Dataset Information

DiGly analysis of autoubiquitinated UBR4 (residues 4730-5183)

ABSTRACT: Human recombinant UBR4 protein, consisting of residues 4730-5183, undergoes in vitro autoubiquitination when incubated with the E2 conjugating enzyme UBE2A and the other reaction components. The autoubiquitinated protein was analysed by mass spectrometry to identify modification sites and the ubiquitin linkage types present. This was achieved by searching for diGly as a variable modification on Ser, Thr, Tyr, Cys and Lys residues. A single autoubiquitination site at K4814 was detected. However, mutation of this residue impaired autoubiquitination, indicating the presence of additional sites. The ubiquitin linkages identified were Lys33, Lys48, Lys63, Lys11 and Lys6, suggesting that UBR4 assembles these ubiquitin linkages on its substrates.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Frederic Lamoliatte

LAB HEAD: Satpal Virdee

PROVIDER: PXD046899 | Pride | 2023-11-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20220822_Virdee_LuBarnsbyGreer_Ubiquitin.fasta	Fasta
	FL_20220802_Lu_SV_InGelUbi_DDA_20220804115104.raw	Raw
	MSMSSpectra.zip	Other
	MaxQuant.zip	Other

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Project description:Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas. It is characterized by the presence of rare Hodgkin and Reed/Sternberg (HRS) cells embedded in an extensive inflammatory infiltrate. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in HRS cells which transcriptionally regulates expression of multiple anti-apoptotic factors and pro-inflammatory cytokines plays a central role in the pathogenesis of cHL (1, 2). In non-stimulated condition, NF-kappaB proteins are rendered inactive by binding to their inhibitors (IkappaB s), which sequester them in the cytoplasm. Stimulation of multiple receptors activates the IkappaB kinase (IKK) complex that phosphorylates IkappaB at two specific serine residues, followed by its ubiquitination and proteasomal degradation, thereby releasing NF-kappaB proteins and allowing their nuclear translocation (3). Recently, two studies provided further insights into the molecular mechanisms of IKK activation upon TNF stimulation (4, 5). Activation of the IKK complex and subsequent NF-kappaB activation requires Lys63 polyubiquitination of RIP1, a kinase which is recruited to the receptor upon TNF stimulation. IKK-ï§ï (NEMO), the regulatory subunit of the IKK complex, specifically recognizes these Lys63-linked polyubiquitins attached to RIP1 and thereby activates IKK and NF-kappaB (4, 5). A20 is an ubiquitin-modifying enzyme that inhibits NF-kappaB activation in succession of tumor necrosis factor (TNF) receptor and Toll-like receptor induced signals (6-8). This enzyme removes Lys63 linked ubiquitin chains from RIP1 and adds Lys48 polyubiquitins to RIP1, thereby targeting this factor for proteasomal degradation, thus explaining the molecular mechanism of NF-kappaB inhibition by A20 (6). A20 likely inhibits NF-kappaB acitivity also by additional means, including interaction with TRAF1 and TRAF2 (9). SNP 6.0 array (Affymetrix) analyses were performed according to the manufacturer's directions on DNA extracted from three Hodgkin cell lines (L1236, HDLM-2, U-HO1), HapMap samples included in the Genotyping Console Software 3.0 were used as references.

Dataset Information

DiGly analysis of autoubiquitinated UBR4 (residues 4730-5183)

Dataset's files

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