Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Spectrin's chimeric E2/E3 enzymatic activity.

ABSTRACT: In this minireview, we cover the discovery of the human erythrocyte ? spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid ? spectrin isoforms (I and II) also have this activity and the importance of testing this hypothesis. If correct, this would suggest that the nonerythroid spectrin isoforms play a major role in protein ubiquitination in all cell types. This would open new fields in experimental biology focused on uncovering the impact that this enzymatic activity has upon protein-protein interactions, protein turnover, cellular signaling, and many other functions impacted by spectrin, including DNA repair.

SUBMITTER: Goodman SR

PROVIDER: S-EPMC4935279 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Spectrin's chimeric E2/E3 enzymatic activity.

Goodman Steven R SR Petrofes Chapa Rachel R Zimmer Warren E WE

Experimental biology and medicine (Maywood, N.J.) 20150801 8

In this minireview, we cover the discovery of the human erythrocyte α spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid α spectrin isoforms (I and II) also have this activity and the ...[more]

PMID: 26283706

Similar Datasets

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation.

Project description:Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.

| S-EPMC4583040 | biostudies-literature

Specificity of the E1-E2-E3 enzymatic cascade for ubiquitin C-terminal sequences identified by phage display.

Project description:Ubiquitin (UB) is a protein modifier that regulates many essential cellular processes. To initiate protein modification by UB, the E1 enzyme activates the C-terminal carboxylate of UB to launch its transfer through the E1-E2-E3 cascade onto target proteins. In this study, we used phage display to profile the specificity of the two human E1 enzymes, Ube1 and Uba6, toward the C-terminal sequence of UB ending with (71)LRLRGG(76). Phage selection revealed that while Arg72 of UB is absolutely required for E1 recognition, UB residues at positions 71, 73, and 74 can be replaced with bulky aromatic side chains, and Gly75 of UB can be changed to Ser, Asp, and Asn for efficient E1 activation. We have thus found that the E1 enzymes have substantial promiscuity regarding the UB C-terminal sequence. The UB variants from phage selection can also be transferred from E1 to E2 enzymes; however, they are blocked from further transfer to the E3 enzymes. This suggests that the C-terminal sequence of UB is important for its discharge from E2 and subsequent transfer to E3. In addition, we observed that the Leu73Phe and Leu73Tyr single mutants of UB are resistant to cleavage by deubiquitinating enzymes (DUBs), although they can be assembled by the E1-E2-E3 cascade into poly-UB chains, thus indicating differences in UB C-terminal specificities between the E1 and DUBs. Consequently these UB mutants may provide stability to UB polymers attached to cellular proteins and facilitate the elucidation of the biological signals encoded in the UB chains.

| S-EPMC4652587 | biostudies-literature

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Project description:Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

| S-EPMC5108872 | biostudies-literature

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Project description:Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

| S-EPMC5042379 | biostudies-literature

Structural insights into E2-E3 interaction for LC3 lipidation.

Project description:The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12-ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2-E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.

| S-EPMC4077891 | biostudies-literature

A rationally-designed chimeric KDM1A/KDM1B histone demethylase tower domain deletion mutant retaining enzymatic activity.

Project description:A target with therapeutic potential, lysine-specific demethylase 1A (KDM1A) is a regulator of gene expression whose tower domain is a protein-protein interaction motif. This domain facilitates the interaction of KDM1A with coregulators and multiprotein complexes that direct its activity to nucleosomes. We describe the design and characterization of a chimeric 'towerless' KDM1A, termed n?150 KDM1A?Tower KDM1B chimera (chKDM1A?Tower), which incorporates a region from the paralog lysine-specific demethylase 1B (KDM1B). This chimera copurifies with FAD and displays demethylase activity, but fails to bind the partner protein corepressor of the RE1-silencing transcription factor (CoREST). We conclude that KDM1A catalysis can be decoupled from tower-dependent interactions, lending chKDM1A?Tower useful for dissecting molecular contributions to KDM1A function.

| S-EPMC4806859 | biostudies-literature

Determinants of E2-ubiquitin conjugate recognition by RBR E3 ligases.

Project description:RING-between-RING (RBR) ubiquitin ligases work with multiple E2 enzymes and function through an E3-ubiquitin thioester intermediate. The RBR module comprises three domains, RING1, IBR and RING2 that collaborate to transfer ubiquitin from the E2~Ub conjugate, recognised by RING1, onto a catalytic cysteine in RING2 and finally onto the substrate in a multi-step reaction. Recent studies have shown that RING1 domains bind E2~Ub conjugates in an open conformation to supress ubiquitin transfer onto lysine residues and promote formation of the E3 thioester intermediate. However, how the nature of the E2 influences the ubiquitin transfer process is currently unclear. We report here a detailed characterization of the RBR/E2-conjugate recognition step that indicates that this mechanism depends on the nature of the E2 enzyme and differs between UbcH5 and UbcH7. In the case of UbcH5~Ub an interaction with ubiquitin is necessary to stabilize the transfer complex while recognition of UbcH7~Ub is driven primarily by E2-RING1 contacts. Furthermore our analysis suggests that RBRs, in isolation and in complex with ubiquitin-loaded E2s, are dynamic species and that their intrinsic flexibility might be a key aspect of their catalytic mechanism.

| S-EPMC5758712 | biostudies-literature

Genes for E1, E2, and E3 small nucleolar RNAs.

Project description:We have found earlier three small nucleolar RNA (snoRNA) species, named E1, E2, and E3, that have unique nucleotide sequences and may participate in ribosome formation. The present report shows that there is a monophosphate at the 5' end of each of these three snoRNAs, suggesting that their 5' termini are formed by RNA processing. E1, E2, and E3 human genomic sequences were isolated. Apparently, the E2 and E3 loci are genes for the main E2 and E3 RNA species, based on their full homology, while the E1 locus is a gene for an E1 RNA sequence variant in HeLa cells. These loci do not have any of the intragenic or flanking sequences known to be functional in other genes. The E1 gene is located within the first intron of the gene for RCC1, a protein that regulates onset of mitosis. There is substantial sequence homology between the human E3 gene and flanking regions, and intron 8 and neighboring exons of the gene for mouse translation initiation factor 4AII. Injection of the human E1, E2, and E3 genes into Xenopus oocytes generated sequence-specific transcripts of the approximate sizes of the respective snoRNAs. We discuss why the available results are compatible with specific transcription and processing occurring in frog oocytes.

| S-EPMC47489 | biostudies-other

EPO-regulated targets in E1, E2, and E3 erythroid progenitors

Project description:Profiling of bone marrow-derived erythroid progenitor cells at E1 (CFU-e), E2 (proerythroblasts), and E3 (maturing erythroblast) stages of development under stress erythropoiesis conditions and in response to EPO challenge. MACS-isolated E1, E2, and E3 stage EPCs were cultured in SP34ex media for 6 hrs in the absence of hematopoietic growth factors and the presence of insulin (to enforce survival and anti-apoptotic effects) and then exposed to rhEPO for 90 minutes. Gene expression analysis was performed using Affymetrix Mouse Genome 430 2.0 arrays; microarray data were analyzed using Bioconductor for R.

2017-06-06 | E-MTAB-5373 | biostudies-arrayexpress

Capturing a substrate in an activated RING E3/E2-SUMO complex.

Project description:Post-translational protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as small ubiquitin-like modifier (SUMO) regulates processes including protein homeostasis, the DNA damage response, and the cell cycle. Proliferating cell nuclear antigen (PCNA) is modified by Ub or poly-Ub at lysine (Lys)164 after DNA damage to recruit repair factors. Yeast PCNA is modified by SUMO on Lys164 and Lys127 during S-phase to recruit the anti-recombinogenic helicase Srs2. Lys164 modification requires specialized E2/E3 enzyme pairs for SUMO or Ub conjugation. For SUMO, Lys164 modification is strictly dependent on the E3 ligase Siz1, suggesting the E3 alters E2 specificity to promote Lys164 modification. The structural basis for substrate interactions in activated E3/E2–Ub/Ubl complexes remains unclear. Here we report an engineered E2 protein and cross-linking strategies that trap an E3/E2–Ubl/substrate complex for structure determination, illustrating how an E3 can bypass E2 specificity to force-feed a substrate lysine into the E2 active site.

| S-EPMC5019495 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data