Ubiquitin-fold Modifier Research Articles

An Enabling Peptide Ligation Induced by Thiol-Salicylaldehyde Ester for Chemical Protein Synthesis.

Chemical protein synthesis by amide-forming ligation of two unprotected peptide segments offers an effective strategy for the preparation of protein derivatives that are not accessible through bioengineering approaches. Herein, an unprecedented chemical ligation between peptides with C-terminal 2-mercaptobenzaldehyde (thiol-salicylaldehyde, TSAL) esters and peptides bearing N-terminal cysteine/penicillamine is reported. Reactive peptide TSAL esters can be obtained from peptide hydrazides in an operationally simple and highly effective manner. This chemoselective peptide ligation enables the rapid production of N,S-benzylidene acetal intermediates, which can readily be converted into native amide bonds even at sterically hindered junctions. In addition, the current method can be applied compatibly in concert with other types of ligations and subsequent desulfurization chemistry, thereby facilitating convergent protein synthesis. The effectiveness of this new method is also showcased by the total synthesis of proteins ubiquitin and hyalomin-3 (Hyal-3), the efficient synthesis of protein ubiquitin-fold modifier 1 (UFM1) via a C-to-N sequential TSAL ester-induced ligation strategy, and the chemical synthesis of protein Mtb CM through a combined strategy of Ser/Thr ligation and TSAL ester-induced ligations.

Panaxatriol exerts anti-senescence effects and alleviates osteoarthritis and cartilage repair fibrosis by targeting UFL1

IntroductionOsteoarthritis (OA), the most common degenerative joint disease, can eventually lead to disability. However, no safe or effective intervention is currently available. Therefore, there is an urgent need to develop effective drugs that reduce cartilage damage and treat OA. ObjectivesThis study aimed to ascertain the potential of panaxatriol, a natural small molecule, as a therapeutic drug for alleviating the progression of OA. MethodsAn in vitro culture of human cartilage explants and C28/I2 human chondrocytes and an in vivo surgically induced OA mouse model were used to evaluate the chondroprotective effect of panaxatriol. The Drug Affinity Responsive Target Stability assay, CRISPR-Cas9 assay, Whole-transcriptome RNA sequencing analysis and agonist or antagonist assays were used to identify the target and potential signaling pathways of panaxatriol. Poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) was used to construct the sustained-release system of panaxatriol. ResultsPanaxatriol protected against OA by regulating chondrocyte metabolism. Ubiquitin-fold modifier 1-specific E3 ligase 1 (UFL1) was identified as a novel target of panaxatriol. Whole transcriptome RNA sequencing showed that UFL1 was closely related to cell senescence. Panaxatriol inhibited chondrocyte senescence through UFL1/forkhead box O1 (FOXO1)/P21 and UFL1/NF-κB/SASPs signaling pathways. It also could inhibit fibrocartilage formation during cartilage repair via the UFL1/FOXO1/Collagen 1 signaling pathway. Finally, we constructed a sustained-release system for panaxatriol based on PLGA-PEG, which reduced the number of intra-articular injections, thereby alleviating joint swelling and injury. ConclusionsPanaxatriol exerts anti-senescence effects and has the potential to delay OA progression and reduce cartilage repair fibrosis by targeting UFL1.

The emerging roles of UFMylation in the modulation of immune responses.

Post-translational modification is a rite of passage for cellular functional proteins and ultimately regulate almost all aspects of life. Ubiquitin-fold modifier 1 (UFM1) system represents a newly identified ubiquitin-like modification system with indispensable biological functions, and the underlying biological mechanisms remain largely undiscovered. The field has recently experienced a rapid growth of research revealing that UFMylation directly or indirectly regulates multiple immune processes. Here, we summarised important advances that how UFMylation system responds to intrinsic and extrinsic stresses under certain physiological or pathological conditions and safeguards immune homeostasis, providing novel perspectives into the regulatory framework and functions of UFMylation system, and its therapeutic applications in human diseases.

Abstract Or109: Regulation of UFM1 modification from ER dynamics to cardiac homeostasis

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. Understanding the molecular mechanism governing CVD has become a major focus in developing new therapeutics. Ubiquitin (Ub) like protein post-translational modifications (PTMs) have been known to play an important role in the normal functioning of the heart. UFM1 (ubiquitin-fold modifier 1) is a novel Ub-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade, named ufmylation. Ufmylation regulates diverse cellular processes and is implicated in various human diseases. However, its importance in the heart is unknown. For the very first time, we have reported dysregulation of ufmylation in dilated cardiomyopathy, hypertrophic human patients, and animal models. It remains unclear whether ufmylation of cardiac proteins is required for heart function and if so, how ufmylation regulates cardiac function. To determine the role of UFM1 modification in cardiomyocytes we have generated cardiac-specific UFM1-knockout (UFM1CKO) mice. The UFM1CKO mice developed dilated cardiomyopathy at resting condition, as indicated by the left ventricle (LV) wall thinning, chamber dilatation, and impaired contractility at 4 months of age, which were further exacerbated by 6 months. It appears that UFM1 modification constrains pathological cardiac remodeling by maintaining ER homeostasis and regulating ER stress response. To test the hypothesis that UFM1 modification, instead of UFM1 itself is required to maintain cardiac function, we replenished UFM1CKO mice with cardiac-specific WT or conjugation-deficient UFM1 via AAV9. By echocardiography, we revealed that, unlike WT UFM1, overexpression of the conjugation-deficient mutant failed to attenuate the cardiac remodeling and dysfunction of the UFM1CKO heart. Interestingly, the conjugation-deficient mutant further exacerbated the cardiac dysfunction of the UFM1CKO mice. Extensive fibrosis and upregulation of ER stress response protein were also noted in conjugation-deficient mutant UFM1 injected UFM1CKO heart. Overall this study indicates that modification of proteins by UFM1 is critical to cardiac function via maintaining ER homeostasis.

Bombyx mori UFL1 facilitates BmNPV proliferation by regulating host cell apoptosis through PERK.

Ubiquitin-fold modifier 1 (UFM1) is attached to protein substrates through the sequential activity of an E1 (UBA5)-E2 (UFC1)-E3 (UFL1) cascade. UFL1 is the E3 ligase for UFMylation in vertebrates. However, there have been no studies on UFL1 in silkworm to date. In this study, we identified a UFL1 ortholog in Bombyx mori genome. Spatio-temporal expression profiles showed that BmUFL1 expression was high in the midgut, epidermis, and testis and in the pupa-adult stage. BmUFL1 knockdown inhibited B. mori nucleopolyhedrovirus (BmNPV)proliferation, while BmUFL1 overexpression promoted BmNPV proliferation. Mechanically, protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling and cell apoptosis are involved in BmUFL1-regulatedBmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm.

The ufmylation cascade controls COPII recruitment, anterograde transport, and sorting of nascent GPCRs at ER.

Ufmylation is implicated in multiple cellular processes, but little is known about its functions and regulation in protein trafficking. Here, we demonstrate that the genetic depletion of core components of the ufmylation cascade, including ubiquitin-fold modifier 1 (UFM1), UFM1 activation enzyme 5, UFM1-specific ligase 1 (UFL1), UFM1-specific protease 2, and UFM1-binding protein 1 (UFBP1) each markedly inhibits the endoplasmic reticulum (ER)-Golgi transport, surface delivery, and recruitment to COPII vesicles of a subset of G protein-coupled receptors (GPCRs) and UFBP1's function partially relies on UFM1 conjugation. We also show that UFBP1 and UFL1 interact with GPCRs and UFBP1 localizes at COPII vesicles coated with specific Sec24 isoforms. Furthermore, the UFBP1/UFL1-binding domain identified in the receptors effectively converts non-GPCR protein transport into the ufmylation-dependent pathway. Collectively, these data reveal important functions for the ufmylation system in GPCR recruitment to COPII vesicles, biosynthetic transport, and sorting at ER via UFBP1 ufmylation and interaction directly.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis in Pancreatic Ductal Adenocarcinoma.

Induction of ferroptosis can inhibit cancer cells in vitro, however, the role of ferroptosis in treatment in vivo is controversial. The immunosuppressive cells activated by the ferroptotic tumor cells can promote the growth of residual tumor cells, hindering the application of ferroptosis stimulation in tumor treatment. In this study, a new strategy is aimed to be identified for effectively triggering immunogenic ferroptosis in pancreatic ductal adenocarcinoma (PDAC) and simultaneously stimulating antitumor immune responses. Toward this, several molecular and biochemical experiments are performed using patient-derived organoid models and a KPC mouse model (LSL-KrasG12D /+, LSL-Trp53R172H/+,Pdx-1-Cre). It is observed that the inhibition of macrophage-capping protein (MCP) suppressed the ubiquitin fold modifier (UFM)ylation of pirin (PIR), a newly identified substrate of UFM1, thereby decreasing the transcription of GPX4, a marker of ferroptosis, and promoting the cytoplasmic transportation of HMGB1, a damage-associated molecular pattern. GPX4 deficiency triggered ferroptosis, and the pre-accumulated cytosolic HMGB1 is released rapidly. This altered release pattern of HMGB1 facilitated the pro-inflammatory M1-like polarization of macrophages. Thus, therapeutic inhibition of MCP yielded dual antitumor effects by stimulating ferroptosis and activating antitumor pro-inflammatory M1-like macrophages. The nanosystem developed for specifically silencing MCP is a promising tool for treating PDAC.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

Inhibition of UFM1 expression suppresses cancer progression and is linked to the dismal prognosis and immune infiltration in oral squamous cell carcinoma.

Ubiquitin fold modifier 1 (UFM1) overexpression is associated with cancer cell proliferation, migration and invasion. However, the roles and pathways of UFM1 in oral squamous cell carcinoma (OSCC) has remained undefined. The expression of UFM1 and the relationship between UFM1 expression and prognosis were investigated using data of OSCC patients from The Cancer Genome Atlas (TCGA) database. The UFM1 co-expressed genes, and the association between the UFM1 expression and immune cells and ubiquitination were explored. The effects of UFM1 expression on the growth and migration of OSCC cells were investigated by siRNA interference, Cell Counting Kit-8 (CCK-8), Transwell, Western blotting, and wound healing experiments. UFM1 was highly expressed in OSCC. UFM1 overexpression was associated with short overall survival, disease-specific survival, and progression-free interval, and was an adverse factor for prognosis in OSCC. UFM1-related nomograms were significantly associated with poor prognosis in OSCC patients. Decreased UFM1 expression could inhibit the proliferation, migration, and invasion of OSCC cells. UFM1 was associated with the immune cells (such as the Th17 cells, T helper cells, and cytotoxic cells) and ubiquitination. Elevated UFM1 expression was associated with poor prognosis, ubiquitination and immune infiltration in OSCC, and inhibition of UFM1 expression delayed OSCC progression, showing that UFM1 could be a biomarker for prognosis and treating OSCC patients.

Abstract 19192: Deciphering the Cardioprotective Role of Ufm1 Modification in Cardiac Remodeling

UFM1 (ubiquitin-fold modifier 1) is a novel Ub-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade. UFM1 modification (ufmylation) controls multiple pathophysiological events and is implicated in various human diseases. However, the significance of ufmylation in cardiac health remains poorly understood. We have previously reported that ufmylation is upregulated in compensated, hypertrophic mouse hearts but decreased in failing human hearts. Inhibition of ufmylation by targeted ablation of the E3 UFM1 ligase 1 (UFL1) in mouse hearts resulted in dilated cardiomyopathy under baseline and stress conditions. Targeted deletion of a putative UFM1 target, UFBP1, also evokes endoplasmic reticulum (ER) stress, leading to dilated cardiomyopathy While these exciting data reveal the pivotal role of ufmylation in the maintenance of ER/cardiac homeostasis, it remains unclear whether ufmylation of cardiac proteins is required for heart function and if so, how ufmylation regulates cardiac function. To determine the role of UFM1 modification in cardiomyocytes we have generated cardiac specific UFM1 CKO mice. The UFM1 CKO mice developed dilated cardiomyopathy at resting condition, as indicated by left ventricle (LV) wall thinning, chamber dilatation and impaired contractility at 4 months of age, which were further exacerbated by 6 months. To test the hypothesis that UFM1 protects the heart by modifying cardiac proteins we replenished UFM1-deficient mice with WT and conjugation deficient UFM1 via AAV9. Restoration of WT-UFM1 expression, but not the conjugation-deficient mutant, attenuated cardiac dysfunction of UFM1CKO hearts. It appears that UFM1 modification constraints pathological cardiac remodeling by maintaining ER homeostasis. Taken together, our data show that Ufmylation is a critical post-translational modification that regulates cardiac homeostasis.

The Post-Translational Role of UFMylation in Physiology and Disease.

Ubiquitin-fold modifier 1 (UFM1) is a newly identified ubiquitin-like protein that has been conserved during the evolution of multicellular organisms. In a similar manner to ubiquitin, UFM1 can become covalently linked to the lysine residue of a substrate via a dedicated enzymatic cascade. Although a limited number of substrates have been identified so far, UFM1 modification (UFMylation) has been demonstrated to play a vital role in a variety of cellular activities, including mammalian development, ribosome biogenesis, the DNA damage response, endoplasmic reticulum stress responses, immune responses, and tumorigenesis. In this review, we summarize what is known about the UFM1 enzymatic cascade and its biological functions, and discuss its recently identified substrates. We also explore the pathological role of UFMylation in human disease and the corresponding potential therapeutic targets and strategies.

Ufmylation on UFBP1 alleviates non-alcoholic fatty liver disease by modulating hepatic endoplasmic reticulum stress

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease characterized by lipid accumulation and endoplasmic reticulum (ER) stress, while effective therapies targeting the specific characteristics of NAFLD are limited. Ufmylation is a newly found post-translational modification process that involves the attachment of the Ubiquitin-fold modifier 1 (UFM1) protein to its substrates via ufmylation modification system. Ufmylation regulates ER stress via modifying UFM1 binding protein 1 (UFBP1), suggesting a potential role for ufmylation in NAFLD pathogenesis. However, the precise role of ufmylation in NAFLD remains unclear. Herein, we aim to elucidate the impact of ufmylation on UFBP1 in NAFLD and explore the underlying mechanisms involved. We observed increased expression of UFM1-conjugated proteins and ufmylation modification system components in livers with steatosis derived from NAFLD patients and NAFLD models. Upregulation of ufmylation on hepatic proteins appeared to be an adaptive response to hepatic ER stress in NAFLD. In vitro, knocking down UFBP1 resulted in increased lipid accumulation and lipogenesis in hepatocytes treated with free fatty acids (FFA), which could be rescued by wild-type UFBP1 (WT UFBP1) but not by a mutant form of UFBP1 lacking the main ufmylation site lys267 (UFBP1 K267R). In vivo, ufmylation on UFBP1 ameliorated obesity, hepatic steatosis, hepatic lipogenesis, dyslipidemia, insulin resistance and liver damage in mice with NAFLD induced by a high fat diet (HFD). We also demonstrated that the downregulation of UFBP1 induced ER stress, whereas the reintroduction or overexpression of UFBP1 alleviated ER stress in a manner dependent on ufmylation in NAFLD. This mechanism could be responsible for the amelioration of aberrant hepatic lipogenesis and insulin resistance in NAFLD. Our data reveal a protective role of ufmylation on UFBP1 against NAFLD and offer a specific target for NAFLD treatment.

Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control.

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.

Ufmylation of UFBP1 Is Dispensable for Endoplasmic Reticulum Stress Response, Embryonic Development, and Cardiac and Intestinal Homeostasis.

Protein modification by ubiquitin fold modifier 1 (UFM1), termed ufmylation, regulates various physiological and pathological processes. Among emerging UFM1 targets, UFM1 binding protein 1 (UFBP1) is the first identified ufmylation substrate. Recent clinical and animal studies have demonstrated the pivotal roles of UFBP1 in development, hematopoiesis, intestinal homeostasis, chondrogenesis, and neuronal development, which has been linked to its function in maintaining endoplasmic reticulum (ER) homeostasis. However, the importance of UFBP1 ufmylation in these cellular and physiological processes has yet to be determined. It has been proposed that ufmylation of lysine 268 (267 in humans) in UFBP1 plays a critical role in mediating the effects of the ufmylation pathway. In this study, we for the first time probe the pathophysiological significance of UFBP1 ufmylation in vivo by creating and characterizing a mouse UFBP1 knockin (KI) model in which the lysine 268 of UFBP1, the amino acid accepting UFM1, was mutated to arginine. Our results showed that the K268R mutation reduced the total ufmylated proteins without altering the expression levels of individual ufmylation enzymes in mouse embryonic fibroblasts. The K268R mutation did not alter ER stress-stimuli-induced ER stress signaling or cell death in mouse embryonic fibroblasts. The homozygous KI mice were viable and morphologically indistinguishable from their littermate wild-type controls up to one year of age. Serial echocardiography revealed no cardiac functional impairment of the homozygous KI mice. Furthermore, the homozygous KI mice exhibited the same susceptibility to dextran sulfate sodium (DSS) -induced colitis as wild-type mice. Taken together, these results suggest that UFBP1 K268 is dispensable for ER stress response, embryonic development, cardiac homeostasis under physiological conditions, and intestinal homeostasis under pathological conditions. Our studies call for future investigations to understand the biological function of UFBP1 ufmylation and offer a new mouse model to determine the roles of UFBP1 ufmylation in different tissues under stress conditions.

Characterization of a silkworm UFM1 homolog in regulating Bombyx mori unfolded protein response and nucleopolyhedrovirus replication

The Ubiquitin (Ub)-like molecules is essential for animal development and the physiopathology of multiple tissues in the vertebrate. Ubiquitin-fold modifier 1 (UFM1) is one of the newly-identified UBL, which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. However, there are no studies on UFMylation and the effect of UFMylation on BmNPV replication in silkworm. In this study, we identified BmUFM1 in the B. mori genome. Spatio-Temporal expression profiles showed that BmUFM1 expression was highly in hemocytes and response to various pathogenic stimuli. Furthermore, BmUFM1 is involved in the regulation of ER stress induced Unfolded Protein Response (UPR) and knockdown of BmUFM1 inhibited BmNPV replication. Overall, these results suggest that BmUFM1 plays an important role in facilitating BmNPV proliferation in silkworm. Our findings advance the understanding of UFM1's conjugation machinery, and also provides a potentially molecular target for BmNPV prevention and silkworm breeding.

The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice

SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.

Loss of Ufl1/Ufbp1 in hepatocytes promotes liver pathological damage and carcinogenesis through activating mTOR signaling

BackgroundUfm1-specific ligase 1 (Ufl1) and Ufm1-binding protein 1 (Ufbp1), as putative targets of ubiquitin-fold modifier 1 (Ufm1), have been implicated in several pathogenesis-related signaling pathways. However, little is known about their functional roles in liver disease.MethodsHepatocyte-specific Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice were used to study their role in liver injury. Fatty liver disease and liver cancer were induced by high-fat diet (HFD) and diethylnitrosamine (DEN) administration, respectively. iTRAQ analysis was employed to screen for downstream targets affected by Ufbp1 deletion. Co-immunoprecipitation was used to determine the interactions between the Ufl1/Ufbp1 complex and the mTOR/GβL complex.ResultsUfl1Δ/Δhep or Ufbp1Δ/Δhep mice exhibited hepatocyte apoptosis and mild steatosis at 2 months of age and hepatocellular ballooning, extensive fibrosis, and steatohepatitis at 6–8 months of age. More than 50% of Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice developed spontaneous hepatocellular carcinoma (HCC) by 14 months of age. Moreover, Ufl1Δ/Δhep and Ufbp1Δ/Δhep mice were more susceptible to HFD-induced fatty liver and DEN-induced HCC. Mechanistically, the Ufl1/Ufbp1 complex directly interacts with the mTOR/GβL complex and attenuates mTORC1 activity. Ablation of Ufl1 or Ufbp1 in hepatocytes dissociates them from the mTOR/GβL complex and activates oncogenic mTOR signaling to drive HCC development.ConclusionsThese findings reveal the potential role of Ufl1 and Ufbp1 as gatekeepers to prevent liver fibrosis and subsequent steatohepatitis and HCC development by inhibiting the mTOR pathway.

The UFM1 conjugation system in mammalian development.

Posttranslational modifications by ubiquitin and ubiquitin-like proteins are important in regulating cellular protein functions. UFM1 (ubiquitin-fold modifier 1), first identified almost two decades ago, is a member of the ubiquitin-like protein family. UFM1 is covalently conjugated to the target proteins in an enzymatic cascade consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. At the molecular level, modification by UFM1 (UFMylation) is an important mediator of the protein function. Dysregulation of the UFM1 system, e.g., the knockout of UFMylation components, disturbs proteome homeostasis and triggers endoplasmic reticulum stress. Such changes are linked to developmental disorders, tumorigenesis, tissue injury, inflammation, and several hereditary neurological syndromes. This review will focus on the role of the UFMylation in animal development and associated congenital disorders. We will cover the hematopoietic system, liver, central nervous system, intestine, heart, kidney, immune, and skeletal system to provide insight into disease pathogenesis and shed light on possible novel therapeutic methods.

Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.

Immunotherapy of PD-L1/PD-1 blockage elicited impressive clinical benefits forcancer treatment. However, the relative low response and therapy resistance highlight the need to better understand the molecular regulation of PD-L1 in tumors. Here, we report that PD-L1 is a target of UFMylation. UFMylation of PD-L1 destabilizes PD-L1 by synergizing its ubiquitination. Inhibition of PD-L1 UFMylation via silencing of UFL1 or Ubiquitin-fold modifier 1 (UFM1), or the defective UFMylation of PD-L1, stabilizes the PD-L1 in multiple human and murine cancer cells, and undermines antitumor immunity invitro and mice, respectively. Clinically, UFL1 expression was decreased in multiple cancers and lower expression of UFL1 negatively correlated with the response of anti-PD1 therapy in melanoma patients. Moreover, we identified a covalent inhibitor of UFSP2 that promoted the UFMylation activity and contributed to the combination therapy with PD-1 blockade. Our findings identified a previously unrecognized regulator of PD-L1 and highlighted UFMylation as a potential therapeutic target.