Crystal structure of the N-terminal domain of the trypanosome flagellar protein BILBO1 reveals a ubiquitin fold with a long structured loop for protein binding.

Trypanosoma brucei is a protist parasite causing sleeping sickness and nagana in sub-Saharan Africa. T. brucei has a single flagellum whose base contains a bulblike invagination of the plasma membrane called the flagellar pocket (FP). Around the neck of the FP on its cytoplasmic face is a structure called the flagellar pocket collar (FPC), which is essential for FP biogenesis. BILBO1 was the first characterized component of the FPC in trypanosomes. BILBO1's N-terminal domain (NTD) plays an essential role in T. brucei FPC biogenesis and is thus vital for the parasite's survival. Here, we report a 1.6-Å resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Results from mutagenesis experiments suggested that another FPC protein, FPC4, interacts with TbBILBO1 by mainly contacting its three conserved aromatic residues Trp-71, Tyr-87, and Phe-89 at the center of this pocket. Our findings disclose the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design targeting BILBO1 to combat T. brucei infections.

The UFM1 Pathway Impacts HCMV US2-Mediated Degradation of HLA Class I.

To prevent accumulation of misfolded proteins in the endoplasmic reticulum, chaperones perform quality control on newly translated proteins and redirect misfolded proteins to the cytosol for degradation by the ubiquitin-proteasome system. This pathway is called ER-associated protein degradation (ERAD). The human cytomegalovirus protein US2 induces accelerated ERAD of HLA class I molecules to prevent immune recognition of infected cells by CD8+ T cells. Using US2-mediated HLA-I degradation as a model for ERAD, we performed a genome-wide CRISPR/Cas9 library screen to identify novel cellular factors associated with ERAD. Besides the identification of known players such as TRC8, p97, and UBE2G2, the ubiquitin-fold modifier1 (UFM1) pathway was found to affect degradation of HLA-I. UFMylation is a post-translational modification resembling ubiquitination. Whereas we observe ubiquitination of HLA-I, no UFMylation was detected on HLA-I or several other proteins involved in degradation of HLA-I, suggesting that the UFM1 pathway impacts ERAD in a different manner than ubiquitin. Interference with the UFM1 pathway seems to specifically inhibit the ER-to-cytosol dislocation of HLA-I. In the absence of detectable UFMylation of HLA-I, UFM1 may contribute to US2-mediated HLA-I degradation by misdirecting protein sorting indirectly. Mass spectrometry analysis of US2-expressing cells showed that ribosomal proteins are a major class of proteins undergoing extensive UFMylation; the role of these changes in protein degradation may be indirect and remains to be established.

Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems.

Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation. The parasite asexual trophozoite stage is susceptible to iron-induced oxidative stress and is subsequently a target for many of the current anti-malarial drugs. Here, we provide evidence that SUMOylation plays a role in the parasite response to oxidative stress during red blood cell stages, indicative of a protective role seen in other organisms. Using x-ray crystallography, we solved the structure of the human SUMO E1 ubiquitin fold domain in complex with the E2, Ubc9. The interface defined in this structure guided in silico modeling, mutagenesis, and in vitro biochemical studies of the P. falciparum SUMO E1 and E2 enzymes, resulting in the identification of surface residues that explain species-specific interactions. Our findings suggest that parasite-specific inhibitors of SUMOylation could be developed and used in combination therapies with drugs that induce oxidative stress.

Autoantibodies binding to ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) are associated with mycobacterial infections.

OBJECTIVES: The diagnosis of extrapulmonary tuberculous infections and nontuberculous mycobacterial (NTM) infections is difficult because the symptoms are nonspecific and suitable specimens for bacterial culture are often not available. Recent publications reported the existence of autoantibodies in tuberculous infections. We screened for specific autoantibodies in mycobacterial infections. METHODS: We screened four in 29 patients with active mycobacterial infections and different controls using protein array technology. We could identify autoantibodies against ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) in all four patients. Subsequently, we designed enzyme-linked immunosorbent assays (ELISAs) for the detection of autoantibodies binding to Ufc1 and Plekhg2. RESULTS: Autoantibodies binding to Ufc1 and Plekhg2 were found in 19 of 29 patients (66%) with active mycobacterial infections. In comparison, we found these autoantibodies in one of 31 patients (3%) with successfully treated mycobacterial infections, in three of 40 (8%) HIV-infected patients not receiving combination antiretorviral therapy (cART) and in six of 134 (5%) blood donors. Interestingly, six of eight (75%) patients with HIV-associated B-cell non-Hodgkin lymphoma (B-NHL) at the onset of disease had autoantibodies against Ufc1 and Plekhg2, but none of nine (0%) patients after treatment of HIV-associated B-NHL, none of seven patients with non-HIV-associated B-NHL and 11 of 115 (10%) patients with other malignant diseases had autoantibodies against both proteins. CONCLUSIONS: In view of the high frequency of these autoantibodies, we postulate that they might be of potential use for additional diagnostics for mycobacterial infections, and further studies may shed light on the pathomechanisms of these two autoantibodies.

Cytoplasmic domain of NCAM140 interacts with ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1).

The neural cell adhesion molecule NCAM is implicated in different neurodevelopmental processes and in synaptic plasticity in adult brain. The cytoplasmic domain of NCAM interacts with several cytoskeletal proteins and signaling molecules. To identify novel interaction partners of the cytosolic domain of NCAM a protein macroarray has been performed. We identified the ubiquitin-fold modifier-conjugating enzyme-1 (Ufc1) as an interaction partner of NCAM140. Ufc1 is one of the enzymes involved in modification of proteins with the ubiquitin-like molecule ubiquitin-fold modifier-1 (Ufm1). We also observed a partial co-localization of NCAM140 with Ufc1 and Ufm1 and increased endocytosis of NCAM140 in the presence of Ufm1 suggesting a possible ufmylation of NCAM140 and a potential novel function of Ufm1 for cell surface proteins.

Modification of ERα by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development.

The post-translational modification (e.g., phosphorylation) of estrogen receptor α (ERα) plays a role in controlling the expression and subcellular localization of ERα as well as its sensitivity to hormone response. Here, we show that ERα is also modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability and transactivity of ERα, which in turn promotes breast cancer development. The elevation of ufmylation via the knockdown of UFSP2 (the UFM1-deconjugating enzyme in humans) dramatically increases ERα stability by inhibiting ubiquitination. In contrast, ERα stability is decreased by the prevention of ufmylation via the silencing of UBA5 (the UFM1-activating E1 enzyme). Lys171 and Lys180 of ERα were identified as the major UFM1 acceptor sites, and the replacement of both Lys residues by Arg (2KR mutation) markedly reduced ERα stability. Moreover, the 2KR mutation abrogated the 17ß-estradiol-induced transactivity of ERα and the expression of its downstream target genes, including pS2, cyclin D1, and c-Myc; this indicates that ERα ufmylation is required for its transactivation function. In addition, the 2KR mutation prevented anchorage-independent colony formation by MCF7 cells. Most notably, the expression of UFM1 and its conjugating machinery (i.e., UBA5, UFC1, UFL1, and UFBP1) were dramatically upregulated in ERα-positive breast cancer cell lines and tissues. Collectively, these findings implicate a critical role attributed to ERα ufmylation in breast cancer development by ameliorating its stability and transactivity.

Ubiquitin fold modifier 1 activates NF-κB pathway by down-regulating LZAP expression in the macrophage of diabetic mouse model.

Inflammatory response is closely related with the development of many serious health problems worldwide including diabetes mellitus (DM). Ubiquitin-fold modifer 1 (Ufm1) is a newly discovered ubiquitin-like protein, while its function remains poorly investigated, especially in inflammatory response and DM. In the present study, we analyzed the role of Ufm1 on inflammatory response in DM, and found that the proinflammatory cytokine levels (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß) and Ufm1 expression were highly increased both in the peritoneal macrophages of db/db mice and Raw264.7 cells induced by lipopolysaccharide (LPS). Western blot and luciferase reporter assay showed that NF-κB pathway was obviously activated in macrophages and the expression of LZAP, an inhibitor of NF-κB pathway, was down-regulated. With the LZAP knockdown plasmid and activation plasmid, we demonstrated that NF-κB/p65 activation was inhibited by LZAP in macrophages. The interaction of Ufm1 and LZAP was further proved with co-immunoprecipitation assay in HEK293 and Raw264.7 cells. The LZAP expression was also related with the presence of Ufm1 demonstrated by Ufm1 knockdown plasmid and activation plasmid. Besides that, we finally proved that the expression and activation of Ufm1 induced by LPS were regulated by JNK/ATF2 and JNK/c-Jun pathway with the use of SP600125. In conclusion, the present study demonstrated that Ufm 1 could activate NF-κB pathway by down-regulating LZAP in macrophage of diabetes, and its expression and activation were regulated by JNK/ATF2 and c-Jun pathway.

Ubiquitin-Fold Modifier-1 Participates in the Diabetic Inflammatory Response by Regulating NF-κB p65 Nuclear Translocation and the Ubiquitination and Degradation of IκBα.

BACKGROUND: Ubiquitin-fold modifier-1 (Ufm1) is a recently identified ubiquitin-like protein. We previously confirmed that Ufm1 expression was increased in diabetic mice. However, its role in the development of diabetes remains undefined. METHODS: Lentivirus-mediated gene knockdown and overexpression techniques were used to observe the effect of Ufm1 on the expression of inflammatory factors, adhesion molecules and chemokines, as well as the transcriptional activity of nuclear factor kappa-B (NF-κB) in macrophages. Western blot and immunofluorescence analyses were used to analyse the mechanism by which Ufm1 affects the transcriptional activity of NF-κB. Finally, the effects of Ufm1 on inflammation and pancreatic, renal and myocardial damage were observed in db/db mice. RESULTS: Knockdown of Ufm1 by lentivirus shRNA targeting Ufm1 (Lv-shUfm1) led to decreased secretion of IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2 in RAW264.7 cells that were exposed to LPS and TNF-α, while lentiviral overexpression of Ufm1 (Lv-Ufm1) caused the opposite effect. Interestingly, further investigation indicated that Ufm1 induced NF-κB p65 nuclear translocation in RAW264.7 cells via increasing the ubiquitination and degradation of IκBα. In an in vivo experiment, pretreatment of db/db mice with Lv-shUfm1 reduced the mRNA levels of TNF-α, IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2 in resident peritoneal macrophages (RPMs) and decreased the plasma levels of TNF-α, IL-6, IL-1ß, ICAM-1, VCAM-1, MCP-1 and CXCL2. Additionally, in Lv-Ufm1-treated mice, the inverse results were observed. Following treatment with Lv-shUfm1 and Lv-Ufm1, NF-κB p65 nuclear translocation in RPMs was decreased and increased, respectively. Importantly, we observed that Lv-shUfm1 injection led to a decrease in plasma glycaemia, a reduction in urinary albuminuria and cardiomyocyte hypertrophy and an improvement in the histopathological appearance of pancreatic, kidney and myocardial tissue. Pretreatment of the mice with Lv-shUfm1 inhibited macrophage infiltration in the pancreas, kidney and myocardial tissue. CONCLUSION: Our data elucidate a new biological function of Ufm1 that mediates inflammatory responses. Ufm1-mediated p65 nuclear translocation occurs by modulating the ubiquitination and degradation of IκBα. Moreover, downregulating Ufm1 is an effective strategy to prevent the development of type 2 diabetes and its complications.

UFL1 attenuates IL-1ß-induced inflammatory response in human osteoarthritis chondrocytes.

Osteoarthritis (OA) is a chronic inflammatory joint disease characterized by degradation of articular cartilage. Ubiquitin-fold modifier 1 (UFM1)-specific ligase 1 (UFL1) is an UFM1 E3 ligase that has been identified as a regulator of inflammatory response. However, the role of UFL1 in OA remains unknown. The aim of the present study was to explore the function of UFL1 in an in vitro OA system in chondrocytes. Our results showed that UFL1 was lowly expressed in both OA articular tissues and chondrocytes with IL-1ß induction. Ectopic expression of UFL1 improved cell viability of IL-1ß-induced chondrocytes. UFL1 suppressed the production of NO and PGE2, as well the expression levels of iNOS and COX-2 in IL-1ß-induced chondrocytes. The IL-1ß-induced increases in TNF-α and IL-6 levels were attenuated by UFL1. Ectopic expression of UFL1 inhibited the production of extracellular matrix (ECM) degrading enzymes including matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4 and ADAMTS-5 in chondrocytes with IL-1ß induction. Additionally, UFL1 suppressed IL-1ß-induced activation of NF-κB signaling pathway in chondrocytes. In conclusion, these findings indicated that UFL1 exerted protective effect on IL-1ß-induced chondrocytes. Thus, UFL1 might be a potential target for the treatment of OA.

Structural basis for Ufm1 recognition by UfSP.

Ubiquitin and ubiquitin-like proteins (Ubls) are involved in a variety of cellular functions, and dysfunction of these proteins often leads to disease, thus requiring the precise molecular recognition of the partner. Here, we report a structural basis for the recognition of Ufm1 by the Ufm1-specific protease (UfSP), both from Caenorhabditis elegans. Ufm1 functions in endoplasmic reticulum homeostasis, cell cycle regulation, and dysfunctions of this protein can result in breast cancer and neurological disorders. The structure reveals that in addition to the extended ß-structure at the C-terminus of cUfm1, the interactions made by the completely conserved residues in Ufm1 orthologs, Pro88-Val92, corresponding to P6-P2 positions from the cleavage site, seem to be important for the specific recognition of Ufm1 by cUfSP.

UFMylation: A Unique & Fashionable Modification for Life.

Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified ubiquitin-like proteins. Similar to ubiquitin, UFM1 is conjugated to its target proteins by a three-step enzymatic reaction. The UFM1-activating enzyme, ubiquitin-like modifier-activating enzyme 5 (UBA5), serves as the E1 to activate UFM1; UFM1-conjugating enzyme 1 (UFC1) acts as the E2 to transfer the activated UFM1 to the active site of the E2; and the UFM1-specific ligase 1 (UFL1) acts as the E3 to recognize its substrate, transfer, and ligate the UFM1 from E2 to the substrate. This process is called ufmylation. UFM1 chains can be cleaved from its target proteins by UFM1-specific proteases (UfSPs), suggesting that the ufmylation modification is reversible. UFM1 cascade is conserved among nearly all of the eukaryotic organisms, but not in yeast, and associated with several cellular activities including the endoplasmic reticulum stress response and hematopoiesis. Furthermore, the UFM1 cascade is closely related to a series of human diseases. In this review, we summarize the molecular details of this reversible modification process, the recent progress of its functional studies, as well as its implication in tumorigenesis and potential therapeutic targets for cancer.

Insights into autophagosome maturation revealed by the structures of ATG5 with its interacting partners.

Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12-ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar α-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.