Universität zu Köln

Seenivasan, Ramkumar, Hermanns, Thomas ORCID: 0000-0001-5350-7479, Blyszcz, Tamara, Lammers, Michael ORCID: 0000-0003-4168-4640, Praefcke, Gerrit J. K. and Hofmann, Kay (2019). Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome. Hum. Mol. Genet., 28 (17). S. 2862 - 2874. OXFORD: OXFORD UNIV PRESS. ISSN 1460-2083

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Abstract

Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Seenivasan, Ramkumar UNSPECIFIED UNSPECIFIED UNSPECIFIED Hermanns, Thomas UNSPECIFIED orcid.org/0000-0001-5350-7479 UNSPECIFIED Blyszcz, Tamara UNSPECIFIED UNSPECIFIED UNSPECIFIED Lammers, Michael UNSPECIFIED orcid.org/0000-0003-4168-4640 UNSPECIFIED Praefcke, Gerrit J. K. UNSPECIFIED UNSPECIFIED UNSPECIFIED Hofmann, Kay UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-142081
DOI:	10.1093/hmg/ddz098
Journal or Publication Title:	Hum. Mol. Genet.
Volume:	28
Number:	17
Page Range:	S. 2862 - 2874
Date:	2019
Publisher:	OXFORD UNIV PRESS
Place of Publication:	OXFORD
ISSN:	1460-2083
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PROTEIN; TRIAD3A; ATAXIA; RECOGNITION Multiple languages Biochemistry & Molecular Biology; Genetics & Heredity Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/14208