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Unique E2-binding specificity of artificial RING fingers in cancer cells.

ABSTRACT: Ubiquitin (Ub)-conjugating enzymes (E2s) are involved in various pathways for Ub transfer and deubiquitinating activities. These enzymes are associated with cancers such as breast cancer which is the second deadliest type of malignancy among women. Here, we revealed the unique E2-binding property and the auto-ubiquitination of artificial RING fingers (ARFs). Circular dichroism spectra showed the characteristic structures of ARFs. The proline, lysine, leucine, threonine and cysteine (PKLTC) sequence of ARF was important for E2-recognition and its mutations induced obvious changes in the E2-binding specificity and the auto-ubiquitination activity of ARF. The ARF mutants were applicable to detection of most of E2 activities. Furthermore, adding the ARF mutant C35A to cancer cells promoted its auto-ubiquitination, leading to the preferential detection of E2 UbcH5b activity. The present work opens up a new avenue for investigating intracellular E2 activities for the fatal diseases.

SUBMITTER: Miyamoto K

PROVIDER: S-EPMC10828389 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Unique E2-binding specificity of artificial RING fingers in cancer cells.

Miyamoto Kazuhide K Tadokoro Takashi T Matsumoto Atsushi A

Scientific reports 20240131 1

Ubiquitin (Ub)-conjugating enzymes (E2s) are involved in various pathways for Ub transfer and deubiquitinating activities. These enzymes are associated with cancers such as breast cancer which is the second deadliest type of malignancy among women. Here, we revealed the unique E2-binding property and the auto-ubiquitination of artificial RING fingers (ARFs). Circular dichroism spectra showed the characteristic structures of ARFs. The proline, lysine, leucine, threonine and cysteine (PKLTC) seque ...[more]

PMID: 38291082

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Unique auto-ubiquitination activities of artificial RING fingers in cancer cells.

Project description:Ubiquitin-conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT-ARF using a cell-penetrating trans-activator protein (TAT) peptide. An in vitro ubiquitination assay without substrates showed auto-ubiquitination of TAT-ARF via its TAT region. TAT-ARF was translocated into MCF7 breast cancer cells, and then TAT-ARF ubiquitinated itself via its ARF. Experiments using confocal laser-scanning microscopy revealed that FAM-labeled TAT-ARF was readily internalized in cells and it remained encapsulated in vesicles. The Cell Counting Kit-8 assay indicated that the TAT-ARF uptake occurred without cytotoxicity in MCF7 cells at concentrations below 5.0 μM. By taking advantage of TAT-ARF, we, for the first time, succeeded in detecting E2 activities in cells. Thus, the present work opens up new avenues in the investigation of protein ubiquitination.

| S-EPMC6194272 | biostudies-literature

Artificial RING finger reveals unique auto-ubiquitination with E2 specificity.

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Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B.

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E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

2009-03-24 | GSE14088 | GEO

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles Ube2m and Ube2f are E2 enzymes that direct protein modification by NEDD8. Here we explore the specific functions of Ube2f and Ube2m by comparing gene expression profiles following knockdown of their function in NIH 3T3 cells. We used microarrays to detail the global programme of gene expression changes following knockdown of Ube2m and Ube2f in NIH 3T3 cells. NIH 3T3 cells were transduced with retroviral constructs containing shRNA directed against Ube2m or Ube2f. Three replicates of each condition were analyzed.

2009-03-23 | E-GEOD-14088 | biostudies-arrayexpress

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification.

| S-EPMC2725360 | biostudies-literature

The ring between ring fingers (RBR) protein family.

Project description:Proteins of the ring between ring fingers (RBR)-domain family are characterized by three groups of specifically clustered (typically eight) cysteine and histidine residues. Whereas the amino-terminal ring domain (N-RING) binds two zinc ions and folds into a classical cross-brace ring finger, the carboxy-terminal ring domain (C-RING) involves only one zinc ion. The three-dimensional structure of the central ring domain, the IBR domain, is still unsolved. About 400 genes coding for RBR proteins have been identified in the genomes of uni- and multicellular eukaryotes and some of their viruses, but the family has not been found in archaea or bacteria. The RBR proteins are classified into 15 major subfamilies (besides some orphan cases) by the phylogenetic relationships of the RBR segments and the conservation of their sequence architecture. The RBR domain mediates protein-protein interactions and a subset of RBR proteins has been shown to function as E3 ubiquitin ligases. RBR proteins have attracted interest because of their involvement in diseases such as parkinsonism, dementia with Lewy bodies, and Alzheimer's disease, and in susceptibility to some intracellular bacterial pathogens. Here, we present an overview of the RBR-domain containing proteins and their subcellular localization, additional domains, function, specificity, and regulation.

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Project description:The conjugation of ubiquitin to substrates requires a series of enzymatic reactions consisting of an activating enzyme (E1), conjugating enzymes (E2) and ligases (E3). Tagging the appropriate substrate with ubiquitin is achieved by specific E2-E3 and E3-substrate interactions. E6AP, a member of the HECT family of E3s, has been previously shown to bind and function with the E2s UbcH7 and UbcH8. To decipher the sequence determinants of this specificity we have developed a quantitative E2-E3 binding assay based on fluorescence polarization and used this assay to measure the affinity of wild-type and mutant E2-E6AP interactions. Alanine scanning of the E6AP-UbcH7 binding interface identified four side-chains on UbcH7 and six side-chains on E6AP that contribute more than 1 kcal/mol to the binding free energy. Two of the hot spot residues from UbcH7 (K96 and K100) are conserved in UbcH8 but vary across other E2s. To determine if these are key specificity determining residues, we attempted to induce a tighter association between the E2 UbcH5b and E6AP by mutating the corresponding positions in UbcH5b to lysine residues. Surprisingly, the mutations had little effect, but rather a mutation at UbcH7 position 4, which is not at a hot spot on the UbcH7-E6AP interface, significantly strengthened UbcH5bs affinity for E6AP. This result indicates that E2-E3 binding specificities are a function of both favorable interactions that promote binding, and unfavorable interactions that prevent binding with unwanted partners.

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