scholarly journals The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1371-1377 ◽  
Author(s):  
M Zhen ◽  
R Heinlein ◽  
D Jones ◽  
S Jentsch ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Elevated Temperatures ◽  
Yeast Cells ◽  
Amino Acid Sequence Similarity ◽  
Western Blots ◽  
Target Proteins ◽  
C Elegans ◽  
Cell Functions ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation

1993 ◽  
Vol 13 (3) ◽  
pp. 1371-1377
Author(s):  
M Zhen ◽  
R Heinlein ◽  
D Jones ◽  
S Jentsch ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Elevated Temperatures ◽  
Yeast Cells ◽  
Amino Acid Sequence Similarity ◽  
Western Blots ◽  
Target Proteins ◽  
C Elegans ◽  
Cell Functions ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

scholarly journals Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin

2019 ◽  
Vol 10 (1) ◽  
pp. 267-280 ◽  
Author(s):  
Michael A. Q. Martinez ◽  
Brian A. Kinney ◽  
Taylor N. Medwig-Kinney ◽  
Guinevere Ashley ◽  
James M. Ragle ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Degradation ◽  
Single Cell ◽  
Water Soluble ◽  
Growth Media ◽  
Synthetic Analog ◽  
Rapid Degradation ◽  
Target Proteins ◽  
C Elegans ◽  
Link Type

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegansSKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

Target Proteins of C/EBPa-p30 in AML: A Critical Role of Ubiquitin-Conjugating Enzyme (Ubc9) in Modulating C/EBPa-p42 Functions.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2219-2219
Author(s):  
M. Geletu ◽  
M. Balkhi ◽  
A. Peer Zada ◽  
A. Trivedi ◽  
J. Pulikkan ◽  
...  
Keyword(s):  
Binding Protein ◽  
Critical Role ◽  
Protein A ◽  
Dominant Negative ◽  
Promoter Assay ◽  
Differentiation Capacity ◽  
Target Proteins ◽  
Calgranulin B ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

Abstract The mutations in the transcription factor CCAAT/enhancer binding protein-a (C/EBPa) occur in 10% of patients with acute myeloid leukemia which often leads to the expression of an N-terminal truncated 30KDa isoform of C/EBPa. The mutated 30KDa isoform has been shown to be dominant negative over wild type isoform that affects most of its biological functions. In the present study, we applied a global proteomics approach to identify the target proteins of C/EBPap30. This analysis revealed that C/EBPap30 modulates the expression of 60 different proteins notably, ubiquitin-conjugating enzyme (Ubc9), Peptidylprolyl isomerase (pin1), hnRNP A2/B1, Cacyclin binding protein, calgranulin B, and Tubulin beta5. Further, we show that in AML patients with C/EBPap30 mutations Ubc9 was predominantly upregulated. We discovered that this ligase was responsible for the enhanced sumolyation of C/EBPap42 on a lysine residue (K161) which results in its reduced transactivation in a promoter assay. When lysine (K) residue at 161 of C/EBPa-p42 was mutated to argenine residue(R), transcriptional activity of C/EBPap42 was restored. This finding was further validated by silencing the expression of Ubc9 expression which completely abrogates the sumoylation at K161 residue and further restored the transactivation and differentiation capacity of C/EBPap42. Our results demonstrate that increasing expression of Ubc9 by C/EBPap30 enhances sumolyation of C/EBPap42 and thereby inhibits its transactivation and differentiation capacity.

scholarly journals Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans

2011 ◽  
Vol 437 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Ida C. Elle ◽  
Karina T. Simonsen ◽  
Louise C. B. Olsen ◽  
Pernille K. Birck ◽  
Sidse Ehmsen ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Unsaturated Fatty Acids ◽  
Expression Patterns ◽  
High Specificity ◽  
Tissue Expression ◽  
Yeast Cells ◽  
Loss Of Function ◽  
C Elegans ◽  
Eukaryotic Species ◽  
Quadruple Mutant

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

scholarly journals Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo

2010 ◽  
Vol 108 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Kenji Kimura ◽  
Akatsuki Kimura
Keyword(s):  
Caenorhabditis Elegans ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Early Embryo ◽  
Organelle Transport ◽  
Animal Cells ◽  
C Elegans ◽  
Cell Functions ◽  
Intracellular Organelles ◽  
Pulling Force

The centrosome is generally maintained at the center of the cell. In animal cells, centrosome centration is powered by the pulling force of microtubules, which is dependent on cytoplasmic dynein. However, it is unclear how dynein brings the centrosome to the cell center, i.e., which structure inside the cell functions as a substrate to anchor dynein. Here, we provide evidence that a population of dynein, which is located on intracellular organelles and is responsible for organelle transport toward the centrosome, generates the force required for centrosome centration in Caenorhabditis elegans embryos. By using the database of full-genome RNAi in C. elegans, we identified dyrb-1, a dynein light chain subunit, as a potential subunit involved in dynein anchoring for centrosome centration. DYRB-1 is required for organelle movement toward the minus end of the microtubules. The temporal correlation between centrosome centration and the net movement of organelle transport was found to be significant. Centrosome centration was impaired when Rab7 and RILP, which mediate the association between organelles and dynein in mammalian cells, were knocked down. These results indicate that minus end-directed transport of intracellular organelles along the microtubules is required for centrosome centration in C. elegans embryos. On the basis of this finding, we propose a model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first model, to our knowledge, providing a mechanical basis for cytoplasmic pulling force for centrosome centration.

scholarly journals Candida albicans Hyphal Formation and Virulence Assessed Using a Caenorhabditis elegans Infection Model

2009 ◽  
Vol 8 (11) ◽  
pp. 1750-1758 ◽  
Author(s):  
Read Pukkila-Worley ◽  
Anton Y. Peleg ◽  
Emmanouil Tampakakis ◽  
Eleftherios Mylonakis
Keyword(s):  
Candida Albicans ◽  
Caenorhabditis Elegans ◽  
Debaryomyces Hansenii ◽  
Yeast Cells ◽  
Infection Model ◽  
Virulence Determinants ◽  
Tissue Destruction ◽  
C Elegans ◽  
Hyphal Formation

ABSTRACT Candida albicans colonizes the human gastrointestinal tract and can cause life-threatening systemic infection in susceptible hosts. We study here C. albicans virulence determinants using the nematode Caenorhabditis elegans in a pathogenesis system that models candidiasis. The yeast form of C. albicans is ingested into the C. elegans digestive tract. In liquid media, the yeast cells then undergo morphological change to form hyphae, which results in aggressive tissue destruction and death of the nematode. Several lines of evidence demonstrate that hyphal formation is critical for C. albicans pathogenesis in C. elegans. First, two yeast species unable to form hyphae (Debaryomyces hansenii and Candida lusitaniae) were less virulent than C. albicans in the C. elegans assay. Second, three C. albicans mutant strains compromised in their ability to form hyphae (efg1Δ/efg1Δ, flo8Δ/flo8Δ, and cph1Δ/cph1Δ efg1Δ/efg1Δ) were dramatically attenuated for virulence. Third, the conditional tet-NRG1 strain, which enables the external manipulation of morphogenesis in vivo, was more virulent toward C. elegans when the assay was conducted under conditions that permit hyphal growth. Finally, we demonstrate the utility of the C. elegans assay in a screen for C. albicans virulence determinants, which identified several genes important for both hyphal formation in vivo and the killing of C. elegans, including the recently described CAS5 and ADA2 genes. These studies in a C. elegans-C. albicans infection model provide insights into the virulence mechanisms of an important human pathogen.

scholarly journals Identification of a Gene Product Induced by Hard-Surface Contact of Colletotrichum gloeosporioidesConidia as a Ubiquitin-Conjugating Enzyme by Yeast Complementation

1998 ◽  
Vol 180 (14) ◽  
pp. 3592-3597 ◽  
Author(s):  
Zhi-Mei Liu ◽  
Pappachan E. Kolattukudy
Keyword(s):  
Early Stage ◽  
Phytopathogenic Fungi ◽  
Yeast Cells ◽  
Reading Frame ◽  
Hard Surface ◽  
Surface Wax ◽  
Surface Contact ◽  
Ubiquitin Conjugating Enzyme ◽  
Dependent Protein ◽  
Conjugating Enzyme

ABSTRACT The germinating conidia of many phytopathogenic fungi on hosts must differentiate into an infection structure called the appressorium in order to penetrate their hosts. Chemical signals, such as the host’s surface wax or fruit ripening hormone, ethylene, trigger germination and appressorium formation of the avocado pathogen Colletotrichum gloeosporioides only after the conidia are in contact with a hard surface. What role this contact plays is unknown. Here, we describe isolation of genes expressed during the early stage of hard-surface treatment by a differential-display method and report characterization of one of these cloned genes, chip1(Colletotrichum hard-surface induced protein 1 gene), which encodes a ubiquitin-conjugating enzyme. RNA blots clearly showed that it is induced by hard-surface contact and that ethylene treatment enhanced this induction. The predicted open reading frame (ubc1 Cg) would encode a 16.2-kDa ubiquitin-conjugating enzyme, which shows 82% identity to theSaccharomyces cerevisiae UBC4-UBC5 E2 enzyme, comprising a major part of total ubiquitin-conjugating activity in stressed yeast cells. UBC1Cg can complement the proteolysis deficiency of the S. cerevisiae ubc4 ubc5 mutant, indicating that ubiquitin-dependent protein degradation is involved in conidial germination and appressorial differentiation.

Metabolism and inactivation of neurotransmitters in nematodes

Parasitology ◽  
1996 ◽  
Vol 113 (S1) ◽  
pp. S157-S173 ◽  
Author(s):  
R. E. Isaac ◽  
D. Macgregor ◽  
D. Coates
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Free Living ◽  
Nervous Systems ◽  
Target Proteins ◽  
C Elegans ◽  
Free Living Nematode ◽  
Increasing Knowledge ◽  
Insight Into

SUMMARYThe nematode nervous system employs many of the same neurotransmitters as are found in higher animals. The inactivation of neurotransmitters is absolutely essential for the correct functioning of the nervous system, In this article we discuss the various mechanisms used generally in animal nervous systems for synaptic inactivation of neurotransmitters and review the evidence for similar mechanisms operating in parasitic and free-living nematodes. The sequencing of the entireCaenorhabditis elegansgenome means that the sequence of nematode genes can be accessed from theC. elegansdatabase (ACeDB) and this wealth of information together with the increasing knowledge of the genetics of this free-living nematode will have great impact on all aspects of nematode neurobiology. The review will provide an insight into how this information may be exploited to identify and characterize target proteins for the development of novel anti-nematode drugs.

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