Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.     

Cloning and characterization of a gene encoding the human putative ubiquitin conjugating enzyme E2Z (UBE2Z)

Ubiquitination, the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins, has been implicated in a number of important physiological processes. ubiquitin conjugating enzyme (E2) plays an important role in the ubiquitin system. Here we report the research about a human putative ubiquitin conjugating enzyme gene, UBE2Z. The length of UBE2Z cDNA is 3054 base pairs and contains an open reading frame encoding 246 amino acids. The UBE2Z gene was mapped to human chromosome 17q21.32 and consisted of 6 exons. RT-PCR showed that UBE2Z was widely expressed in human tissues, especially high in placenta, pancreas, spleen and testis. The UBE2Z-GFP fusion protein was located in both nucleus and cytoplasm of AD293 cells.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

Binding to E1 and E3 is mutually exclusive for the human autophagy E2 Atg3

Ubiquitin‐like proteins (UBLs) are activated, transferred and conjugated by E1‐E2‐E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3‐mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12～Atg5‐Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3‐binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.     

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.     

E6AP AZUL interaction with UBQLN1/2 in cells,condensates, and an AlphaFold-NMR integrated structure

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Effete,an E2 ubiquitin-conjugating enzyme with multiple roles in Drosophila development and chromatin organization

The Drosophila effete gene encodes an extremely conserved class I E2 ubiquitin-conjugating enzyme. Growing evidence indicates that Eff is involved in many cellular processes including eye development, maintenance of female germline stem cells, and regulation of apoptosis. Eff is also a major component of Drosophila chromatin and it is particularly enriched in chromatin with repressive properties. In addition, Eff is required for telomere protection and to prevent telomere fusion. Consistent with its multiple roles in chromatin maintenance, Eff is also one of the rare factors that modulate both telomere-induced and heterochromatin-induced position effect variegation.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Subcellular dynamics of proteins and metabolites under abiotic stress reveal deferred response of the Arabidopsis thaliana hexokinase‐1 mutant gin2‐1 to high light

Stress responses in plants imply spatio‐temporal changes in enzymes and metabolites, including subcellular compartment‐specific re‐allocation processes triggered by sudden changes in environmental parameters. To investigate interactions of primary metabolism with abiotic stress, the gin2‐1 mutant, defective in the sugar sensor hexokinase 1 (HXK1) was compared with its wildtype Landsberg erecta (Ler) based on time resolved, compartment‐specific metabolome and proteome data obtained over a full diurnal cycle. The high light sensitive gin2‐1 mutant was substantially delayed in subcellular re‐distribution of metabolites upon stress, and this correlated with a massive reduction in proteins belonging to the ATP producing electron transport chain under high light, while fewer changes occurred in the cold. In the wildtype, compounds specifically protecting individual compartments could be identified, e.g., maltose and raffinose in plastids, myo‐inositol in mitochondria, but gin2‐1 failed to recruit these substances to the respective compartments, or responded only slowly to high irradiance. No such delay was obtained in the cold. At the whole cell level, concentrations of the amino acids, glycine and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure, and different subcellular allocation of serine may contribute to the slow growth of the gin2‐1 mutant under high irradiance.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Immobilization of (1S,2R)-(+)-2-amino-1,2-diphenylethanol derivates on aminated silica gel with different linkages as chiral stationary phases and their enantioseparation evaluation by HPLC

A chiral selector was prepared through the reaction between (1S,2R)-(+)-2-amino-1,2-diphenylethanol and phenyl isocyanate. This selector was immobilized on aminated silica gel, respectively, with bifunctional group linkers of 1,4-phenylene diisocyanate, methylene-di-p-phenyl diisocyanate, and terephthaloyl chloride to produce corresponding three chiral stationary phases. The prepared compounds and chiral stationary phases were characterized by FT-IR, elemental analysis, (1)H NMR, and solid-state (1)H NMR. The enantioseparation ability of these chiral stationary phases was evaluated with structurally various chiral compounds. The chiral stationary phase prepared with 1,4-phenylene diisocyanate as linker showed excellent enantioseparation ability. The influence of different linkages on the enantioseparation was discussed.     

Regulation of base excision repair proteins by ubiquitylation

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.     

Drug screening assay based on the interaction of intact Keap1 and Nrf2 proteins in cancer cells

Background

The Nrf2–Keap1 interaction is the major regulatory pathway for cytoprotective responses against oxidative and electrophilic stresses. Keap1, a substrate protein of a Cul3-dependent E3 ubiquitin ligase complex, is a negative regulator of Nrf2. The use of chemicals to regulate the interaction between Keap1 and Nrf2 has been proposed as a strategy for the chemoprevention of degenerative diseases and cancers.

Biochemical characterization of TRIM72 E3 ligase and its interaction with the insulin receptor substrate 1

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.