scholarly journals Exostosin-1 Glycosyltransferase Regulates Endoplasmic Reticulum Architecture and Dynamics

2020 ◽  
Author(s):  
Despoina Kerselidou ◽  
Bushra Saeed Dohai ◽  
David R. Nelson ◽  
Sarah Daakour ◽  
Nicolas De Cock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Super Resolution ◽  
High Energy ◽  
Structural Proteins ◽  
Post Translational Modifications ◽  
Nucleotide Sugars ◽  
Resolution Imaging ◽  
The Impact ◽  
Hydrolysis Of

SUMMARYThe endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including ER shape-dynamics-function relationships in mammalian cells. We have observed that, inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated to ER network extension. Our findings suggest that EXT1 drives glycosylation reactions involving ER structural proteins and high-energy nucleotide sugars, which might also apply to other organelles.

scholarly journals Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells

2021 ◽  
Vol 7 (19) ◽  
pp. eabe8349
Author(s):  
Despoina Kerselidou ◽  
Bushra Saeed Dohai ◽  
David R. Nelson ◽  
Sarah Daakour ◽  
Nicolas De Cock ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Secretion ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Cell Enlargement ◽  
Guanosine Triphosphate ◽  
Superresolution Imaging ◽  
Functional Aspects ◽  
The Impact ◽  
Hydrolysis Of

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.

scholarly journals Heterologous expression and purification of recombinant human protoporphyrinogen oxidase IX: A comparative study

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259837
Author(s):  
Zora Novakova ◽  
Daria Khuntsaria ◽  
Marketa Gresova ◽  
Jana Mikesova ◽  
Barbora Havlinova ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Eukaryotic Cells ◽  
Post Translational Modifications ◽  
Protoporphyrinogen Oxidase ◽  
Intracellular Targeting ◽  
Alternative Systems ◽  
Protein Functions ◽  
The Impact

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

scholarly journals A Simple Probe for Super-Resolution Imaging of the Endoplasmic Reticulum in Living Cells

2018 ◽  
Vol 101 (11) ◽  
pp. e1800165 ◽  
Author(s):  
Elias A. Halabi ◽  
Salome Püntener ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Endoplasmic Reticulum ◽  
Super Resolution ◽  
Living Cells ◽  
Resolution Imaging

scholarly journals Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy

2019 ◽  
Vol 116 (11) ◽  
pp. 4804-4809 ◽  
Author(s):  
Felipe Moser ◽  
Vojtěch Pražák ◽  
Valerie Mordhorst ◽  
Débora M. Andrade ◽  
Lindsay A. Baker ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Low Dose ◽  
Super Resolution ◽  
Biological Application ◽  
High Laser ◽  
Environmental Background ◽  
Special Sample Preparation ◽  
Reduce Image Quality ◽  
Resolution Imaging

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

scholarly journals Differential Effects of Endoplasmic Reticulum Stress-induced Autophagy on Cell Survival

2006 ◽  
Vol 282 (7) ◽  
pp. 4702-4710 ◽  
Author(s):  
Wen-Xing Ding ◽  
Hong-Min Ni ◽  
Wentao Gao ◽  
Yi-Feng Hou ◽  
Melissa A. Melan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Endoplasmic Reticulum Stress ◽  
Cell Survival ◽  
Cancer Cells ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Brefeldin A ◽  
Human Colon ◽  
The Impact

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.

scholarly journals Super-Resolution Imaging of Higher-Order Chromatin Structures at Different Epigenomic States in Single Mammalian Cells

Cell Reports ◽  
2018 ◽  
Vol 24 (4) ◽  
pp. 873-882 ◽  
Author(s):  
Jianquan Xu ◽  
Hongqiang Ma ◽  
Jingyi Jin ◽  
Shikhar Uttam ◽  
Rao Fu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Higher Order ◽  
Resolution Imaging

scholarly journals 2P265 Super-resolution imaging of chromatin domains in living mammalian cells(21B. Genome biology:Genome structure,Poster)

2014 ◽  
Vol 54 (supplement1-2) ◽  
pp. S239
Author(s):  
Tadasu Nozaki ◽  
Tomomi Tani ◽  
Sachiko Tamura ◽  
Takeharu Nagai ◽  
Kazuhiro Maeshima
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Chromatin Domains ◽  
Resolution Imaging

scholarly journals Characterization of X- box binding protein 1(XBP1) and BiP functions and investigation their roles in YHV replication in P. monodon

2021 ◽  
Vol 20 (4) ◽  
Author(s):  
Piyawadee Rugsanit ◽  
◽  
Pongsopee Attasart ◽  
Apinunt Udomkit ◽  
Witoon Tirasophon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Amino Acid Residues ◽  
Total Change ◽  
Yellow Head Virus ◽  
Black Tiger Shrimp ◽  
Tiger Shrimp ◽  
The Impact

Abstract Eukaryotic cells have mechanisms to cope with stress in endoplasmic reticulum (ER) called unfolded protein response (UPR). In this study, we characterized putative X-box DNA binding protein1 (XBP1) and Binding protein (BiP) cDNAs from black tiger shrimp (Peneaus monodon). When the shrimp were infected with Yellow head virus (YHV), the levels of XBP1 and BiP mRNA transcripts were elevated approximately 8 and 55 folds, respectively. In normal shrimp, the putative XBP1 (XBP1u) was predicted to encode a protein with 283 amino acid residues. When shrimp were infected with YHV, portion of cDNA with 17 nucleotides intron elimination (XBP1s) was observed. The elimination caused alteration of its translational frame. The predicted protein from this is 469 amino acids in length with total change in its amino acid sequence downstream of the intron. Functional analysis of these XBP1 proteins in mammalian cells clearly showed that overexpression of P. monodon XBP1s was capable of activating the transcription rate of mammalian UPR responsive genes (BiP, ERdj4 and P58IPK). Finally, the impact of XBP1 and BiP on YHV multiplication in black tiger shrimp was investigated by RNAi approach. Knocking down either XBP1 or BiP expression efficiently inhibited YHV replication. Therefore, these two components in the UPR pathway may be an interesting target for anti YHV development in the future. Keywords: Endoplasmic reticulum, siRNA, Stress-inducible genes, Transcriptional regulation

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