HRD1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination.

The endoplasmic reticulum-associated protein degradation (ERAD) is responsible for recognizing and retro-translocating protein substrates, misfolded or not, from the ER for cytosolic proteasomal degradation. HMG-CoA Reductase (HMGCR) Degradation protein-HRD1-was initially identified as an E3 ligase critical for ERAD. However, its physiological functions remain largely undefined. Herein, we discovered that hepatic HRD1 expression is induced in the postprandial condition upon mouse refeeding. Mice with liver-specific HRD1 deletion failed to repress FGF21 production in serum and liver even in the refeeding condition and phenocopy the FGF21 gain-of-function mice showing growth retardation, female infertility, and diurnal circadian behavior disruption. HRD1-ERAD facilitates the degradation of the liver-specific ER-tethered transcription factor CREBH to downregulate FGF21 expression. HRD1-ERAD catalyzes polyubiquitin conjugation onto CREBH at lysine 294 for its proteasomal degradation, bridging a multi-organ crosstalk in regulating growth, circadian behavior, and female fertility through regulating the CREBH-FGF21 regulatory axis.

Inflammasome-Derived Exosomes Activate NF-κB Signaling in Macrophages.

Exosomes are secreted small vesicles that mediate various biological processes, such as tumorigenesis and immune response. However, whether the inflammasome signaling leads to the change of constituent of exosomes and its roles in immune response remains to be determined. We isolated the exosomes from macrophages with treatment of mock, endotoxin, or endotoxin/nigericin. A label-free quantification method by MS/MS was used to identify the components of exosomes. In total, 2331 proteins were identified and 513 proteins were exclusively detected in exosomes with endotoxin and nigericin treatment. The differentially expressed proteins were classified by Gene Ontology and KEGG pathways. The immune response-related proteins and signaling pathways were specifically enriched in inflammasome-derived exosomes. Moreover, we treated macrophages with the exosomes from different stimulation. We found that inflammasome-derived exosomes directly activate NF-κB signaling pathway, while the control or endotoxin-derived exosomes have no effect. The inflammatory signaling was amplified in neighbor cells in an exosome-dependent way. The inflammasome-derived exosomes might be used to augment the immune response in disease treatment, and preventing the transfer of these exosomes might ameliorate autoimmune diseases.

The GTPase-activating protein GIT2 protects against colitis by negatively regulating Toll-like receptor signaling.

G protein-coupled receptor kinase-interactor 2 (GIT2) regulates thymocyte positive selection, neutrophil-direction sensing, and cell motility during immune responses by regulating the activity of the small GTPases ADP ribosylation factors (Arfs) and Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we show that Git2-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis, Escherichia coli, or endotoxin-shock challenge, and a dramatic increase in proinflammatory cytokines was observed in Git2 knockout mice and macrophages. GIT2 is a previously unidentified negative regulator of Toll-like receptor (TLR)-induced NF-κB signaling. The ubiquitination of TNF receptor associated factor 6 (TRAF6) is critical for the activation of NF-κB. GIT2 terminates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis to inhibit the ubiquitination of TRAF6. Finally, we show that the susceptibility of Git2-deficient mice to DSS-induced colitis depends on TLR signaling. Thus, we show that GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation and severe organ damage.

Interactome map reveals phospholipid scramblase 1 as a novel regulator of hepatitis B virus x protein.

HBV X protein plays crucial roles during viral infection and hepatocellular carcinoma (HCC) development through interaction with various host factors. Here, we mapped the interactome of HBx using a yeast two-hybrid screen. Nine human proteins were identified as novel interacting partners of HBx, one of which is phospholipid scramblase 1 (PLSCR1). PLSCR1 is an interferon-inducible protein that mediates antiviral activity against DNA and RNA viruses. However, the molecular mechanisms of PLSCR1 activity against HBV remain unclear. Here, we reported that PLSCR1 promotes HBx degradation by a proteasome- and ubiquitin-dependent mechanism. Furthermore, we found that PLSCR1 inhibits HBx-mediated cell proliferation. After HBV infection, the protein level of PLSCR1 in plasma is elevated, and chronic hepatitis B patients with low plasma levels of PLSCR1 have a high risk of developing HCC. These results suggest that the nuclear trafficking of PLSCR1 mediates the antiviral activity and anticarcinogenesis against HBV by regulating HBx stability.

A proteomics strategy for the identification of FAT10-modified sites by mass spectrometry.

The ubiquitin-like protein FAT10 (HLA-F adjacent transcript 10) is uniquely expressed in mammals. The fat10 gene is encoded in the MHC class I locus in the human genome and is related to some specific processes, such as apoptosis, immune response, and cancer. However, biological knowledge of FAT10 is limited, owing to the lack of identification of its conjugates. FAT10 covalently modifies proteins in eukaryotes, but only a few substrates of FAT10 have been reported until now, and no FATylated sites have been identified. Here, we report the proteome-scale identification of FATylated proteins by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 175 proteins with high confidence as FATylated candidates. A total of 13 modified sites were identified for the first time by a modified search of the raw MS data. The modified sites were highly enriched with hydrophilic amino acids. Furthermore, the FATylation processes of hnRNP C2, PCNA, and PDIA3 were verified by a coimmunoprecipitation assay. We confirmed that most of the substrates were covalently attached to a FAT10 monomer. The functional distribution of the FAT10 targets suggests that FAT10 participates in various biological processes, such as translation, protein folding, RNA processing, and macromolecular complex assembly. These results should be very useful for investigating the biological functions of FAT10.

Hepatitis B virus encoded X protein suppresses apoptosis by inhibition of the caspase-independent pathway.

Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV.

Toward an understanding of the protein interaction network of the human liver.

Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26.

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2-p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26-HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.

19q13.12 KRAB zinc-finger protein ZNF383 represses p53 signaling pathway by interacting with p53.

As one of the most important tumor suppressors, the activity of p53 is precisely regulated. However, the mechanism of p53 regulation is still being elucidated and new regulatory molecules for p53 have also been frequently identified. Our previous works revealed that two members of the KRAB zinc-finger protein (KZFP) family Apak and PISA, which are located on human 19q13.12, participated in the regulation of p53 signaling pathway. KZFPs genes are mainly amplified via tandem in situ duplication during evolution, which indicates that similar sequences and functions may be conserved in evolutionarily and physically close KZFPs. Here, we revealed that ZNF383, another member of the KZFPs mapped at 19q13.12, could inhibit p53-mediated apoptosis and the activation of IFN-ß pathway by decreasing the H3K36me2 level at p53's binding sites and the attenuating the binding of p53 to its target genes. We further explored the effect of other KZFPs clustered on 19q13.12 on p53, and found that 85% of these KZFPs exerted p53-repressive activity. Intriguingly, an acidic amino acid-enriched sequence, the SAcL motif in the zinc-finger domains of these KZFPs, was found to be critical for p53 binding. Taken together, our findings revealed the KZFPs cluster located at 19q13.12 not only was involved in p53 regulation but also exhibited different features in the selective regulation of p53 and functional mechanisms, and for the first time, confirmed a motif in KZFPs that mediates the interaction of KZFPs and p53. These results would enrich the knowledge on the role, sequence features, and functional mechanisms of the KZFP family in p53 regulation.

Bursaphelenchus xylophilus detection and analysis system based on CRISPR - Cas12.

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

ZNF498 promotes hepatocellular carcinogenesis by suppressing p53-mediated apoptosis and ferroptosis via the attenuation of p53 Ser46 phosphorylation.

BACKGROUND: Dysfunctional p53 signaling is one of the major causes of hepatocellular carcinoma (HCC) tumorigenesis and development, but the mechanisms underlying p53 inactivation in HCC have not been fully clarified. The role of Krüppel-associated box (KRAB)-type zinc-finger protein ZNF498 in tumorigenesis and the underlying mechanisms are poorly understood. METHODS: Clinical HCC samples were used to assess the association of ZNF498 expression with clinicopathological characteristics and patient outcomes. A mouse model in which HCC was induced by diethylnitrosamine (DEN) was used to explore the role of ZNF498 in HCC initiation and progression. ZNF498 overexpression and knockdown HCC cell lines were employed to examine the effects of ZNF498 on cellular proliferation, apoptosis, ferroptosis and tumor growth. Western blotting, immunoprecipitation, qPCR, luciferase assays and flow cytometry were also conducted to determine the underlying mechanisms related to ZNF498 function. RESULTS: ZNF498 was found to be highly expressed in HCC, and increased ZNF498 expression was positively correlated with advanced pathological grade and poor survival in HCC patients. Furthermore, ZNF498 promoted DEN-induced hepatocarcinogenesis and progression in mice. Mechanistically, ZNF498 directly interacted with p53 and suppressed p53 transcriptional activation by inhibiting p53 Ser46 phosphorylation. ZNF498 competed with p53INP1 for p53 binding and suppressed PKCδ- and p53INP1-mediated p53 Ser46 phosphorylation. In addition, functional assays revealed that ZNF498 promoted liver cancer cell growth in vivo and in vitro in a p53-dependent manner. Moreover, ZNF498 inhibited p53-mediated apoptosis and ferroptosis by attenuating p53 Ser46 phosphorylation. CONCLUSIONS: Our results strongly suggest that ZNF498 suppresses apoptosis and ferroptosis by attenuating p53 Ser46 phosphorylation in hepatocellular carcinogenesis, revealing a novel ZNF498-PKCδ-p53INP1-p53 axis in HCC cells that would enrich the non-mutation p53-inactivating mechanisms in HCC.

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping.

Genome-wide physical protein-protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.

Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP.

Tandem affinity purification and identification of the human TSC1 protein complex.

Mutations in the TSC1 and TSC2 genes lead to tuberous sclerosis complex (TSC), which is characterized clinically by mental retardation, epilepsy, and benign tumors affecting multiple tissues. Numerous components of the TSC protein complex remain uncharacterized. Here we report the purification of the TSC1 complex under physiological conditions using a proteomic strategy. We purified the TSC1 protein complex using a tandem affinity purification method and identified a protein complex containing 139 components. Two known binding proteins of TSC1 (TSC2 and DOCK7) were identified along with other new potential partners, which cover reported and novel TSC1 functional categories. Bioinformatics and biochemical methods were used to evaluate the observed protein-protein interactions. A comparative analysis with a published expression proteomics/genomics study of TSC1 revealed more than 20 common candidates that might be functionally relevant. The data set provides new directions in which to expand our knowledge of the functions of TSC1 and the mechanisms of TSC. The results are highly reliable, which is reflected by the identification of a few reported partners of TSC1 and many TSC1/2-regulated proteins. Interestingly, many new functional categories were identified, such as DNA repair, which provide novel hints to the function of TSC1. Moreover, a few neuronal disease-related proteins that might regulate the normal functions of neurons were identified. Thus, the results suggest that many of the new interactions should be biologically significance. It will be interesting to further investigate the regulatory mechanisms of these components.

Nuclear protein IkappaB-zeta inhibits the activity of STAT3.

STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IkappaB-zeta that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IkappaB-zeta. Overexpression of IkappaB-zeta inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IkappaB-zeta is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-kappaB signaling pathway inhibits the activation of STAT3.

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity.

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

ER-associated ubiquitin ligase HRD1 programs liver metabolism by targeting multiple metabolic enzymes.

The HMG-CoA reductase degradation protein 1 (HRD1) has been identified as a key enzyme for endoplasmic reticulum-associated degradation of misfolded proteins, but its organ-specific physiological functions remain largely undefined. Here we show that mice with HRD1 deletion specifically in the liver display increased energy expenditure and are resistant to HFD-induced obesity and liver steatosis and insulin resistance. Proteomic analysis identifies a HRD1 interactome, a large portion of which includes metabolic regulators. Loss of HRD1 results in elevated ENTPD5, CPT2, RMND1, and HSD17B4 protein levels and a consequent hyperactivation of both AMPK and AKT pathways. Genome-wide mRNA sequencing revealed that HRD1-deficiency reprograms liver metabolic gene expression profiles, including suppressing genes involved in glycogenesis and lipogenesis and upregulating genes involved in glycolysis and fatty acid oxidation. We propose HRD1 as a liver metabolic regulator and a potential drug target for obesity, fatty liver disease, and insulin resistance associated with the metabolic syndrome.

The ubiquitin ligase TRAF6 negatively regulates the JAK-STAT signaling pathway by binding to STAT3 and mediating its ubiquitination.

STAT3 is a key transcription factor that mediates various cellular and organismal processes, such as cell growth, apoptosis, immune response and cancer. However, the molecular mechanisms of STAT3 regulation remain poorly understood. Here, we identified TRAF6 as a new STAT3 interactor. TRAF6 augmented the ubiquitination of STAT3 and deactivated its transcriptional activity induced by IFNα stimulation or overexpressed with JAK2. Both the RING domain and the TRAF-type zinc finger domain of TRAF6 were indispensable for STAT3 deactivation. Accordingly, TRAF6 also down-regulated the expression of two known STAT3 target genes, CRP and ACT. Therefore, we showed that TRAF6 is a new regulator of JAK/STAT signaling and provide a new mechanistic explanation for the crosstalk between the NF-κB and the JAK-STAT pathways.

Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells.

Hepatitis C virus (HCV) infects human hepatocytes through several host factors. However, other prerequisite factors for viral entry remain to be identified. Using a yeast two-hybrid screen, we found that human phospholipid scramblase 1 interacts with HCV envelope proteins E1 and E2. These physical interactions were confirmed by co-immunoprecipitation and GST pull-down assays. Knocking down the expression of PLSCR1 inhibited the entry of HCV pseudoparticles. Moreover, PLSCR1 was required for the initial attachment of HCV onto hepatoma cells, where it specifically interacted with entry factor OCLN. We show that PLSCR1 is a novel attachment factor for HCV entry.