Effect of L-azetidine-2-carboxylic acid on glycosylations of collagen in chick-embryo tendon cells.
The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid. (+info)
Intracellular retention of procollagen within the endoplasmic reticulum is mediated by prolyl 4-hydroxylase.
The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly. (+info)
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. (+info)
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. (+info)
Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance.
We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae. (+info)
Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficiency barley roots: a possible role for phytosiderophore production.
To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 188.8.131.52) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1-sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed. (+info)
Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III).
Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition. (+info)
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. (+info)