Solution structure of the ubiquitin‐associated domain of human BMSC‐UbP and its complex with ubiquitin

Self Cite

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A 2 -activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3, has been proposed to be able to bind with Ub, which plays important roles in endoplasmic reticulum-associated degradation, vesicle formation, and DNA damage response. We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA (residues 386 -465, namely PFUC) that can bind Ub and elucidated its solution structure and Ubbinding mode by NMR approaches. The PFUC domain possesses equal population of two conformers in solution by cis/ trans-isomerization, whereas the two isomers exhibit almost equivalent Ub binding abilities. This domain structure takes a novel fold consisting of four ␤-strands and two ␣-helices, and the Ub-binding site on PFUC locates in the surface of ␣2-helix, which is to some extent analogous to those of UBA, CUE, and UIM domains. This study provides structural basis and biochemical information for Ub recognition of the novel PFU domain from a PLAA family protein that may connect ubiquitination and degradation in endoplasmic reticulum-associated degradation.The eukaryotic secreted proteins are translocated into the endoplasmic reticulum after synthesis in cytosol. Misfolded or abnormally assembled proteins should be targeted for degradation through the endoplasmic reticulum-associated degradation (ERAD) 3 pathway (1, 2). This pathway involves many molecular steps: unfolded protein response in the endoplasmic reticulum lumen, retrotranslocation back into the cytosol, ubiquitin (Ub) conjugation, delivery of ubiquitinated proteins to proteasome, and degradation of the substrates by proteases (3, 4).A yeast protein DOA1/UFD3 has been shown to bind to CDC48 by both indirect and direct ways (5-7), suggesting that DOA1 may be involved in ERAD. Evidence indicates that DOA1 directly competes with UFD2 at the same docking site on CDC48, which determines whether a substrate is multiubiquitinated and routed to the proteasome for degradation or deubiquitinated and released for other purposes (8). The direct interaction between DOA1 and Ub was suggested by recent studies (7, 9). Moreover, DOA1 also plays roles in the monoubiquitination of histone H2B and proliferating cell nuclear antigen (10) and in sorting ubiquitinated membrane proteins into multivesicular bodies (11).The mammalian homolog of DOA1 is called phospholipase A 2 -activating protein (PLAA), which can bind to P97/VCP (a CDC48 homolog) with its C-terminal domain PUL (7). Having high sequence similarity (31% identity) with DOA1, PLAA is proposed to possess similar function of DOA1. Like DOA1, PLAA has an N-terminal WD40 domain with yet unknown function. The central region of PLAA contains a putative PLAA family ubiquitin-binding (PFU) domain, which is supposed to bind with Ub as observed in yeast DOA1 (7). Although the mechanism underlying the function of PLAA remains unclear, Ub binding of PLAA might be the central role that connects ubiquitin...

Section: Discussionmentioning

confidence: 99%

Structural Basis for Ubiquitin Recognition by a Novel Domain from Human Phospholipase A2-activating Protein

Zhou

Gao

et al. 2009

Self Cite

“…P97-N213 (residues 1-213) was titrated to GB1-NUB1L-VBM at different molar ratios, and the 1 H, 15 N HSQC spectra were acquired to monitor the chemical-shift changes upon titration. The NMR titration of 15 N-labeled NEDD8 with GB1-UBA2 or GB1-PEST followed this procedure (57).…”

Section: Methodsmentioning

confidence: 99%

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

Liu

Zhao

et al. 2013

Self Cite

“…The GB1 fusion protein was applied for the purpose of enhancing the solubility of NS1B-1-103 (22). The sequence-specific assignment for 95% backbone resonances of the amino acid residues of ISG15 was achieved by analyzing a set of two-dimensional and threedimensional NMR spectra (supplemental Fig.…”

Section: Ns1b-1-103 Binds Specifically To the N-terminal Domain Of Ismentioning

confidence: 99%

Different Roles for Two Ubiquitin-like Domains of ISG15 in Protein Modification

Chang

Yan

Xie

et al. 2008

Self Cite

ISG15 (interferon-stimulated gene 15) is a novel ubiquitinlike (UbL) modifier with two UbL domains in its architecture.We investigated different roles for the two UbL domains in protein modification by ISG15 (ISGylation) and the impact of Influenza B virus NS1 protein (NS1B) on regulation of the pathway. The results show that, although the C-terminal domain is sufficient to link ISG15 to UBE1L and UbcH8, the N-terminal domain is dispensable in the activation and transthiolation steps but required for efficient E3-mediated transfer of ISG15 from UbcH8 to its substrates. NS1B specifically binds to the N-terminal domain of ISG15 but does not affect ISG15 linkage via a thioester bond to its activating and conjugating enzymes. However, it does inhibit the formation of cellular ISG15 conjugates upon interferon treatment. We propose that the N-terminal UbL domain of ISG15 mainly functions in the ligation step and NS1B inhibits ISGylation by competing with E3 ligases for binding to the N-terminal domain.ISG15 (interferon-stimulated gene 15) is highly induced and conjugated to a large number of cellular proteins upon Type I interferon (IFN) 3 treatment (1, 2). ISG15 protein belongs to the family of ubiquitin-like (UbL) modifiers whose members are capable of forming conjugates to cellular proteins (3). Similar to the ubiquitination pathway, the ISG15 modification or ISGylation process involves the concerted action of a set of enzymes: the activating (UBE1L), conjugating (UbcH8), and ligating enzymes (4 -8). However, ISG15 is distinct from other members of the UbLs, such as SUMO and NEDD8 that contain one single UbL domain, in that it possesses two tandem UbL domains. Another two-UbL domain-containing protein, FAT10, can modify a very limited number of as-yet-unidentified proteins and has been associated with apoptosis (9, 10). In contrast, ISG15 in response to Type I IFN stimulation is potent in modifying a wide array of cellular proteins that have such diverse roles as RNA splicing, antiviral ability, cytoskeleton regulation, and signal transduction (7,11,12). Recently, ISG15 has been shown to be a critical component in IFN-mediated inhibition of human immunodeficiency virus, type 1 release (13). Furthermore, it has also been demonstrated to be a critical antiviral molecule against influenza, herpes, and Sindbis viruses (14, 15). Given that ISG15 possesses the antiviral property, it is not surprising that the Influenza B virus has developed a strategy to block the ISG15 modification of cellular proteins through its nonstructural protein NS1 (NS1B) (4).To explore the respective roles of the two UbL domains of ISG15, we carried out a series of biochemical experiments to study their specific functions in ISGylation pathway as well as to elucidate the molecular mechanism by which NS1B inhibits ISG15 conjugation to cellular proteins. EXPERIMENTAL PROCEDURES Generation of Expression Constructs and Protein Purification-The encoding sequences for UBE1L, NS1B-1-103, NS1B-1-90, NS1B-91-103, ISG15, and its two separate domains, I...