Classification and interaction modes of 40 rice E2 ubiquitin-conjugating enzymes with 17 rice ARM-U-box E3 ubiquitin ligases

Rice, a monocot model crop, contains at least 48 putative E2 ubiquitin (Ub)-conjugating enzymes. Based on homology comparisons with 40 Arabidopsis E2 proteins and 35 human E2s, 48 rice E2s were classified into 15 different groups. Yeast two-hybrid analyses using the U-box-domain regions of armadillo (ARM)-U-box E3 Ub-ligases and the Ub-conjugating (UBC) domains of E2s showed that, among 40 rice E2s, 11 E2s accounted for 70% of the interactions with 17 ARM-U-box E3s. Thus, a single E2 could interact with multiple ARM-U-box E3s, suggesting the presence of E2 hubs for E2–E3 interactions in rice. Rice SPL11 ARM-U-box E3 displayed distinct self-ubiquitination patterns, including poly-ubiquitination, mono-ubiquitination, or no ubiquitination, depending on different E2 partners. This suggests that the mode of ubiquitination of SPL11 E3 is critically influenced by individual E2s.     

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.     

Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway

Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the α-NH₂ group of the N terminus and the ε-NH₂ groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling.     

The N-Terminal Extension of UBE2E Ubiquitin-Conjugating Enzymes Limits Chain Assembly

Protein ubiquitylation depends upon the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). All E2s have a conserved ubiquitin-conjugating (UBC) domain but many have variable extensions N- and C-terminal to the UBC domain. For many E2s, the function of the extension is not well understood. Here, we show that the N-terminal extension of the UBE2E proteins regulates formation of polyubiquitin chains by the processive UBC domain. Target proteins are therefore monoubiquitylated by full-length UBE2E, whereas the UBC domain alone polyubiquitylates proteins. Although the N-terminal extension of UBE2E1 is largely disordered in solution, these residues have a critical role in limiting chain building, and when fused to the highly processive E2, UBE2D2, ubiquitylation is limited. For some E2s, interaction of ubiquitin with the ‘backside’ of the UBC domain promotes polyubiquitylation. However, interaction of ubiquitin with the backside of the UBC domain of UBE2E1 does not appear to be important for processivity. This study underscores the importance of studying full-length E2 proteins and not just the highly conserved core domain.     

Atypical binding of the Swa2p UBA domain to ubiquitin

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix α1 and the N-terminal part of helix α2 bind to Ub. Mutation of Ala148, a key residue in helix α1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices α1 and α3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix α1 of UBA domains involved in membrane protein trafficking.     

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1).

Structured summary

MINT-6797182:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with CDC37 (uniprotkb:Q16543) and HSP90 (uniprotkb:P07900) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797194:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB1 (uniprotkb:Q15750), HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797248:

TAK1 (uniprotkb:Q62073) physically interacts (MI:0218) with HSP90 (uniprotkb:P07901), CDC37 (uniprotkb:Q61081), TAB2 (uniprotkb:Q99K90) and TAB1 (uniprotkb:Q8CF89) by anti bait coimmunoprecipitation (MI:0006)

MINT-6797232:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with HSP90 (uniprotkb:P07900) and CDC37 (uniprotkb:Q16543) by pull down (MI:0096)

MINT-6797216:

TAK1 (uniprotkb:O43318-2) physically interacts (MI:0218) with TAB2 (uniprotkb:Q9NYJ8), CDC37 (uniprotkb:Q16543), HSP90 (uniprotkb:P07900) and TAB1 (uniprotkb:Q15750) by anti bait coimmunoprecipitation (MI:0006)

     

TRAF-interacting protein (TRIP) is a RING-dependent ubiquitin ligase

TRAF-interacting protein (TRIP) was initially identified as a TRAF1- and TRAF2-binding partner that inhibited NF-kappaB activation without a known mechanism. Inspection of the TRIP sequence revealed an N-terminal RING domain, which is found in many E3 ubiquitin (Ub) ligases. We show that TRIP is a RING-dependent Ub ligase that undergoes auto-ubiquitination and requires an intact RING domain. Both TRIP and its RING mutant interact with TRAF1, 2, 3, 5, and 6, but failed to interact with CYLD and NIK. Stable expression of TRIP or a RING mutant did not affect IKK activation induced by TNF or IL-1 and had no affect on TNF-induced apoptosis. Similarly, RANKL-induced signaling and osteoclastogenesis were not affected by TRIP or its RING mutant. Interestingly, TRIP expression was down regulated during the late stages of osteoclastogenesis. Taken together, our results demonstrate that TRIP is a novel RING-dependent Ub ligase and a binding partner for TRAFs.     

Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: insights into an evolutionarily conserved RING fold

The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a betabetaalpha RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C(2)H(2)) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short alpha-helix and a structured loop with two short anti-parallel beta-strands and adopts a tertiary structure similar to the B-box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a 'cross-brace' pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor.     

The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5

Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the circadian oscillator, and DET1, a negative regulator of light-regulated gene expression. We also found that SINAT5 has E3 ubiquitination activity for LHY but not for DET1. Interestingly, LHY ubiquitination by SINAT5 was inhibited by DET1. Late flowering of sinat5 mutants indicates that flowering time can be controlled by DET1 through regulation of LHY stability by SINAT5.