In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

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Functional Domains of the TOL Plasmid Transcription Factor XylS

Journal of Bacteriology ◽

10.1128/jb.182.4.1118-1126.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1118-1126 ◽

Cited By ~ 33

Author(s):

Niilo Kaldalu ◽

Urve Toots ◽

Victor de Lorenzo ◽

Mart Ustav

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Dependent Manner ◽

Tol Plasmid ◽

Terminal Fragment ◽

Central Regions ◽

Basic Features ◽

Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

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Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

Journal of Cell Science ◽

10.1242/jcs.108.12.3775 ◽

1995 ◽

Vol 108 (12) ◽

pp. 3775-3786 ◽

Cited By ~ 9

Author(s):

C. Ruppert ◽

J. Godel ◽

R.T. Muller ◽

R. Kroschewski ◽

J. Reinhard ◽

...

Keyword(s):

Subcellular Localization ◽

Brush Border ◽

Subcellular Fractionation ◽

Actin Binding ◽

Resting Cells ◽

Medium Density ◽

Subcellular Targeting ◽

Myosin I ◽

Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

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Superagonist CD28 Antibody Preferentially Expanded Foxp3-Expressing nTreg Cells and Prevented Graft-Versus-Host Diseases

Cell Transplantation ◽

10.1177/096368970901805-619 ◽

2009 ◽

Vol 18 (5-6) ◽

pp. 627-638 ◽

Cited By ~ 35

Author(s):

Yusuke Kitazawa ◽

Masayuki Fujino ◽

Xiao-Kang Li ◽

Lin Xie ◽

Naotsugu Ichimaru ◽

...

Keyword(s):

Treg Cells ◽

Fourfold Increase ◽

Graft Versus Host ◽

Th2 Cytokine ◽

Potent Inhibition ◽

Treg Subset ◽

Organ Specific ◽

Regulatory Functions ◽

Regulatory Lymphocytes

Regulatory lymphocytes play a pivotal role in preventing organ-specific autoimmune disease and in induction and maintenance of tolerance in various experimental transplantation models. The enhancement of the number and activity of peripheral CD4+CD25+ Treg cells is an obvious goal for the treatment of autoimmunity and for the suppression of alloreactions. The present study demonstrates that naturally occurring CD4+CD25+ Treg (nTreg) cells preferentially proliferate to a fourfold increase within 3 days in response to the administration of a single superagonistic CD28-specific monoclonal antibody (supCD28 mAb). The appearance of increased Foxp3 molecules was accompanied with polarization toward a Th2 cytokine profile with decreased production of IFN-γ and increased production of IL-4 and IL-10 in the expanded Treg subset. Adoptive transfer of supCD28 mAb-expanded cells in a graft-versus-host disease (GvHD) model induced a potent inhibition of lethality. These results suggest that this therapeutic effect is mediated by the in vivo expansion of nTreg cells. Taken together, these data demonstrate that supCD28-mAb may target nTreg cells in vivo and maintain and enhance their potent regulatory functions for the treatment GvHD.

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Identification of Novel Plant Peroxisomal Targeting Signals by a Combination of Machine Learning Methods and in Vivo Subcellular Targeting Analyses

The Plant Cell ◽

10.1105/tpc.111.084095 ◽

2011 ◽

Vol 23 (4) ◽

pp. 1556-1572 ◽

Cited By ~ 74

Author(s):

Thomas Lingner ◽

Amr R. Kataya ◽

Gerardo E. Antonicelli ◽

Aline Benichou ◽

Kjersti Nilssen ◽

...

Keyword(s):

Machine Learning ◽

Subcellular Targeting ◽

Learning Methods ◽

Machine Learning Methods ◽

Peroxisomal Targeting ◽

Targeting Signals

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Tenovin-6 impairs autophagy by inhibiting autophagic flux

Cell Death and Disease ◽

10.1038/cddis.2017.25 ◽

2017 ◽

Vol 8 (2) ◽

pp. e2608-e2608 ◽

Cited By ~ 12

Author(s):

Hongfeng Yuan ◽

Brandon Tan ◽

Shou-Jiang Gao

Keyword(s):

Cell Types ◽

Inhibitory Effect ◽

Lymphocytic Leukemia ◽

Autophagic Flux ◽

Dependent Manner ◽

Autophagy Pathway ◽

Sarcoma Cells ◽

Regulatory Functions

Abstract Tenovin-6 has attracted significant interest because it activates p53 and inhibits sirtuins. It has anti-neoplastic effects on multiple hematopoietic malignancies and solid tumors in both in vitro and in vivo studies. Tenovin-6 was recently shown to impair the autophagy pathway in chronic lymphocytic leukemia cells and pediatric soft tissue sarcoma cells. However, whether tenovin-6 has a general inhibitory effect on autophagy and whether there is any involvement with SIRT1 and p53, both of which are regulators of the autophagy pathway, remain unclear. In this study, we have demonstrated that tenovin-6 increases microtubule-associated protein 1 light chain 3 (LC3-II) level in diverse cell types in a time- and dose-dependent manner. Mechanistically, the increase of LC3-II by tenovin-6 is caused by inhibition of the classical autophagy pathway via impairing lysosomal function without affecting the fusion between autophagosomes and lysosomes. Furthermore, we have revealed that tenovin-6 activation of p53 is cell type dependent, and tenovin-6 inhibition of autophagy is not dependent on its regulatory functions on p53 and SIRT1. Our results have shown that tenovin-6 is a potent autophagy inhibitor, and raised the precaution in interpreting results where tenovin-6 is used as an inhibitor of SIRT1.

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Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.00067-09 ◽

2009 ◽

Vol 8 (7) ◽

pp. 922-932 ◽

Cited By ~ 37

Author(s):

Jens Boesger ◽

Volker Wagner ◽

Wolfram Weisheit ◽

Maria Mittag

Keyword(s):

Chlamydomonas Reinhardtii ◽

Protein Phosphorylation ◽

Proteome Analysis ◽

Physiological Status ◽

Phosphorylation Sites ◽

Cell Organelles ◽

Flagellar Proteins ◽

Reversible Protein Phosphorylation ◽

Cilia And Flagella

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

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circAMOTL1L Suppresses Renal Cell Carcinoma Growth by Modulating the miR-92a-2-5p/KLLN Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/9970272 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Ling Gao ◽

Xian Shao ◽

Qingqing Yue ◽

Weifei Wu ◽

Xuejuan Yang ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Therapeutic Strategy ◽

Underlying Mechanism ◽

Tumor Stage ◽

Circular Rnas ◽

The Novel ◽

Regulatory Functions

Accumulating evidence indicates that the dysregulation of circular RNAs (circRNAs) contributes to tumor progression; however, the regulatory functions of circRNAs in renal cell carcinoma (RCC) remain largely unknown. In this study, the function and underlying mechanism of circAMOTL1L in RCC progression were explored. qRT-PCR showed the downregulation of circAMOTL1L in RCC tissues and cell lines. The decrease in circAMOTL1L expression correlated with the tumor stage, metastasis, and poor prognosis in patients with RCC. Functional experiments revealed that circAMOTL1L inhibited cell proliferation and increased apoptosis in RCC cells. Subcutaneous implantation with circAMOTL1L-overexpressing cells in nude mice decreased the growth ability of the xenograft tumors. Mechanistically, circAMOTL1L served as a sponge for miR-92a-2-5p in upregulating KLLN (killin, p53-regulated DNA replication inhibitor) expression validated by bioinformatics analysis, oligo pull-down, and luciferase assays. Further, reinforcing the circAMOTL1L–miR-92a-2-5p–KLLN axis greatly reduced the growth of RCC in vivo. Conclusively, our findings demonstrate that circAMOTL1L has an antioncogenic role in RCC growth by modulating the miR-92a-2-5p–KLLN pathway. Thus, targeting the novel circAMOTL1L–miR-92a-2-5p–KLLN regulatory axis might provide a therapeutic strategy for RCC.

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Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma

NAR Cancer ◽

10.1093/narcan/zcab009 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Verónica Freire-Benéitez ◽

Nicola Pomella ◽

Thomas O Millner ◽

Anaëlle A Dumas ◽

Maria Victoria Niklison-Chirou ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Protein Composition ◽

Mrna Splicing ◽

Cholesterol Transport ◽

Normal Brain ◽

Regulatory Functions ◽

Polycomb Repressive Complex 1 ◽

Normal Brain Development

Abstract Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.

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Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽

10.3390/plants9070892 ◽

2020 ◽

Vol 9 (7) ◽

pp. 892

Author(s):

Alexandre Augusto Pereira Firmino ◽

Michal Gorka ◽

Alexander Graf ◽

Aleksandra Skirycz ◽

Federico Martinez-Seidel ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Gradient Centrifugation ◽

Leaf Tissue ◽

Proteome Analysis ◽

Sucrose Density Gradient ◽

Cross Linking ◽

Sucrose Density Gradient Centrifugation ◽

Cytoplasmic Ribosome ◽

Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

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