Purification and functional characterization of the DUB domain of SdeA.
Methods Enzymol
; 618: 343-355, 2019.
Article
em En
| MEDLINE
| ID: mdl-30850059
ABSTRACT
Intracellular pathogens like Legionella pneumophila hijack the host ubiquitination network in order to create a facultative niche for their survival by means of effector molecules secreted into the host cell. Some of these effectors function as ubiquitin ligases or deubiquitinases, among other types of enzymes. Deubiquitinating enzymes (DUBs) remove ubiquitin or ubiquitin-like modifiers from conjugated substrates to regulate various cellular processes. Members of the SidE effector family from the L. pneumophila pathogen harbor multiple functional domains that possess discrete biochemical activities impinging on host ubiquitin signaling. At the N-terminal end of these ~1500-residue proteins is a ~200-residue conserved DUB domain capable of recognizing both ubiquitin and the NEDD8 Ubl. SdeA, a member of the SidE family, plays an important role in intracellular bacterial replication. Downstream domains in this protein also catalyze substrate ubiquitination via a phosphoribosyl linkage. Several mammalian Rab proteins (Rab1, Rab30, and Rab33) have been shown to be targeted. The novel mechanism is independent of the classical E1 and E2 ubiquitin ligation machinery and does not require ATP. The N-terminal DUB domain, which does not appear to affect this ubiquitination activity, but it catalyzes cleavage of three different types of polyubiquitination chains (K11, K48, and K63) commonly found in host cells. This chapter describes methods, including purification of recombinant SdeA (full-length and DUB domain alone), and enzymatic assays that have been utilized to characterize the deubiquitination activity of SdeA.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Legionella pneumophila
/
Enzimas Desubiquitinantes
/
Proteínas de Membrana
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article