Linked Life Data

12054879

Source:http://linkedlifedata.com/resource/pubmed/id/12054879

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2002-6-10
pubmed:abstractText
Polyubiquitination is a death signal for proteins and condemns proteins to subsequent degradation by the 26S proteasome. However, recent studies imply that monoubiquitination and polyubiquitination of proteins do not necessarily result in protein degradation but play an important role in the execution of various biological events such as signal transduction and transcription. Ubiquitin was originally identified as a moiety attached to histones, and this as well as other histone modifications may play an important role for transcription and various other DNA-dependent processes. Considerable progress has been made in linking several histone modifications with chromatin dynamics in transcription. Acetylation of histones has been intimately linked to activation of transcription, while deacetylation is concomitant with repression of transcription. Although other histone modifications such as methylation, phosphorylation, and ubiquitination have been correlated with transcriptionally competent or inactive chromatin, the enzymes that mediate these modifications are only now being discovered. The identification of these histone-modifying enzymes may provide valuable insights into the role and function of histone modifications such as ubiquitination in transcription as well as other DNA-dependent processes. Recently, we have used various in vitro assays to show that the coactivator TAF(II)250 possesses both ubiquitin-activating and ubiquitin-conjugating activities, which monoubiquitinate histone H1. Here, we describe the methods used to identify this bifunctional enzyme: (1) in-gel activity assay; (2) protein-transfer membrane activity assay; and (3) in-solution activity assay. These methods have been successfully used to identify various histone-modifying enzymes and protein kinases. In this article we contribute a short review of the history of the methods used to study ubiquitination of proteins and histone modification. We provide protocols for in-gel, protein-transfer membrane, and in-solution ubiquitination assays. A discussion of the general use of the provided protocols, their limitations, and future perspectives are presented. The described methods provide useful tools for the identification of not only novel histone-modifying enzymes but also other protein-modifying enzymes that act in a variety of biological events.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1046-2023
pubmed:author
pubmed:issnType
Print
pubmed:volume
26
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
233-44
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
In vitro assays to study protein ubiquitination in transcription.
pubmed:affiliation
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't