SEL1L-HRD1 interaction is required to form a functional HRD1 ERAD complex.

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD). Despite recent advances in both mouse models and humans, in vivo evidence for the importance of SEL1L in the ERAD complex formation and its (patho-)physiological relevance in mammals remains limited. Here we report that SEL1L variant p.Ser658Pro (SEL1LS658P) is a pathogenic hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Biochemical analyses reveal that SEL1LS658P variant not only reduces the protein stability of SEL1L, but attenuates the SEL1L-HRD1 interaction, likely via electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes reveal that SEL1L-HRD1 interaction is a prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L is required for the recruitment of E2 enzyme UBE2J1 as well as DERLIN to HRD1. These data not only establish the disease relevance of SEL1L-HRD1 ERAD, but also provide additional insight into the formation of a functional HRD1 ERAD complex.

Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins.

Hypomorphic variants of SEL1L-HRD1 ER-associated degradation are associated with neurodevelopmental disorders.

Recent studies using cell type-specific knockout mouse models have improved our understanding of the pathophysiological relevance of suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) endoplasmic reticulum-associated (ER-associated) degradation (ERAD); however, its importance in humans remains unclear, as no disease variant has been identified. Here, we report the identification of 3 biallelic missense variants of SEL1L and HRD1 (or SYVN1) in 6 children from 3 independent families presenting with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, hypotonia, and/or ataxia. These SEL1L (p.Gly585Asp, p.Met528Arg) and HRD1 (p.Pro398Leu) variants were hypomorphic and impaired ERAD function at distinct steps of ERAD, including substrate recruitment (SEL1L p.Gly585Asp), SEL1L-HRD1 complex formation (SEL1L p.Met528Arg), and HRD1 activity (HRD1 p.Pro398Leu). Our study not only provides insights into the structure-function relationship of SEL1L-HRD1 ERAD, but also establishes the importance of SEL1L-HRD1 ERAD in humans.

Biallelic Cys141Tyr variant of SEL1L is associated with neurodevelopmental disorders, agammaglobulinemia, and premature death.

Suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) ER-associated degradation (ERAD) plays a critical role in many physiological processes in mice, including immunity, water homeostasis, and energy metabolism; however, its relevance and importance in humans remain unclear, as no disease variant has been identified. Here, we report a biallelic SEL1L variant (p. Cys141Tyr) in 5 patients from a consanguineous Slovakian family. These patients presented with not only ERAD-associated neurodevelopmental disorders with onset in infancy (ENDI) syndromes, but infantile-onset agammaglobulinemia with no mature B cells, resulting in frequent infections and early death. This variant disrupted the formation of a disulfide bond in the luminal fibronectin II domain of SEL1L, largely abolishing the function of the SEL1L-HRD1 ERAD complex in part via proteasomal-mediated self destruction by HRD1. This study reports a disease entity termed ENDI-agammaglobulinemia (ENDI-A) syndrome and establishes an inverse correlation between SEL1L-HRD1 ERAD functionality and disease severity in humans.

SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex.

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD); however, definitive evidence for the importance of SEL1L in HRD1 ERAD is lacking. Here we report that attenuation of the interaction between SEL1L and HRD1 impairs HRD1 ERAD function and has pathological consequences in mice. Our data show that SEL1L variant p.Ser658Pro ( SEL1L S 658 P ) previously identified in Finnish Hound suffering cerebellar ataxia is a recessive hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Mechanistically, SEL1L S 658 P variant attenuates the SEL1L-HRD1 interaction and causes HRD1 dysfunction by generating electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes revealed that the SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L recruits not only the lectins OS9 and ERLEC1, but the E2 UBE2J1 and retrotranslocon DERLIN, to HRD1. These data underscore the pathophysiological importance and disease relevance of the SEL1L-HRD1 complex, and identify a key step in organizing the HRD1 ERAD complex.

The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes.

Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.

SEL1L-HRD1 endoplasmic reticulum-associated degradation controls STING-mediated innate immunity by limiting the size of the activable STING pool.

Stimulator of interferon genes (STING) orchestrates the production of proinflammatory cytokines in response to cytosolic double-stranded DNA; however, the pathophysiological significance and molecular mechanism underlying the folding and maturation of nascent STING protein at the endoplasmic reticulum (ER) remain unknown. Here we report that the SEL1L-HRD1 protein complex-the most conserved branch of ER-associated degradation (ERAD)-is a negative regulator of the STING innate immunity by ubiquitinating and targeting nascent STING protein for proteasomal degradation in the basal state. SEL1L or HRD1 deficiency in macrophages specifically amplifies STING signalling and immunity against viral infection and tumour growth. Mechanistically, nascent STING protein is a bona fide substrate of SEL1L-HRD1 in the basal state, uncoupled from ER stress or its sensor inositol-requiring enzyme 1α. Hence, our study not only establishes a key role of SEL1L-HRD1 ERAD in innate immunity by limiting the size of the activable STING pool, but identifies a regulatory mechanism and therapeutic approach to targeting STING.

Interleukin-37 promotes DMBA/TPA skin cancer through SIGIRR-mediated inhibition of glycolysis in CD103+DC cells.

Interleukin 37 (IL-37), a member of the IL-1 family, is considered a suppressor of innate and adaptive immunity and, hence is a regulator of tumor immunity. However, the specific molecular mechanism and role of IL-37 in skin cancer remain unclear. Here, we report that IL-37b-transgenic mice (IL-37tg) treated with the carcinogenic 7,12-dimethylbenzoanthracene (DMBA)/12-o-tetradecylphorbol-13-acetate (TPA) exhibited enhanced skin cancer and increased tumor burden in the skin by inhibiting the function of CD103+ dendritic cells (DCs). Notably, IL-37 induced rapid phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and via single immunoglobulin IL-1-related receptor (SIGIRR), inhibited the long-term Akt activation. Specifically, by affecting the SIGIRR-AMPK-Akt signaling axis, which is related to the regulation of glycolysis in CD103+DCs, IL-37 inhibited their anti-tumor function. Our results show that a marked correlation between the CD103+DC signature (IRF8, FMS-like tyrosine kinase 3 ligand, CLEC9A, CLNK, XCR1, BATF3, and ZBTB46) and chemokines C-X-C motif chemokine ligand 9, CXCL10, and CD8A in a mouse model with DMBA/TPA-induced skin cancer. In a word, our results highlight that IL-37 as an inhibitor of tumor immune surveillance through modulating CD103+DCs and establishing an important link between metabolism and immunity as a therapeutic target for skin cancer.

Hepatic SEL1L-HRD1 ER-associated degradation regulates systemic iron homeostasis via ceruloplasmin.

Iron homeostasis is critical for cellular and organismal function and is tightly regulated to prevent toxicity or anemia due to iron excess or deficiency, respectively. However, subcellular regulatory mechanisms of iron remain largely unexplored. Here, we report that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) in hepatocytes controls systemic iron homeostasis in a ceruloplasmin (CP)-dependent, and ER stress-independent, manner. Mice with hepatocyte-specific Sel1L deficiency exhibit altered basal iron homeostasis and are sensitized to iron deficiency while resistant to iron overload. Proteomics screening for a factor linking ERAD deficiency to altered iron homeostasis identifies CP, a key ferroxidase involved in systemic iron distribution by catalyzing iron oxidation and efflux from tissues. Indeed, CP is highly unstable and a bona fide substrate of SEL1L-HRD1 ERAD. In the absence of ERAD, CP protein accumulates in the ER and is shunted to refolding, leading to elevated secretion. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD is responsible for the degradation of a subset of disease-causing CP mutants, thereby attenuating their pathogenicity. Together, this study uncovers the role of SEL1L-HRD1 ERAD in systemic iron homeostasis and provides insights into protein misfolding-associated proteotoxicity.

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

SerpinB7 deficiency contributes to development of psoriasis via calcium-mediated keratinocyte differentiation dysfunction.

Defective execution of proteases and protease inhibitors that mediate abnormal signaling cascades is emerging as a key contributor to skin diseases, such as psoriasis. SerpinB7 is identified as a skin-specific endogenous protease inhibitor, but the role and underlying mechanism in psoriasis are poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patients and imiquimod-induced psoriatic lesions in mice. SerpinB7-/- mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to SerpinB7+/+ mice after Imiquimod treatment. Mechanistically, SerpinB7 deficiency results in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in normal human epidermal keratinocyte and mouse primary keratinocyte. Transcriptomics and proteomics results showed that the SeprinB7 deficiency affected keratinocyte differentiation and proinflammatory cytokines, possibly by affecting the calcium ion channel-related proteins. Notably, we demonstrated that SerpinB7 deficiency prevented the increase in intracellular Ca2+ influx, which was partly eliminated by the intracellular Ca2+ chelator BAPTA-AM. Our findings first described the critical role of SerpinB7 in the regulation of keratinocyte differentiation and psoriatic microenvironment mediated via keratinocytes' intracellular calcium flux, proposing a new candidate for therapeutic targets in psoriasis.

Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner.

Interleukin-38 (IL-38) is strongly associated with chronic inflammatory diseases; however, its role in tumorigenesis is poorly understood. We demonstrated that expression of IL-38, which exhibits high expression in the skin, is downregulated in human cutaneous squamous cell carcinoma and 7,12-dimethylbenzanthracene/12-O-tetradecanoyl phorbol-13-acetate-induced mouse skin tumorigenesis. IL-38 keratinocyte-specific knockout mice displayed suppressed skin tumor formation and malignant progression. Keratinocyte-specific deletion of IL-38 was associated with reduced expression of inflammatory cytokines, leading to reduced myeloid cell infiltration into the local tumor microenvironment. IL-38 is dispensable for epidermal mutagenesis, but IL-38 keratinocyte-specific deletion reduces proliferative gene expression along with epidermal cell proliferation and hyperplasia. Mechanistically, we first demonstrated that IL-38 activates the c-Jun N-terminal kinase (JNK)/activator protein 1 signal transduction pathway to promote the expression of cancer-related inflammatory cytokines and proliferation and migration of tumor cells in an IL-1 receptor-related protein 2 (IL-1Rrp2)-dependent manner. Our findings highlight the role of IL-38 in the regulation of epidermal cell hyperplasia and pro-tumorigenic microenvironment through IL-1Rrp2/JNK and suggest IL-38/IL-1Rrp2 as a preventive and potential therapeutic target in skin cancer.

Interleukin-37 promotes colitis-associated carcinogenesis via SIGIRR-mediated cytotoxic T cells dysfunction.

Interleukin-37b (hereafter called IL-37) was identified as fundamental inhibitor of natural and acquired immunity. The molecular mechanism and function of IL-37 in colorectal cancer (CRC) has been elusive. Here, we found that IL-37 transgenic (IL-37tg) mice were highly susceptible to colitis-associated colorectal cancer (CAC) and suffered from dramatically increased tumor burdens in colon. Nevertheless, IL-37 is dispensable for intestinal mutagenesis, and CRC cell proliferation, apoptosis, and migration. Notably, IL-37 dampened protective cytotoxic T cell-mediated immunity in CAC and B16-OVA models. CD8+ T cell dysfunction is defined by reduced retention and activation as well as failure to proliferate and produce cytotoxic cytokines in IL-37tg mice, enabling tumor evasion of immune surveillance. The dysfunction led by IL-37 antagonizes IL-18-induced proliferation and effector function of CD8+ T cells, which was dependent on SIGIRR (single immunoglobulin interleukin-1 receptor-related protein). Finally, we observed that IL-37 levels were significantly increased in CRC patients, and positively correlated with serum CRC biomarker CEA levels, but negatively correlated with the CD8+ T cell infiltration in CRC patients. Our findings highlight the role of IL-37 in harnessing antitumor immunity by inactivation of cytotoxic T cells and establish a new defined inhibitory factor IL-37/SIGIRR in cancer-immunity cycle as therapeutic targets in CRC.

EDIL3 deficiency ameliorates adverse cardiac remodelling by neutrophil extracellular traps (NET)-mediated macrophage polarization.

AIMS: After myocardial infarction (MI), injured cardiomyocytes recruit neutrophils and monocytes/macrophages to myocardium, which in turn initiates inflammatory and reparative cascades, respectively. Either insufficient or excessive inflammation impairs cardiac healing. As an endogenous inhibitor of neutrophil adhesion, EDIL3 plays a crucial role in inflammatory regulation. However, the role of EDIL3 in MI remains obscure. We aimed to define the role of EDIL3 in cardiac remodelling after MI. METHODS AND RESULTS: Serum EDIL3 levels in MI patients were negatively associated with MI biomarkers. Consistently, WT mice after MI showed low levels of cardiac EDIL3. Compared with WT mice, Edil3-/- mice showed improvement of post-MI adverse remodelling, as they exhibited lower mortality, better cardiac function, shorter scar length, and smaller LV cavity. Accordingly, infarcted hearts of Edil3-/- mice contained fewer cellular debris and lower amounts of fibrosis content, with decreased collagen I/III expression and the percentage of α-smooth muscle actin myofibroblasts. Mechanistically, EDIL3 deficiency did not affect the recruitment of monocytes or T cells, but enhanced neutrophil recruitment and following expansion of pro-inflammatory Mertk-MHC-IIlo-int (myeloid-epithelial-reproductive tyrosine kinase/major histocompatibility complex II) macrophages. The injection of neutrophil-specific C-X-C motif chemokine receptor 2 antagonist eliminated the differences in macrophage polarization and cardiac function between WT and Edil3-/- mice after MI. Neutrophil extracellular traps (NETs), which were more abundant in the hearts of Edil3-/- mice, contributed to Mertk-MHC-IIlo-int polarization via Toll-like receptor 9 pathway. The inhibition of NET formation by treatment of neutrophil elastase inhibitor or DNase I impaired macrophage polarization, increased cellular debris and aggravated cardiac adverse remodelling, thus removed the differences of cardiac function between WT and Edil3-/- mice. Totally, EDIL3 plays an important role in NET-primed macrophage polarization and cardiac remodelling during MI. CONCLUSION: We not only reveal that EDIL3 deficiency ameliorates adverse cardiac healing via NET-mediated pro-inflammatory macrophage polarization but also discover a new crosstalk between neutrophil and macrophage after MI.

IL-30 ameliorates imiquimod and K14-VEGF induced psoriasis-like disease by inhibiting both innate and adaptive immunity disorders.

Psoriasis is a severe skin disease with significant physical and psychological health consequences. As a typical type of immune disease, both innate and adaptive immunity disorders play key roles in the development of psoriasis. Interleukin (IL)-30 was thought as a natural antagonist of gp130-mediated signaling that affects T helper type 1 and 17 cell polarization by inhibiting IL-6 and IL-27 signaling pathways. Here, we found that, in vitro, IL-30 reduced cytokine levels of HaCaT keratinocytes and dendritic cells (DCs), weakened the maturationS of DCs, inhibited DC-mediated T cell proliferation, and blocked the activation of nuclear factor-κB. In vivo, IL-30 inhibited the development of skin disease in two animal models: Krt14-Vegfa and imiquimod (IMQ)-induced psoriasis-like skin disease. Thus, IL-30 may be useful as a therapeutic agent for controlling psoriasis.

Establishing Transcription Profile of Psoriasiform Cutaneous In Vitro using HaCaT Cells Stimulated with Combination of Cytokines.

Psoriasis is a common chronic inflammatory skin disease mediated by innate and adaptive immune systems, characterized by abnormal proliferation and differentiation of epidermal keratinocytes and infiltration of inflammatory cells. Skin-specific keratinocytes are key participants in innate immunity, responding to immune cells and environmental stimulation, thereby serving an important role in the immunopathogenesis of psoriasis. Here, we present a method for inducing psoriasiform keratinocytes inflammation at transcription level with HaCaT cell line using five proinflammatory cytokines combination (M5 combination), including IL-17A, IL-22, IL-1α, TNF-α, and oncostatin M. Results demonstrate that M5 combination induced HaCaT cells showed increased levels of antimicrobial peptides (BD2, S100A7, S100A8, and S100A9), chemokines, and cytokines (CXCL1, CXCL2, CXCL8, CCL20, IL-1ß, IL-6 and, IL-18). The mRNA levels of keratinocytes differentiation markers (Keratin1, Keratin10, Filaggrin, and Loricrin) were down regulated, which was consistent with the transcriptome data derived from psoriasis-like keratinocytes. The method described here, therefore, establishes an in vitro psoriasiform cutaneous inflammation at transcription level and contributes to the research for molecular pathogenesis of psoriasis.

Endoplasmic reticulum-associated degradation is required for nephrin maturation and kidney glomerular filtration function.

Podocytes are key to the glomerular filtration barrier by forming a slit diaphragm between interdigitating foot processes; however, the molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here, we show that the SEL1L-HRD1 protein complex of ER-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome with an impaired slit diaphragm shortly after weaning and die prematurely, with a median lifespan of approximately 3 months. We show mechanistically that nephrin, a type 1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuated the maturation of nascent nephrin, leading to its retention in the ER. We also show that various autosomal-recessive nephrin disease mutants were highly unstable and broken down by SEL1L-HRD1 ERAD, which attenuated the pathogenicity of the mutants toward the WT allele. This study uncovers a critical role of SEL1L-HRD1 ERAD in glomerular filtration barrier function and provides insights into the pathogenesis associated with autosomal-recessive disease mutants.

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation.

The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco's modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1ß: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f: mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

Detecting Gene Ontology misannotations using taxon-specific rate ratio comparisons.

MOTIVATION: Many protein function databases are built on automated or semi-automated curations and can contain various annotation errors. The correction of such misannotations is critical to improving the accuracy and reliability of the databases. RESULTS: We proposed a new approach to detect potentially incorrect Gene Ontology (GO) annotations by comparing the ratio of annotation rates (RAR) for the same GO term across different taxonomic groups, where those with a relatively low RAR usually correspond to incorrect annotations. As an illustration, we applied the approach to 20 commonly studied species in two recent UniProt-GOA releases and identified 250 potential misannotations in the 2018-11-6 release, where only 25% of them were corrected in the 2019-6-3 release. Importantly, 56% of the misannotations are 'Inferred from Biological aspect of Ancestor (IBA)' which is in contradiction with previous observations that attributed misannotations mainly to 'Inferred from Sequence or structural Similarity (ISS)', probably reflecting an error source shift due to the new developments of function annotation databases. The results demonstrated a simple but efficient misannotation detection approach that is useful for large-scale comparative protein function studies. AVAILABILITY AND IMPLEMENTATION: https://zhanglab.ccmb.med.umich.edu/RAR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.