scholarly journals The Microsporidian Encephalitozoon Hellem Secretes A Host Nucleus-Targeting Protein (Ehhntp1) to Upregulate Endoplasmic Reticulum-Associated Degradation and Promote Protein Ubiquitination

2021 ◽  
Author(s):  
Yinze Han ◽  
Hailong Gao ◽  
Bing Han ◽  
Jinzhi Xu ◽  
Jian Luo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Western Blotting ◽  
Hek293 Cells ◽  
Rna Seq ◽  
Protein Subcellular Localization ◽  
Transfected Cells ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Host Nucleus

Abstract Background: Microsporidia, a group of obligate intracellular parasites that can infect humans and nearly all animals, have lost the pathways for de novo amino acid, lipid and nucleotide synthesis and instead evolved strategies to manipulate host metabolism and immunity. The endoplasmic reticulum (ER) is a vital organelle for producing and processing proteins and lipids and is often hijacked by intracellular pathogens. However, little is known about how microsporidia modulate host ER pathways. Herein, we identified a secreted protein of Encephalitozoon hellem, EhHNTP1, and characterized its subcellular localization and functions in host cells.Methods: A polyclonal antibody against EhHNTP1 was produced to verify the protein subcellular localization in E. hellem-infected cells using indirect immunofluorescence assay (IFA) and Western blotting. HEK293 cells were transfected with wild-type or mutant EhHNTP1 fused with HA-EGFP, and the impacts on pathogen proliferation, protein subcellular localization and sequence functions were assessed. RNA sequencing of EhHNTP1-transfected cells was conducted to identify differentially expressed genes (DEGs) and pathway responses by bioinformatics analysis mainly with R packages. The DEGs in the transfected cells were experimentally confirmed with RT-qPCR and Western blotting. The regulatory effects of candidate DEGs were analyzed via RNA interference and cell transfection, and the effects were determined with RT-qPCR and Western blotting.Results: EhHNTP1 is secreted into the host nucleus, and its translocation depends on a nuclear localization signal sequence (NLS) at the C-terminus from amino acids 239 to 250. Transfection and overexpression of EhHNTP1 in HEK293 cells significantly promoted pathogen proliferation. RNA-seq of the transfected cells showed that genes involved in ER-associated degradation (ERAD), a quality control mechanism that allows for the targeted degradation of proteins in the ER, were prominently upregulated. Upregulation of the ERAD genes PDIA4, HERP, HSPA5 and Derlin3 determined by RNA-seq data was verified using RT-qPCR and Western blotting. Protein ubiquitination in the transfected cells was then assayed and found to be markedly increased, confirming the activation of ERAD. PDIA4 knockdown with RNAi significantly suppressed the expression of HERP, indicating that PDIA4 is a vital ERAD component exploited by EhHNTP1. Moreover, EhHNTP1ΔHRD, a deletion mutant lacking the histidine-rich domain (HRD) in the C-terminus, predominantly suppressed the upregulation of ERAD genes, indicating that the HRD is essential for EhHNTP1 functions.Conclusion: This study is the first report on a microsporidian secretory protein that targets the host nucleus to upregulate the ERAD pathway and subsequently promote protein ubiquitination. Our work provides new insights into microsporidia-host interactions.

scholarly journals The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

2007 ◽  
Vol 401 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Matthew P. A. Henderson ◽  
Yeen Ting Hwang ◽  
John M. Dyer ◽  
Robert T. Mullen ◽  
David W. Andrews
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Lipid Bilayers ◽  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Cytochrome B5 ◽  
Terminal Sequence ◽  
C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

scholarly journals A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression

1998 ◽  
Vol 330 (2) ◽  
pp. 909-914 ◽  
Author(s):  
D. Lucille BURTON ◽  
G. Andree KIPPENBERGER ◽  
Bettina LINGEN ◽  
Michael BRÜSS ◽  
Heinz BÖNISCH ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Cdna Clones ◽  
Similar Treatment ◽  
Noradrenaline Transporter ◽  
C Terminus ◽  
Golgi Region ◽  
Uptake Activity ◽  
High Level

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3ʹ-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

pLoc_bal-mPlant: Predict Subcellular Localization of Plant Proteins by General PseAAC and Balancing Training Dataset

2019 ◽  
Vol 24 (34) ◽  
pp. 4013-4022 ◽  
Author(s):  
Xiang Cheng ◽  
Xuan Xiao ◽  
Kuo-Chen Chou
Keyword(s):  
Subcellular Localization ◽  
Basic Research ◽  
Training Dataset ◽  
Sequence Information ◽  
Plant Proteins ◽  
Protein Subcellular Localization ◽  
Computational Tools ◽  
Validation Tests ◽  
User Friendly ◽  
Better Than

Knowledge of protein subcellular localization is vitally important for both basic research and drug development. With the avalanche of protein sequences emerging in the post-genomic age, it is highly desired to develop computational tools for timely and effectively identifying their subcellular localization based on the sequence information alone. Recently, a predictor called “pLoc-mPlant” was developed for identifying the subcellular localization of plant proteins. Its performance is overwhelmingly better than that of the other predictors for the same purpose, particularly in dealing with multi-label systems in which some proteins, called “multiplex proteins”, may simultaneously occur in two or more subcellular locations. Although it is indeed a very powerful predictor, more efforts are definitely needed to further improve it. This is because pLoc-mPlant was trained by an extremely skewed dataset in which some subsets (i.e., the protein numbers for some subcellular locations) were more than 10 times larger than the others. Accordingly, it cannot avoid the biased consequence caused by such an uneven training dataset. To overcome such biased consequence, we have developed a new and bias-free predictor called pLoc_bal-mPlant by balancing the training dataset. Cross-validation tests on exactly the same experimentconfirmed dataset have indicated that the proposed new predictor is remarkably superior to pLoc-mPlant, the existing state-of-the-art predictor in identifying the subcellular localization of plant proteins. To maximize the convenience for the majority of experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc_bal-mPlant/, by which users can easily get their desired results without the need to go through the detailed mathematics.

Integrating Second-order Moving Average and Over-sampling Algorithm to Predict Apoptosis Protein Subcellular Localization

2020 ◽  
Vol 15 (6) ◽  
pp. 517-527
Author(s):  
Yunyun Liang ◽  
Shengli Zhang
Keyword(s):  
Subcellular Localization ◽  
Moving Average ◽  
Subcellular Location ◽  
Second Order ◽  
Test Method ◽  
Support Vector ◽  
Protein Subcellular Localization ◽  
Protein Subcellular Location ◽  
Apoptosis Protein ◽  
Leibler Divergence

Background: Apoptosis proteins have a key role in the development and the homeostasis of the organism, and are very important to understand the mechanism of cell proliferation and death. The function of apoptosis protein is closely related to its subcellular location. Objective: Prediction of apoptosis protein subcellular localization is a meaningful task. Methods: In this study, we predict the apoptosis protein subcellular location by using the PSSMbased second-order moving average descriptor, nonnegative matrix factorization based on Kullback-Leibler divergence and over-sampling algorithms. This model is named by SOMAPKLNMF- OS and constructed on the ZD98, ZW225 and CL317 benchmark datasets. Then, the support vector machine is adopted as the classifier, and the bias-free jackknife test method is used to evaluate the accuracy. Results: Our prediction system achieves the favorable and promising performance of the overall accuracy on the three datasets and also outperforms the other listed models. Conclusion: The results show that our model offers a high throughput tool for the identification of apoptosis protein subcellular localization.

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