in depletion of ER calcium, showing that such depletion was not necessary for up-regulation of SERCA2b expression or UPR activation in general. We conclude that the SERCA2b expression can be controlled by the UPR pathway independently of ER Ca(2+) depletion.
Animals↗
Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock.
Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.
Animals↗