Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases

2002 ◽  
Vol 282 (4) ◽  
pp. E739-E745 ◽  
Author(s):  
Venkatesh Rajapurohitam ◽  
Nathalie Bedard ◽  
Simon S. Wing
Keyword(s):  
Rat Tissues ◽  
Deubiquitinating Enzymes ◽  
Ubiquitinated Proteins ◽  
Tissue Extracts ◽  
Ubiquitin Conjugation ◽  
Tissue Differences ◽  
Conjugating Enzymes ◽  
Rat Tissue ◽  
Ubiquitin Conjugating Enzymes

The activity of the ubiquitin-dependent proteolytic system in differentiated tissues under basal conditions remains poorly explored. We measured rates of ubiquitination in rat tissue extracts. Accumulation of ubiquitinated proteins increased in the presence of ubiquitin aldehyde, indicating that deubiquitinating enzymes can regulate ubiquitination. Rates of ubiquitination varied fourfold, with the highest rate in the testis. We tested whether ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzymes (E2s) could be limiting for conjugation. Immunodepletion of the E2s UBC2 or UBC4 lowered rates of conjugation similarly. Supplementation of extracts with excess UBC2 or UBC4, but not E1, stimulated conjugation. However, UBC2-stimulated rates of ubiquitination still differed among tissues, indicating that tissue differences in E3s or substrate availability may also be rate controlling. UBC2 and UBC4 stimulated conjugation half-maximally at concentrations of 10–50 and 28–44 nM, respectively. Endogenous tissue levels of UBC2, but not UBC4, appeared saturating for conjugation, suggesting that in vivo modulation of UBC4 levels can likely control ubiquitin conjugation. Thus the pool of ubiquitin conjugates and therefore the rate of degradation of proteins by this system may be controlled by E2s, E3s, and isopeptidases. The regulation of the ubiquitin pathway appears complex, but precise.

Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor

Cell ◽  
1993 ◽  
Vol 74 (2) ◽  
pp. 357-369 ◽  
Author(s):  
Ping Chen ◽  
Phoebe Johnson ◽  
Thomas Sommer ◽  
Stefan Jentsch ◽  
Mark Hochstrasser
Keyword(s):  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Vivo Degradation

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

scholarly journals Two Ubiquitin-Conjugating Enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, Have Distinct Functions for Ubiquitination of Mitotic Cyclin

2003 ◽  
Vol 23 (10) ◽  
pp. 3497-3505 ◽  
Author(s):  
Hiroaki Seino ◽  
Tsutomu Kishi ◽  
Hideo Nishitani ◽  
Fumiaki Yamao
Keyword(s):  
Fission Yeast ◽  
Ubiquitin Proteasome System ◽  
High Expression ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Mutant Cells ◽  
Conjugating Enzyme

ABSTRACT Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4 + cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11 + cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

The effects of various pharmacological agents on the distribution of antihistamine activity of rat tissue extracts

1969 ◽  
Vol 47 (9) ◽  
pp. 755-762 ◽  
Author(s):  
M. Lefcort ◽  
L. E. Francis ◽  
K. I. Melville
Keyword(s):  
Mast Cell ◽  
Guinea Pig ◽  
Rat Tissues ◽  
Guinea Pig Ileum ◽  
Pharmacological Agents ◽  
Abdominal Skin ◽  
Tissue Extracts ◽  
Antihistamine Activity ◽  
Rat Tissue ◽  
Different Tissues

Extracts of rat tissues (ear and abdominal skin, heart, cerebrum, spleen, kidney, lung, liver, stomach, and jejunum) were tested for their ability to antagonize histamine on the guinea pig ileum preparation. Antihistamine activity varied considerably between tissues; it was found to be highest in the ear skin and lowest in the jejunum and stomach. After pretreatment with semicarbazide (plus a pyridoxine-free diet) or reserpine, or after adrenalectomy, the antihistamine activity was reduced in some tissues but was increased in others. The variation in the behavior of the different tissues with these treatments made it impossible to interpret the distribution of antihistamine activity in terms of known sites of amine formation. After treatment with compound 48/80, however, there was an apparent parallelism between loss of antihistamine activity and depletion of mast cell histamine.

scholarly journals A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

1996 ◽  
Vol 16 (8) ◽  
pp. 4064-4072 ◽  
Author(s):  
S S Wing ◽  
N Bédard ◽  
C Morales ◽  
P Hingamp ◽  
J Trasler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Amino Acid Identity ◽  
Cellular Function ◽  
Rat Tissues ◽  
Regulation Of Expression ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
First Time

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins.

scholarly journals Characterization of OTUB1 activation and inhibition by different E2 enzymes

2019 ◽  
Author(s):  
Lauren T. Que ◽  
Marie E. Morrow ◽  
Cynthia Wolberger
Keyword(s):  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Kinetics And Thermodynamics ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

scholarly journals Regulation of p53 by the Ubiquitin-conjugating Enzymes UbcH5B/C in Vivo

2004 ◽  
Vol 279 (40) ◽  
pp. 42169-42181 ◽  
Author(s):  
Mark K. Saville ◽  
Alison Sparks ◽  
Dimitris P. Xirodimas ◽  
Julie Wardrop ◽  
Lauren F. Stevenson ◽  
...  
Keyword(s):  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3288-3293 ◽  
Author(s):  
Sven G. Gehrke ◽  
Hans-Dieter Riedel ◽  
Thomas Herrmann ◽  
Boris Hadaschik ◽  
Karin Bents ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Hereditary Hemochromatosis ◽  
Regulatory Region ◽  
Iron Chelation ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

Abstract SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5′ regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5′ regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

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