Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Kurkela, Mika; Garcı́a-Horsman, J. Arturo; Luukkanen, Leena; Mörsky, Saila; Taskinen, Jyrki; Baumann, Marc; Kostiainen, Risto; Hirvonen, Jouni; Finel, Moshe

doi:10.1074/jbc.m206136200

Cited by 139 publications

(44 citation statements)

References 27 publications

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“…Homodimerization analysis of other UGT1A isoforms by FRET also revealed that UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 all have the capacity to homodimerize. Homodimerization of UGT1A9 by FRET supports previously published biochemical data that UGT1A9 forms a stable homodimer (34). The evaluation of UGT1A1 protein-protein interactions with other UGT1A proteins by FRET analysis also revealed the promiscuous nature of UGT1A1 to heterodimerize with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.…”

Section: Discussionsupporting

confidence: 65%

Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

Operaña

Tukey

2007

Journal of Biological Chemistry

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UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of ␤-D-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

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Section: Discussionsupporting

confidence: 65%

Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

Operaña

Tukey

2007

Journal of Biological Chemistry

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“…All 64 UDP-glucuronosyltransferases deposited in the SwissProt database (16) are annotated as monotopic membrane proteins, and no natural soluble form is known. However, an engineered water-soluble form is reported (22). If expressed at the protein level, isoform 002 would be the first naturally encoded soluble UDP-glucuronosyltransferase.…”

Section: Resultsmentioning

confidence: 99%

The implications of alternative splicing in the ENCODE protein complement

Tress

Martelli

Frankish

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

201

187

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Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.function ͉ human ͉ isoforms ͉ splice ͉ structure A lternative mRNA splicing, the generation of a diverse range of mature RNAs, has considerable potential to expand the cellular protein repertoire (1-3), and recent studies have estimated that 40-80% of multiexon human genes can produce differently spliced mRNAs (4, 5). The importance of alternative splicing in processes such as development (6) has long been recognized, and proteins coded by alternatively spliced transcripts have been implicated in a number of cellular pathways (7-9). The extent of alternative splicing in eukaryotic genomes has lead to suggestions that alternative splicing is key to understanding how human complexity can be encoded by so few genes (10).The pilot project of the Encyclopedia of DNA Elements (ENCODE) (11), which aims to identify all the functional elements in the human genome, has undertaken a comprehensive analysis of 44 selected regions that make up 1% of the human genome. One valuable element of the project has been the detailing of a reference set of manually annotated splice variants by the GENCODE consortium (12). The annotation by the GENCODE consortium is an extension of the manually curated annotation by the Havana team at The Sanger Institute.Although a full understanding of the functional implications of alternative splicing is still a long way off, the GENCODE set has provided us with the material to make an in-depth assessment of a systematically collected reference set of splice variants. ResultsAlternative Splicing Frequency. The GENCODE set is made up of 2,608 annotated transcripts for 487 distinct loci. A total of 1,097 transcripts from 434 loci are predicted to be protein coding. There are on average 2.53 protein coding variants per locus; 182 loci have only one variant, whereas one locus, RP1-309K20.2 (CPNE1) has 17 coding variants.A total of 57.8% of the loci are annotated with alternatively spliced transcripts, although there are differences between target re...

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“…Nearest neighbor crosslinking studies followed by gel filtration provided evidence that UGTs form dimers in microsomes [19,20]. Using a variety of techniques evidence was obtained that oligomers may function as homo- [20,21] or hetero-oligomers [21][22][23][24][25].…”

Section: Monoglucuronide Formation By Ugt Dimersmentioning

confidence: 99%

“…For example, in live cells intermolecular interactions among UGT1A proteins was demonstrated by fluorescently tagged UGT1A proteins and homo-and heterodimerization by co-immunoprecipitation analysis [21]. Cotranslational insertion of UGTs into the membrane appears to be a requirement for oligomerization [21].…”

Section: Using Mutants and Chimeric Constructs Meech And Mackenzie Dementioning

confidence: 99%

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Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions

Bock

Köhle

2009

Biochemical Pharmacology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64

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Expression and Characterization of Recombinant Human UDP-glucuronosyltransferases (UGTs)

Cited by 139 publications

References 27 publications

Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

The implications of alternative splicing in the ENCODE protein complement

Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions

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