scholarly journals Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A

2005 ◽  
Vol 170 (5) ◽  
pp. 745-755 ◽  
Author(s):  
Parker L. Andersen ◽  
Honglin Zhou ◽  
Landon Pastushok ◽  
Trevor Moraes ◽  
Sean McKenna ◽  
...  
Keyword(s):  
Nuclear Factor Κb ◽  
Cysteine Residue ◽  
Physical Interaction ◽  
Polyubiquitin Chains ◽  
Active Site Cysteine ◽  
Cellular Processes ◽  
Damage Repair ◽  
Ubiquitin Conjugating Enzyme ◽  
Active Site Cysteine Residue ◽  
Conjugating Enzyme

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13–Mms2 is required for DNA damage repair but not nuclear factor κB (NF-κB) activation, whereas Ubc13–Uev1A is involved in NF-κB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

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 Analysis of the Active Site Cysteine Residue of the Sacrificial Sulfur Insertase LarE from Lactobacillus plantarum

Biochemistry ◽  
2018 ◽  
Vol 57 (38) ◽  
pp. 5513-5523 ◽  
Author(s):  
Matthias Fellner ◽  
Joel A. Rankin ◽  
Benoît Desguin ◽  
Jian Hu ◽  
Robert P. Hausinger
Keyword(s):  
Lactobacillus Plantarum ◽  
Active Site ◽  
Cysteine Residue ◽  
Active Site Cysteine ◽  
Active Site Cysteine Residue

scholarly journals Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols

2006 ◽  
Vol 398 (2) ◽  
pp. 197-206 ◽  
Author(s):  
Jingmin Zeng ◽  
Rachael A. Dunlop ◽  
Kenneth J. Rodgers ◽  
Michael J. Davies
Keyword(s):  
Active Site ◽  
Cysteine Proteases ◽  
Cysteine Residue ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Active Site Cysteine ◽  
Reactive Aldehydes ◽  
Modified Proteins ◽  
Active Site Cysteine Residue

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259–269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine–lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

scholarly journals Peroxiredoxin III Protects Tumor Suppressor PTEN from Oxidation by 15-Hydroperoxy-eicosatetraenoic Acid

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Ying Zhang ◽  
Jiyoung Park ◽  
Seong-Jeong Han ◽  
Yongwoon Lim ◽  
Iha Park ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Lipid Peroxide ◽  
Lipid Peroxides ◽  
Mouse Embryonic Fibroblast ◽  
Cysteine Residue ◽  
Eicosatetraenoic Acid ◽  
Phosphatase And Tensin Homolog ◽  
Active Site Cysteine ◽  
Cellular Processes ◽  
Tensin Homolog

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase that coordinates various cellular processes. Its activity is regulated by the reversible oxidation of an active-site cysteine residue by H2O2 and thioredoxin. However, the potential role of lipid peroxides in the redox regulation of PTEN remains obscure. To evaluate this, 15-hydroperoxy-eicosatetraenoic acid (15s-HpETE), a lipid peroxide, was employed to investigate its effect on PTEN using molecular and cellular-based assays. Exposure to 15s-HpETE resulted in the oxidation of recombinant PTEN. Reversible oxidation of PTEN was also observed in mouse embryonic fibroblast (MEF) cells treated with a 15s-HpETE and Lipofectamine mixture. The oxidative dimerization of thioredoxin was found simultaneously. In addition, the absence of peroxiredoxin III aggravated 15s-HpETE-induced PTEN oxidation in MEF cells. Our study provides novel insight into the mechanism linking lipid peroxidation to the etiology of tumorigenesis.

Bortezomib Impairs Myeloma Cells (MM) Homologous Recombination Through Inhibition of the E2-Ubiquitin-Conjugating Enzyme UBC13

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1834-1834
Author(s):  
Paola Neri ◽  
Li Ren ◽  
Jordan Johnson ◽  
Kathy J Gratton ◽  
Erin Stebner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Proteasome Inhibition ◽  
Nuclear Accumulation ◽  
Transcriptional Level ◽  
Damage Repair ◽  
Dna Dsbs ◽  
Ubiquitin Conjugating Enzyme ◽  
Repair Genes ◽  
Conjugating Enzyme

Abstract Abstract 1834 Background: Chromosomal instability is a defining feature of clonal MM plasma cells resulting in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis the ubiquitin-proteasome system is also involved in the regulation of DNA damage repair proteins. We have recently reported that proteasome inhibition induces a BRCAness state in MM cells (MM) resulting in a contextual synthetic lethality when combined with PARP inhibitors. We now report on the mechanisms by which bortezomib impairs homologous recombination (HR) mediated repair of DNA double stranded breaks (DNA-DSBs). Methods and results: Using the DR-GFP/SceI reporter assay in which cells are transfected with a plasmid tandemly expressing mutated GFP genes (5'-Sce/GFP – truncated IGFP-3') and followed by infection with an adenovirus (AdNGUS24i) expressing the yeast Sce endonuclease, we have confirmed that treatment with bortezomib does severely impair HR in MM cells. At the transcriptional level, proteasome inhibition transiently (6–12 hours post treatment) reduced the mRNA expression of several HR and DNA damage repair genes (BRCA1, BRCA2, RAD51 and FANCD2) followed by recovery to baseline levels by 24 hours. This transient downregulation of these DNA damage repair genes was also confirmed in a promoter luciferase reporter screen. At the post-trancriptional level, examining the dynamics and kinetics of DNA-DSBs repair induced by radiation therapy or PARP inhibition, we have demonstrated that bortezomib does not alter the initial phase of DNA damage sensing (MRN complex recruitment, ATM and H2AX phosphorylation and MDC1 activation) but does impair the Lys63-poly-ubiquitylation of histones γH2AX and H2A. This histone ubiquitylation is a required modification for the second wave of DNA repair proteins recruitment and retention of BRCA1 and RAD51 at the sites of DNA-DSBs. Furthermore, we have shown that bortezomib treatment depleted the nuclear pools of ubiquitin and abrogated the poly-ubiquitylation (lack of co-localization poly-Ub FK2 foci with γH2AX), but not the phosphorylation and foci formation of histone H2AX. Therefore, bortezomib appears to impair histones (H2AX and H2A) poly-ubiquitylation, a process that is dependent on the activation and recruitment of the E3 ubiquitin-protein ligases RNF8 and RNF-168 as well as the ubiquitin-E2 conjugating enzyme UBC13 to the sites of DNA-DSBs. Western blot analysis of the nuclear and cytoplasmic fractions of cellular extracts, revealed that bortezomib dramatically reduced the nuclear accumulation of UBC13 in response to DNA-DSBs as well as the formation of UBC13-Ubiquitin thiolester bonds formation and hence impaired the transfer of ubiquitin to nucleosomal histones. Conclusion: Our studies demonstrate that proteasome inhibition with bortezomib severely impairs homology-mediated repair of DNA breaks in MM cells at the post-transcriptional level by altering the nuclear accumulation and function of the E2-ubiquitin conjugating enzyme UBC13. These results explain the observed clinical synergy between bortezomib and several DNA damaging agents and support further clinical investigation of the combination of these class of drugs in MM patients. Disclosures: Neri: Celgene: Honoraria, Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.

Sign in / Sign up

Export Citation Format

Share Document