scholarly journals Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases

2000 ◽  
Vol 4 (2) ◽  
pp. 147-153 ◽  
Author(s):  
LILIANA E. SCARAFIA ◽  
ANDREAS WINTER ◽  
DAVID C. SWINNEY
Keyword(s):  
Cell Types ◽  
Jurkat Cells ◽  
Lung Fibroblasts ◽  
Absolute Quantitation ◽  
Hect Domain ◽  
E3 Ligases ◽  
Quantitative Expression ◽  
C Terminus ◽  
Cellular Specificity ◽  
Carboxy Terminal

We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is “homologous to E6AP C-terminus”). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20–100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.

scholarly journals Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

2002 ◽  
Vol 76 (14) ◽  
pp. 6893-6899 ◽  
Author(s):  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
David T. Curiel
Keyword(s):  
Heparan Sulfate ◽  
Capsid Protein ◽  
Minor Component ◽  
Cell Types ◽  
Binding Motif ◽  
Flag Epitope ◽  
C Terminus ◽  
Protein Ix ◽  
A Minor ◽  
Carboxy Terminal

ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

scholarly journals Pirh2, an E3 ligase, regulates the AIP4–p73 regulatory pathway by modulating AIP4 expression and ubiquitination

2021 ◽  
Author(s):  
Rami Abou Zeinab ◽  
H Helena Wu ◽  
Yasser Abuetabh ◽  
Sarah Leng ◽  
Consolato Sergi ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Ectopic Expression ◽  
E3 Ligase ◽  
Regulatory Pathway ◽  
Hect Domain ◽  
E3 Ligases ◽  
Multiple Substrates ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Negative Regulatory Pathway

Abstract Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

scholarly journals Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo

2004 ◽  
Vol 377 (2) ◽  
pp. 459-467 ◽  
Author(s):  
Jose M. LAPLAZA ◽  
Magnolia BOSTICK ◽  
Derek T. SCHOLES ◽  
M. Joan CURCIO ◽  
Judy CALLIS
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein A ◽  
Fold Increase ◽  
Lysine Residue ◽  
Reading Frame ◽  
E3 Ligases ◽  
C Terminus ◽  
Dependent Modification ◽  
F Box Protein

In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.

scholarly journals Selective expression of TLQP-21 and other VGF peptides in gastric neuroendocrine cells and modulation by feeding

2010 ◽  
Vol 207 (3) ◽  
pp. 329-341 ◽  
Author(s):  
Carla Brancia ◽  
Cristina Cocco ◽  
Filomena D'Amato ◽  
Barbara Noli ◽  
Fabrizio Sanna ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Mucosal Cell ◽  
Clear Cut ◽  
Ecl Cell ◽  
C Terminus ◽  
Gene Knockout Mice ◽  
Definition Of ◽  
Fasted Rats

Although vgf gene knockout mice are hypermetabolic, administration of the VGF peptide TLQP-21 itself increased energy consumption. Agonist–antagonist roles are thus suggested for different VGF peptides, and the definition of their tissue heterogeneity is mandatory. We studied the rat stomach using antisera to C- or N-terminal sequences of known or predicted VGF peptides in immunohistochemistry and ELISA. TLQP (rat VGF556–565) peptide/s were most abundant (162±11 pmol/g, mean±s.e.m.) and were brightly immunostained in enterochromaffin-like (ECL) cells and somatostatin cells. A peptide co-eluting with TLQP-21 was revealed in HPLC of gastric and hypothalamic extracts, while the extended TLQP-62 form was restricted to the hypothalamus. Novel PGH (rat VGF422–430) peptide/s were revealed in ghrelin cells, mostly corresponding to low MW forms (0.8–1.5 kDa), while VGF C-terminus peptides were confined to neurons. VGF mRNA was present in the above gastric endocrine cell types, and was prominent in chief cells, in parallel with low-intensity staining for further cleaved products from the C-terminal region of VGF (HVLL peptides: VGF605–614). In swine stomach, a comparable profile of VGF peptides was revealed by immunohistochemistry. When fed and fasted rats were studied, a clear-cut, selective decrease on fasting was observed for TLQP peptides only (162±11 vs 74±5.3 pmol/g, fed versus fasted rats, mean±s.e.m., P<0.00001). In conclusion, specific VGF peptides appear to be widely represented in different gastric endocrine and other mucosal cell populations. The selective modulation of TLQP peptides suggests their involvement in peripheral neuro-endocrine mechanisms related to feeding responses and/or ECL cell regulation.

scholarly journals The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

2021 ◽  
Author(s):  
John H. Henson ◽  
Bakary Samasa ◽  
Charles B. Shuster ◽  
Athula H. Wikramanayake
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Super Resolution ◽  
Cell Types ◽  
Early Embryo ◽  
Ultrastructural Organization ◽  
C Terminus ◽  
Pathway Activation ◽  
Vegetal Cortex ◽  
Canonical Wnt

AbstractWnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

scholarly journals A Cell Atlas of Microbe-Responsive Processes in the Zebrafish Intestine

2020 ◽  
Author(s):  
Reegan J. Willms ◽  
Jennifer C. Hocking ◽  
Edan Foley
Keyword(s):  
Transcriptional Activity ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Host Responses ◽  
Cellular Specificity ◽  
Direct Growth ◽  
A Cell ◽  
Microbe Interactions ◽  
Regulatory Effects ◽  
Host Microbe Interactions

ABSTRACTGut microbial products direct growth, differentiation and development in the animal host. Disruptions to host-microbe interactions have profound health consequences, that include onset of chronic inflammatory illnesses. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled transcriptional activity in individual cells from the intestine, and associated tissue, of zebrafish larvae that we raised in the presence, or absence, of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome, that included regulatory effects on patterning, metabolic and immune activity. For example, we showed that removal of microbes hindered transduction of vascular endothelial growth factor-dependent signals in the developing vasculature, resulting in impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type.

Nuclear Morphology and the Ultra-Structural Localization of Deoxyribonucleic Acid Synthesis during Interphase

1969 ◽  
Vol 4 (3) ◽  
pp. 569-582
Author(s):  
GILLIAN R. MILNER
Keyword(s):  
Epithelial Cells ◽  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Ultrastructural Localization ◽  
Cell Types ◽  
Acid Synthesis ◽  
Lung Fibroblasts ◽  
Nuclear Morphology ◽  
Structural Localization ◽  
Electron Microscope Autoradiography

The ultrastructural localization of deoxyribonucleic acid (DNA) synthesis was studied by electron-microscope autoradiography in human transforming lymphocytes, embryonic lung fibroblasts, epithelial cells and normoblasts. Euchromatin was found to be active in DNA synthesis in all cell types studied, whereas heterochromatin was inactive. However, DNA synthesis was also prominent in the regions where heterochromatin was thought to be decondensing to form euchromatin. Analysis of sequential changes in nuclear morphology of the transforming lymphocyte suggested that there is decondensation of heterochromatin during the S-phase until none is left. In nuclei with no heterochromatin a prominent localization of DNA synthesis was at the nuclear membrane. This sequence of complete decondensation of heterochromatin also seemed likely for fibroblasts and epithelial cells. Normoblasts however showed no stage where the nucleus was wholly euchromatic and it is suggested that in this cell decondensation of heterochromatin for replication is localized and transient.

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph

1988 ◽  
Vol 8 (9) ◽  
pp. 3770-3776
Author(s):  
Y Maru ◽  
H Hirai ◽  
M C Yoshida ◽  
F Takaku
Keyword(s):  
Tyrosine Kinase ◽  
Blot Analysis ◽  
Protein Tyrosine Kinase ◽  
Normal Liver ◽  
Cell Types ◽  
Kinase Domain ◽  
Kinase Gene ◽  
Preferential Expression ◽  
Cdna Sequences ◽  
Carboxy Terminal

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.

scholarly journals Molecular cloning of MADM: a catalytically active mammalian disintegrin-metalloprotease expressed in various cell types

1996 ◽  
Vol 317 (1) ◽  
pp. 45-50 ◽  
Author(s):  
Linda HOWARD ◽  
Xiaohong LU ◽  
Sally MITCHELL ◽  
Susan GRIFFITHS ◽  
Paul GLYNN
Keyword(s):  
Snake Venom ◽  
Transmembrane Helix ◽  
Cell Types ◽  
Bovine Brain ◽  
Peptide Sequence ◽  
Cdna Libraries ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Mammalian Cell Lines ◽  
C Terminus

A peptide sequence of a metalloprotease purified from bovine brain [Chantry, Gregson and Glynn (1989) J. Biol. Chem. 264, 21603–21607] was used to design an oligonucleotide probe for screening a bovine brain cDNA library. A contig of the two overlapping cDNA clones that were isolated encoded a 748-amino-acid polypeptide with similarity to the disintegrin–metalloprotease precursor proteins of haemorrhagic snake venom. The bovine protein has been named MADM, for mammalian disintegrin–metalloprotease. The predicted mature protein has 534 amino acids arrayed as extracellular metalloprotease and disintegrin (potential integrin-binding) domains, a transmembrane helix and a basic/proline-rich cytoplasmic C-terminus. Highly conserved homologues of bovine MADM were found in cDNA libraries of rat brain and a human U937 histiocytic lymphoma cell line. A wide variety of mammalian cell lines expressed low levels of MADM mRNA (4.5 and 3.2 kb transcripts) and mature polypeptide (Mr 62000), as assessed by Northern analysis and Western blotting with an antiserum raised to a peptide within the disintegrin domain. MADM appears to be a rather distantly related member of the reprolysin protein family, which includes both the snake venom disintegrin–metalloproteases and a number of predicted cell-surface disintegrin-containing mammalian proteins.

scholarly journals Expression of a naturally occurring angiotensin AT1 receptor cleavage fragment elicits caspase-activation and apoptosis

2011 ◽  
Vol 301 (5) ◽  
pp. C1175-C1185 ◽  
Author(s):  
Julia L. Cook ◽  
Akannsha Singh ◽  
Dawn deHaro ◽  
Jawed Alam ◽  
Richard N. Re
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Fold Increase ◽  
Muscle Cells ◽  
Cell Types ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Breast Adenocarcinoma ◽  
Protein Fusion ◽  
Cleavage Fragment ◽  
Carboxy Terminal

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT1R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT1R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase ( P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase ( P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold ( P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.

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