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 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 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 IMMU-43. POLYAMINE METABOLISM REGULATES MYELOID IMMUNE SUPPRESSION IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi128-vi128
Author(s):  
Jason Miska ◽  
Catalina Lee Chang ◽  
Aida Rashidi ◽  
Yu Han ◽  
Aurora Lopez-Rosas ◽  
...  
Keyword(s):  
Immune Suppression ◽  
De Novo ◽  
Specific Inhibitor ◽  
Suppressor Cells ◽  
Polyamine Metabolism ◽  
Arginase 1 ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract Tumor-associated myeloid cells, which consist of tumor associated macrophages and myeloid-derived suppressor cells (MDSCs), make up a majority of cellular infiltrates in glioma. Glioma infiltrating MDSCs highly express arginase-1 (Arg-1), a catabolic enzyme thought to deplete arginine from the tumor microenvironment. Despite being a well-known marker of immunosuppressive cells, the metabolic reasons for this choice are not clear. Examination of MDSC phenotype in murine glioma models using: RNA-seq, bulk metabolomics, and Carbon-13 arginine flux revealed that two separate pathways of arginine catabolism converge on the generation of ornithine. Ornithine is the prerequisite substrate for the de-novo generation of polyamines, a group of nitrogen-rich metabolites with foundational importance to all mammalian, bacterial, and plant biology. Importantly, we found that the rate-limiting step of polyamine generation, ornithine decarboxylase 1 (ODC1), is dramatically upregulated by glioma infiltrating MDSCs, suggesting de-novo polyamine generation is important for MDSC function. Treatment with a specific inhibitor of de-novo polyamine synthesis, difluoromethylornithine (DMFO), inhibited the immunosuppressive function of in-vitro generated glioma associated MDSCs. However, DFMO only exerted effects before differentiation, as DFMO treatment post-generation did not change their suppressive functions. This suggests that the generation of the polyamine pool is critical to immune suppression by MDSCs. Interestingly the expression of the rate limiting step of polyamine degradation (SAT1) is inversely correlated with (ODC1) during MDSC differentiation, suggesting that utilization of this polyamine pool may be required for the suppressive functions of these cells. Inhibition of SAT1 after MDSC generation blunted MDSC mediated T-cell suppression. The results of this study show that the role of arginine metabolism in tumor infiltrating MDSCs is to generate pools of polyamines which maintain MDSC function in glioma. Therapeutic targeting of this pathway may be a novel and powerful tool to combat immunosuppression in glioma.

Glutathione and Glutathione Utilizing Enzymes Are Key Determinants in the Sensitivity of Myeloma Cells to Arsenic Trioxide as Well as Its Mechanism of Action.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5143-5143
Author(s):  
Delia Gutman ◽  
Alejo Morales ◽  
Jennifer M. McCafferty ◽  
Emilio Volz ◽  
Tatiana Eguizabal ◽  
...  
Keyword(s):  
Cell Death ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
De Novo ◽  
Single Agent ◽  
Building Blocks ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Abstract Arsenic trioxide (ATO, Trisenox) is currently being tested in clinical trials as a single agent or in combination with other agents that have activity in multiple myeloma (MM). We and others have demonstrated that glutathione (GSH) levels can influence the ability of ATO to induce cell death in MM cell lines/patient samples and based on these data have initiated a trial to test the safety and efficacy of the combination of ATO and ascorbic acid for the treatment of refractory/relapsed MM. Therefore we performed expression profiling on 4 MM cell lines treated with ATO over a 48 timecourse. Affymetrix Hu133 2.0 plus arrays were hybridized and up to 7035 out of 54,675 probe sets displayed a change and up to 1546 probes sets displayed 2 fold or greater changes compared to untreated cells at 6 hrs. By only looking at genes that increased in all four cell lines we restricted our search to less than 365 probes sets at any given time point. Interestingly the cells appear to have initiated several pathways that are consistent with an attempt to enhance GSH synthesis. Upregulation of transporters of cysteine (xCT) and glycine (Glyt1) as well as enzymes that convert methionine to cysteine (cystathionase) and serine to glycine (serine hydroxymethyl transferase-1) was observed. Moreover the rate-limiting step of the glutathione salvage pathway gamma-glutamyltransferase is also upregulated. Together this suggests an increase in the building blocks for GSH that can be used for de novo synthesis. The rate limiting step for this reaction is performed by gamma-glutamate cysteine ligase which both the catalytic and modifier subunits are upregulated. GSH can also be regenerated from GSSG by glutathione reductase (GR) in an NADPH-dependent fashion. Both GR and the NADPH generating malic enzyme are also upregulated following treatment with ATO. While consistent with our previous findings the data do not provide much insight as to how the GSH is utilized. The only GSH utilizing enzyme that was observed to be upregulated were the cytosolic and mitochondrial forms of glutaredoxin. Glutathione peroxidase (GPx) activity is not altered by ATO treatment. However GPx baseline expression and activity do correlate with sensitivity of MM cell lines to ATO. We also determined GSTP1 activity in the cells and found that it was expressed in 4/5 MM cell lines tested. In contrast to GPx, GSTP1 baseline expression did not correlate with sensitivity to ATO. However this pattern of expression correlated with our previous findings regarding these cells demonstrating differences in caspase dependence of ATO-induced cell death. The one line that did not express GSTP1, RPMI 8226, also utilizes caspase-independent mechanisms of cell death. Transfection of the GSTP1A allele into these cells could render cells more resistant to ATO-induced apoptosis at concentrations of ATO that are not likely to be achieved in patients. Interestingly transfection of the GSTP1B allele could not render cells more resistant, however like GSTP1A it resulted in inhibition of the caspase-independent pathway. Taken together these data confirm that GSH is an important modulator of ATO therapy and that GPx expression may determine the sensitivity of cells to ATO while GSTP1 can affect the mechanism of action by which ATO-induces apoptosis.

scholarly journals Limited Environmental Serine Confers Sensitivity to PHGDH Inhibition in Brain Metastasis

2020 ◽  
Author(s):  
Bryan Ngo ◽  
Eugenie Kim ◽  
Victoria Osorio-Vasquez ◽  
Sophia Doll ◽  
Sophia Bustraan ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Cancer Metabolism ◽  
Triple Negative ◽  
De Novo ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Catalytically Active ◽  
Phosphoglycerate Dehydrogenase ◽  
Rate Limiting ◽  
The Brain

AbstractA hallmark of metastasis is the adaptation of tumor cells to new environments. Although it is well established that the metabolic milieu of the brain is severely deprived of nutrients, particularly the amino acids serine and its catabolite glycine, how brain metastases rewire their metabolism to survive in the nutrient-limited environment of the brain is poorly understood. Here we demonstrate that cell-intrinsic de novo serine synthesis is a major determinant of brain metastasis. Whole proteome comparison of triple-negative breast cancer (TNBC) cells that differ in their capacity to colonize the brain reveals that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is the most significantly upregulated protein in cells that efficiently metastasize to the brain. Genetic silencing or pharmacological inhibition of PHGDH attenuated brain metastasis and improved overall survival in mice, whereas expression of catalytically active PHGDH in a non-brain trophic cell line promoted brain metastasis. Collectively, these findings indicate that nutrient availability determines serine synthesis pathway dependence in brain metastasis, and suggest that PHGDH inhibitors may be useful in the treatment of patients with cancers that have spread to the brain.Statement of SignificanceOur study highlights how limited serine and glycine availability within the brain microenvironment potentiates tumor cell sensitivity to serine synthesis inhibition. This finding underscores the importance of studying cancer metabolism in physiologically-relevant contexts, and provides a rationale for using PHGDH inhibitors to treat brain metastasis.

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.

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