Systematic HOIP interactome profiling reveals critical roles of linear ubiquitination in tissue homeostasis.

Linear ubiquitination catalyzed by HOIL-1-interacting protein (HOIP), the key component of the linear ubiquitination assembly complex, plays fundamental roles in tissue homeostasis by executing domain-specific regulatory functions. However, a proteome-wide analysis of the domain-specific interactome of HOIP across tissues is lacking. Here, we present a comprehensive mass spectrometry-based interactome profiling of four HOIP domains in nine mouse tissues. The interaction dataset provides a high-quality HOIP interactome resource with an average of approximately 90 interactors for each bait per tissue. HOIP tissue interactome presents a systematic understanding of linear ubiquitination functions in each tissue and also shows associations of tissue functions to genetic diseases. HOIP domain interactome characterizes a set of previously undefined linear ubiquitinated substrates and elucidates the cross-talk among HOIP domains in physiological and pathological processes. Moreover, we show that linear ubiquitination of Integrin-linked protein kinase (ILK) decreases focal adhesion formation and promotes the detachment of Shigella flexneri-infected cells. Meanwhile, Hoip deficiency decreases the linear ubiquitination of Smad ubiquitination regulatory factor 1 (SMURF1) and enhances its E3 activity, finally causing a reduced bone mass phenotype in mice. Overall, our work expands the knowledge of HOIP-interacting proteins and provides a platform for further discovery of linear ubiquitination functions in tissue homeostasis.

A Golgi-resident GPR108 cooperates with E3 ubiquitin ligase Smurf1 to suppress antiviral innate immunity.

The regulation of antiviral immunity is crucial in maintaining host immune homeostasis, a process that involves dynamic modulations of host organelles. The Golgi apparatus is increasingly perceived as a host organelle functioning as a critical platform for innate immunity, but the detailed mechanism by which it regulates antiviral immunity remains elusive. Here, we identify the Golgi-localized G protein-coupled receptor 108 (GPR108) as a regulator of type Ι interferon responses by targeting interferon regulatory factor 3 (IRF3). Mechanistically, GPR108 enhances the ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1)-mediated K63-linked polyubiquitination of phosphorylated IRF3 for nuclear dot 10 protein 52 (NDP52)-dependent autophagic degradation, leading to suppression of antiviral immune responses against DNA or RNA viruses. Taken together, our study provides insight into the crosstalk between the Golgi apparatus and antiviral immunity via a dynamic and spatiotemporal regulation of GPR108-Smurf1 axis, thereby indicating a potential target for treating viral infection.

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1's E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.

OTUB2 exerts tumor-suppressive roles via STAT1-mediated CALML3 activation and increased phosphatidylserine synthesis.

Oral and esophageal squamous cell carcinomas (SCCs) are associated with high mortality, yet the molecular mechanisms underlying these malignancies are largely unclear. We show that DNA hypermethylation of otubain 2 (OTUB2), a previously recognized oncogene, drives tongue and esophageal SCC initiation and drug resistance. Mechanistically, OTUB2 promotes the deubiquitination and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and subsequently regulates the transcription of calmodulin-like protein 3 (CALML3). Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation (OXPHOS) and the synthesis of phosphatidylserine (PS). In mouse models, orally administered soybean-derived PS inhibits SCC initiation in cells with low OTUB2 expression and increases their sensitivity to chemotherapy. Our study indicates that the OTUB2/STAT1/CALML3/PS axis plays tumor-suppressive roles and shows the potential of PS administration as a strategy for the treatment and prevention of tongue and esophageal SCCs.

The deubiquitinase OTUD1 inhibits colonic inflammation by suppressing RIPK1-mediated NF-κB signaling.

The E3 ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination processes are closely associated with the occurrence and development of colonic inflammation. Ovarian tumor deubiquitinase 1 (OTUD1) is involved in immunoregulatory functions linked to infectious diseases. However, the effect of OTUD1 on intestinal immune responses during colonic inflammatory disorders such as inflammatory bowel disease (IBD) remains unclear. Here, we show that loss of OTUD1 in mice contributes to the pathogenesis of dextran sulfate sodium (DSS)-induced colitis via excessive release of proinflammatory cytokines. In addition, bone marrow transplantation experiments revealed that OTUD1 in hematopoietic cells plays a dominant role in protection against colitis. Mechanistically, OTUD1 physically interacts with receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and selectively cleaves K63-linked polyubiquitin chains from RIPK1 to inhibit the recruitment of NF-κB essential modulator (NEMO). Moreover, the expression of OTUD1 in mucosa samples from ulcerative colitis (UC) patients was lower than that in mucosa samples from healthy controls. Furthermore, we demonstrate that the UC-associated OTUD1 G430V mutation abolishes the ability of OTUD1 to inhibit RIPK1-mediated NF-κB activation and intestinal inflammation. Taken together, our study unveils a previously unexplored role of OTUD1 in moderating intestinal inflammation by inhibiting RIPK1-mediated NF-κB activation, suggesting that the OTUD1-RIPK1 axis could be a potential target for the treatment of IBD.

Global Screening of LUBAC and OTULIN Interacting Proteins by Human Proteome Microarray.

Linear ubiquitination is a reversible posttranslational modification, which plays key roles in multiple biological processes. Linear ubiquitin chain assembly complex (LUBAC) catalyzes linear ubiquitination, while the deubiquitinase OTULIN (OTU deubiquitinase with linear linkage specificity, FAM105B) exclusively cleaves the linear ubiquitin chains. However, our understanding of linear ubiquitination is restricted to a few substrates and pathways. Here we used a human proteome microarray to detect the interacting proteins of LUBAC and OTULIN by systematically screening up to 20,000 proteins. We identified many potential interacting proteins of LUBAC and OTULIN, which may function as regulators or substrates of linear ubiquitination. Interestingly, our results also hint that linear ubiquitination may have broad functions in diverse pathways. In addition, we recognized lymphocyte activation gene-3 (LAG3, CD223), a transmembrane receptor that negatively regulates lymphocyte functions as a novel substrate of linear ubiquitination in the adaptive immunity pathway. In conclusion, our results provide searchable, accessible data for the interacting proteins of LUBAC and OTULIN, which broaden our understanding of linear ubiquitination.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

The functions and regulation of Otubains in protein homeostasis and diseases.

OTU domain-containing ubiquitin aldehyde-binding proteins Otubain1 (OTUB1) and Otubain2 (OTUB2) were initially identified as OTU deubiquitinases (DUBs). Recently, Otubains have emerged as essential regulators of diverse physiological processes, such as immune signaling and DNA damage response. Dysregulation of those processes is likely to increase the risk in multiple aspects of aging-related diseases, including cancers, neurodegenerative disorders, chronic kidney diseases, bone dysplasia and pulmonary fibrosis. Consistently, Otubains are aberrantly expressed in cancers and have been identified to be both tumor suppressors and tumor promoters in different types of cancers. Therefore, the regulatory mechanism of the activity and expression of Otubains is very important for better understanding of Otubains-associated biological networks and human diseases. This review provides a comprehensive description of functions and regulatory axis of Otubains, highlighting experimental evidences indicating Otubains as potential therapeutic targets against aging-related disorders.

CKIP-1 limits foam cell formation and inhibits atherosclerosis by promoting degradation of Oct-1 by REGγ.

Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. Macrophages uptake modified lipoproteins and transform into foam cells, triggering an inflammatory response and thereby promoting plaque formation. Here we show that casein kinase 2-interacting protein-1 (CKIP-1) is a suppressor of foam cell formation and atherosclerosis. Ckip-1 deficiency in mice leads to increased lipoprotein uptake and foam cell formation, indicating a protective role of CKIP-1 in this process. Ablation of Ckip-1 specifically upregulates the transcription of scavenger receptor LOX-1, but not that of CD36 and SR-A. Mechanistically, CKIP-1 interacts with the proteasome activator REGγ and targets the transcriptional factor Oct-1 for degradation, thereby suppressing the transcription of LOX-1 by Oct-1. Moreover, Ckip-1-deficient mice undergo accelerated atherosclerosis, and bone marrow transplantation reveals that Ckip-1 deficiency in hematopoietic cells is sufficient to increase atherosclerotic plaque formation. Therefore, CKIP-1 plays an essential anti-atherosclerotic role through regulation of foam cell formation and cholesterol metabolism.

NEDL2 is an essential regulator of enteric neural development and GDNF/Ret signaling.

Although glial cell line-derived neurotrophic factor (GDNF)/Ret signaling is essential for enteric nervous system (ENS) development, the positive regulators regulating GDNF/Ret signaling and controlling ENS development are poorly understood. Here, we show that Nedd4-related E3 ubiquitin ligase-2 (NEDL2) plays an essential and positive physiological role in regulating ENS development and GDNF/Ret signaling. All of the NEDL2-deficient mice die within 2weeks after birth, showing low body weight. These mice showed a progressive bowel motility defect resulting from intestinal aganglionosis. We show that NEDL2 positively regulates enteric neural precursor proliferation through the GDNF/Akt signaling pathway. Together, these findings unveil the physiological function of NEDL2 in vivo.

The HECT type ubiquitin ligase NEDL2 is degraded by anaphase-promoting complex/cyclosome (APC/C)-Cdh1, and its tight regulation maintains the metaphase to anaphase transition.

NEDD4-like ubiquitin ligase 2 (NEDL2) is a HECT type ubiquitin ligase. NEDL2 enhances p73 transcriptional activity and degrades ATR kinase in lamin misexpressed cells. Compared with the important functions of other HECT type ubiquitin ligase, there is less study concerning the function and regulation of NEDL2. Using primary antibody immunoprecipitation and mass spectrometry, we identify a list of potential proteins that are putative NEDL2-interacting proteins. The candidate list contains many of mitotic proteins, especially including several subunits of anaphase-promoting complex/cyclosome (APC/C) and Cdh1, an activator of APC/C. Cdh1 can interact with NEDL2 in vivo and in vitro. Cdh1 recognizes one of the NEDL2 destruction boxes (R(740)GSL(743)) and targets it for degradation in an APC/C-dependent manner during mitotic exit. Overexpression of Cdh1 reduces the protein level of NEDL2, whereas knockdown of Cdh1 increases the protein level of NEDL2 but has no effect on the NEDL2 mRNA level. NEDL2 associates with mitotic spindles, and its protein level reaches a maximum in mitosis. The function of NEDL2 during mitosis is essential because NEDL2 depletion prolongs metaphase, and overexpression of NEDL2 induces chromosomal lagging. Elevated expression of NEDL2 protein and mRNA are both found in colon cancer and cervix cancer. We conclude that NEDL2 is a novel substrate of APC/C-Cdh1 as cells exit mitosis and functions as a regulator of the metaphase to anaphase transition. Its overexpression may contribute to tumorigenesis.

[Changes of granulocyte distribution induced by thyroxin and epinephrine: experiment with rabbits].

OBJECTIVE: To investigate the distribution of granulocyte during thyrotoxicosis and the relation of granulopenia and the gathering of circulating granulocytes into the marginal pool. METHODS: Forty-five rabbits were randomly divided into 3 equal groups: epinephrine group fed with levo-thyroxin (LT4) for 14 days, injected subcutaneously with epinephrine 0.07 mg/kg, and undergoing collection of peripheral blood samples for white blood cell and granulocyte counts, free blood triiodothyroxine (FT3) and free blood thyroxine (FT4), electrocardiography, and body weight measurement; labeled group fed with LT4 for 14 days, injected subcutaneously with 131I-labeled anti-human granulocyte monoclonal antibody, and killed 6 hours later to calculate the ratio of radioactivity of heart, liver, spleen, and muscle tissues to blood (CPM/g), and control group, not fed with L-T4 but injected with 131I-labeled anti-human granulocyte monoclonal antibody on the day 14, and killed 6 hours later to calculate the ratio of radioactivity of tissues to blood. RESULTS: After 14-day feeding of L-T4, the heart rates of the epinephrine and labeled groups were significantly higher than those before injection, and the FT3 and FT4 levels were significantly increased;the venous WBC and granulocyte counts were significantly reduced. But the granulocyte count of 13 of the 15 rabbits in the epinephrine group increased 20 minutes after the injection of epinephrine, even becoming 2-4.8 times as high as those before the injection in 7 rabbits. The heart, liver, spleen, and muscle tissues to blood CPM/g ratios of the labeled group were all significantly higher than those of the control group (all P<0.01). CONCLUSION: During thyrotoxicosis the circulating granulocytes are reduced and the distribution of circulating granulocytes is abnormal with a gathering phenomenon of the granulocytes into the marginal pool.