Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5' truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.
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PMID:The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. 842 9

A family of highly-conserved 70 kDa stress proteins is localized in various intracellular compartments of Saccharomyces cerevisiae. Their gene expression is specifically and/or sometimes cooperatively regulated at the transcriptional level by cis-acting elements found in their respective promoters. Here, we find that depletion of cytosolic Ssa1p induced BiP(Kar2p) in the endoplasmic reticulum at the transcriptional level. By analyzing internal deletion mutants of the KAR2 promoter, we determined that the heat shock element (HSE) is necessary for KAR2 gene induction in response to the depletion of Ssa1p. Furthermore, either the KAR2HSE or SSA1HSE is sufficient for gene activation, as assayed using HSE-CYC1-lacZ fusion reporter plasmids. Finally, temperature-sensitive ssa1 mutants transformed with an HSE-CYC1-lacZ fusion vector exhibited strong induction of beta-galactosidase activity when shifted to a restrictive temperature. These results show that loss of functional Ssa1p from the cytosol up-regulates KAR2 gene expression through an HSE-mediated pathway and also support the idea that SSA1 gene expression is autoregulated.
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PMID:Saccharomyces cerevisiae KAR2 (BiP) gene expression is induced by loss of cytosolic HSP70/Ssa1p through a heat shock element-mediated pathway. 913 28

New secretory signals and strategies can be attempted to improve the secretion of heterologous proteins of biotechnological interest which encounter difficulties being exported in yeast. The GGPI gene of Saccharomyces cerevisiae codes for a 125 kDa glycoprotein transported through the secretory pathway and anchored to the plasma membrane by means of a glycosylphosphatidylinositol. The regions coding for the secretory signal or also for the first 46 amino acids were tested for efficiency in secretion by fusion to the lacZ gene of Escherichia coli resulting in the synthesis of the endoplasmic reticulum-targeted 1-22- and 1-68-GgpIp/beta-gal hybrids. A cytoplasmic form was also examined. The 1-22 beta gal is partially transported to the cell surface and in the medium in an unglycosylated form. The 1-68 beta gal is completely retained in the intracellular membranes and is N-glycosylated in the GgpIp moiety. The amount of hybrid protein produced is similar and independent from its targeted site, suggesting that translocation through endoplasmic reticulum is not a limiting step, whereas the amount of active enzyme is from 50 to 80% lower for the endoplasmic reticulum forms compared with the cytoplasmic form. BiP/Kar2p putative precursor is accumulated in cells expressing the endoplasmic reticulum-targeted forms but not in those producing the cytosolic beta-galactosidase or over-expressing an endogenous secretory protein. Thus, glycosylation and abnormal folding rather than over-expression are among the factors responsible for the decreased activity and exit of beta-galactosidase from the endoplasmic reticulum and for induction of BiP. The results obtained indicate that the sole secretory signal of GgpIp is suitable to drive secretion of foreign products with complex folding and point to the importance of the endoplasmic reticulum quality control in the secretion of heterologous proteins in yeast.
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PMID:Expression and secretion of beta-galactosidase in Saccharomyces cerevisiae using the signal sequences of GgpI, the major yeast glycosylphosphatidylinositol-containing protein. 956 2

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.
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PMID:GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis. 1535 Feb 19

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.
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PMID:Molecular consequences of the pathogenic mutation in feline GM1 gangliosidosis. 1835 97

Calnexin is a type I integral endoplasmic reticulum (ER) membrane chaperone involved in folding of newly synthesized (glycol)proteins. In this study, we used beta-galactosidase reporter gene knock-in and reverse transcriptase polymerase chain reaction (RT-PCR) to investigate activation of the calnexin gene during embryonic development. We showed that the calnexin gene was activated in neuronal tissue at the early stages of embryonic development but remained low in the heart, intestine, and smooth muscle. At early stages of embryonic development, large quantities of calnexin messenger RNA (mRNA) were also found in neuronal tissue and liver. There was no detectable calnexin mRNA in the heart, lung, and intestine. The absence of calnexin had no significant effect on ER stress response (unfolded protein response, UPR) at the tissue level as tested by IRE1-dependent splicing of Xbp1 mRNA. In contrast, non-stimulated calnexin-deficient cells showed increased activation of IRE1, as measured by RT-PCR and luciferase reporter gene analysis of splicing of Xbp1 mRNA and activation of the BiP promoter. This indicates that cnx (-/-) cells have increased constitutively active UPR. Importantly, cnx (-/-) cells have significantly increased proteasomal activity, which may play a role in the adaptive mechanisms addressing the acute ER stress observed in the absence of calnexin.
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PMID:Endoplasmic reticulum stress in the absence of calnexin. 1852 84

Copper sulfate-induced premature senescence (CuSO4-SIPS) consistently mimetized molecular mechanisms of replicative senescence, particularly at the endoplasmic reticulum proteostasis level. In fact, disruption of protein homeostasis has been associated to age-related cell/tissue dysfunction and human disorders susceptibility. Resveratrol is a polyphenolic compound with proved antiaging properties under particular conditions. In this setting, we aimed to evaluate resveratrol ability to attenuate cellular senescence induction and to unravel related molecular mechanisms. Using CuSO4-SIPS WI-38 fibroblasts, resveratrol is shown to attenuate typical senescence alterations on cell morphology, senescence-associated beta-galactosidase activity, and cell proliferation. The mechanisms implicated in this antisenescence effect seem to be independent of senescence-associated genes and proteins regulation but are reliant on cellular proteostasis improvement. In fact, resveratrol supplementation restores copper-induced increased protein content, attenuates BiP level, and reduces carbonylated and polyubiquitinated proteins by autophagy induction. Our data provide compelling evidence for the beneficial effects of resveratrol by mitigating CuSO4-SIPS stressful consequences by the modulation of protein quality control systems. These findings highlight the importance of a balanced cellular proteostasis and add further knowledge on molecular mechanisms mediating resveratrol antisenescence effects. Moreover, they contribute to identifying specific molecular targets whose modulation will prevent age-associated cell dysfunction and improve human healthspan.
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PMID:Resveratrol Attenuates Copper-Induced Senescence by Improving Cellular Proteostasis. 2914 66