scholarly journals EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

2014 ◽  
Vol 206 (3) ◽  
pp. 347-356 ◽  
Author(s):  
Satoshi Ninagawa ◽  
Tetsuya Okada ◽  
Yoshiki Sumitomo ◽  
Yukiko Kamiya ◽  
Koichi Kato ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Human Cell Line ◽  
Transcription Activator ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Glycoprotein Degradation ◽  
Rate Limiting

Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease–mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2.

scholarly journals Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

2020 ◽  
Vol 3 (2) ◽  
pp. e201800161 ◽  
Author(s):  
Mainak Bose ◽  
Susanta Chatterjee ◽  
Yogaditya Chakrabarty ◽  
Bahnisikha Barman ◽  
Suvendra N Bhattacharyya
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
De Novo ◽  
Mirna Biogenesis ◽  
Argonaute 2 ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cellular Context ◽  
Subcellular Compartmentalization ◽  
Rate Limiting

microRNAs are short regulatory RNAs in metazoan cells. Regulation of miRNA activity and abundance is evident in human cells where availability of target messages can influence miRNA biogenesis by augmenting the Dicer1-dependent processing of precursors to mature microRNAs. Requirement of subcellular compartmentalization of Ago2, the key component of miRNA repression machineries, for the controlled biogenesis of miRNPs is reported here. The process predominantly happens on the polysomes attached with the endoplasmic reticulum for which the subcellular Ago2 trafficking is found to be essential. Mitochondrial tethering of endoplasmic reticulum and its interaction with endosomes controls Ago2 availability. In cells with depolarized mitochondria, miRNA biogenesis gets impaired, which results in lowering of de novo–formed mature miRNA levels and accumulation of miRNA-free Ago2 on endosomes that fails to interact with Dicer1 and to traffic back to endoplasmic reticulum for de novo miRNA loading. Thus, mitochondria by sensing the cellular context regulates Ago2 trafficking at the subcellular level, which acts as a rate-limiting step in miRNA biogenesis process in mammalian cells.

scholarly journals The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 597-605
Author(s):  
A S Waldman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Insertion Mutant ◽  
Insertion Mutation ◽  
Homologous Pairing ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Homology Searching ◽  
Rate Limiting

Abstract Mouse LTK- cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini.

scholarly journals Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.

1988 ◽  
Vol 106 (6) ◽  
pp. 1853-1861 ◽  
Author(s):  
B Stieger ◽  
K Matter ◽  
B Baur ◽  
K Bucher ◽  
M Höchli ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Colon Adenocarcinoma ◽  
Subcellular Fractionation ◽  
Adenocarcinoma Cell Line ◽  
Dipeptidylpeptidase Iv ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.

scholarly journals A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

2020 ◽  
Author(s):  
Chris R. Hackley
Keyword(s):  
Homologous Recombination ◽  
Long Range ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dsb Repair ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Attractive Option ◽  
End Resection ◽  
Rate Limiting

AbstractCRISPR/Cas9 has revolutionized genetic engineering, however, the inability to control double strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice, however particularly in the case of larger edits, none have been able to bypass the rate-limiting step of Homologous Recombination (HR): long-range 5’ end resection. Here we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Cas9-HRs can increase the rate HR by 2-2.5 fold and decrease cellular toxicity by 2-4 fold compared to Cas9 in mammalian cells with minimal apparent editing site bias, thus making Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.SummaryA novel Cas9 fusion protein designed to increase HDR rates through bypassing the rate limiting step of homologous recombination repair.

scholarly journals Xanthomonas translucens commandeers the host rate-limiting step in ABA biosynthesis for disease susceptibility

2019 ◽  
Vol 116 (42) ◽  
pp. 20938-20946 ◽  
Author(s):  
Zhao Peng ◽  
Ying Hu ◽  
Junli Zhang ◽  
Jose C. Huguet-Tapia ◽  
Anna K. Block ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Disease Susceptibility ◽  
Susceptibility Gene ◽  
Abiotic Stress Response ◽  
Transcription Activator ◽  
Gene Encoding ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Xanthomonas Translucens ◽  
Rate Limiting

Plants are vulnerable to disease through pathogen manipulation of phytohormone levels, which otherwise regulate development, abiotic, and biotic responses. Here, we show that the wheat pathogen Xanthomonas translucens pv. undulosa elevates expression of the host gene encoding 9-cis-epoxycarotenoid dioxygenase (TaNCED-5BS), which catalyzes the rate-limiting step in the biosynthesis of the phytohormone abscisic acid and a component of a major abiotic stress-response pathway, to promote disease susceptibility. Gene induction is mediated by a type III transcription activator-like effector. The induction of TaNCED-5BS results in elevated abscisic acid levels, reduced host transpiration and water loss, enhanced spread of bacteria in infected leaves, and decreased expression of the central defense gene TaNPR1. The results represent an appropriation of host physiology by a bacterial virulence effector.

scholarly journals AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

1994 ◽  
Vol 14 (12) ◽  
pp. 7984-7995 ◽  
Author(s):  
C A Lagnado ◽  
C Y Brown ◽  
G J Goodall
Keyword(s):  
Binding Site ◽  
Mammalian Cells ◽  
Degradation Rate ◽  
Degradation Pathway ◽  
Untranslated Regions ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Two Phases ◽  
The Many ◽  
Rate Limiting

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A)

1994 ◽  
Vol 14 (12) ◽  
pp. 7984-7995
Author(s):  
C A Lagnado ◽  
C Y Brown ◽  
G J Goodall
Keyword(s):  
Binding Site ◽  
Mammalian Cells ◽  
Degradation Rate ◽  
Degradation Pathway ◽  
Untranslated Regions ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Two Phases ◽  
The Many ◽  
Rate Limiting

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

scholarly journals Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110

2016 ◽  
Vol 113 (19) ◽  
pp. 5424-5428 ◽  
Author(s):  
Maria Nagy ◽  
Wayne A. Fenton ◽  
Di Li ◽  
Krystyna Furtak ◽  
Arthur L. Horwich
Keyword(s):  
Motor Neurons ◽  
Mammalian Cells ◽  
Superoxide Dismutase 1 ◽  
Nucleotide Exchange ◽  
Native Form ◽  
Transgenic Expression ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Extended Survival

Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoter-driven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ∼5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery.

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