Neuroimmune modulation by tryptophan derivatives in neurological and inflammatory disorders.

The nervous and immune systems are highly developed, and each performs specialized physiological functions. However, they work together, and their dysfunction is associated with various diseases. Specialized molecules, such as neurotransmitters, cytokines, and more general metabolites, are essential for the appropriate regulation of both systems. Tryptophan, an essential amino acid, is converted into functional molecules such as serotonin and kynurenine, both of which play important roles in the nervous and immune systems. The role of kynurenine metabolites in neurodegenerative and psychiatric diseases has recently received particular attention. Recently, we found that hyperactivity of the kynurenine pathway is a critical risk factor for septic shock. In this review, we first outline neuroimmune interactions and tryptophan derivatives and then summarized the changes in tryptophan metabolism in neurological disorders. Finally, we discuss the potential of tryptophan derivatives as therapeutic targets for neuroimmune disorders.

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA.

Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.

Curcumin partly prevents ISG15 activation via ubiquitin-activating enzyme E1-like protein and decreases ISGylation.

The expression of the ubiquitin-like molecule interferon-stimulated gene 15 kDa (ISG15) and post-translational protein modification by ISG15 (ISGylation) are strongly activated by interferons or pathogen infection, suggesting that ISG15 and ISGylation play an important role in innate immune responses. More than 400 proteins have been found to be ISGylated. ISG15 is removed from substrates by interferon-induced ubiquitin-specific peptidase 18 or severe acute respiratory syndrome coronavirus 2‒derived papain-like protease. Therefore, maintaining strong ISGylation may help prevent the spread of coronavirus disease 2019 (COVID-19). However, it is unknown whether nutrients or chemicals affect ISGylation level. Curcumin is the major constituent of turmeric and functions as an immunomodulator. Here, we investigated the effect of curcumin on ISGylation. MCF10A and A549 cells were treated with interferon α and curcumin after which the expression levels of various proteins were determined. The effect of curcumin on ubiquitylation was also determined. Curcumin treatment was found to reduce ISGylation in a dose-dependent manner. The findings suggested that curcumin partly prevents disulfide bond-mediated ISG15 dimerization directly or indirectly, thereby increasing monomer ISG15 levels. Reduced ISGylation may also occur via the prevention of ISG15 activation by ubiquitin-activating enzyme E1-like protein. In conclusion, curcumin treatment was found to reduce ISGylation, suggesting that it may contribute to severe COVID-19. This is the first study to report a relationship between ISGylation and a food component.

TRIM22 negatively regulates MHC-II expression.

The development of cancer treatment has recently achieved a remarkable breakthrough, and checkpoint blockade immunotherapy has received much attention. To enhance the therapeutic efficacy of checkpoint blockade immunotherapy, recent studies have revealed the importance of activation of CD4+ T cells via an increase in major histocompatibility complex (MHC) class II molecules in cancer cells. Here, we demonstrate that tripartite motif-containing (TRIM) 22, negatively regulates MHC-II expression. Gene knockout of TRIM22 using Cas9-sgRNAs led to an increase of MHC-II proteins, while TRIM22 overexpression remarkably decreased MHC-II proteins. mRNA levels of MHC-II and class II transactivator (CIITA), which plays an essential role in the regulation of MHC-II transcription, were not affected by TRIM22. Furthermore, TRIM22 knockout did not suppress the degradation of MHC-II protein but rather promoted it. These results suggest that TRIM22 decreases MHC-II protein levels through a combination of multiple mechanisms other than transcription or degradation. We showed that inhibition of TRIM22 can increase the amount of MHC-II expression in cancer cells, suggesting a possibility of providing the biological basis for a possible therapeutic target to potentiate checkpoint blockade immunotherapy.

Loss of FAM83H promotes cell migration and invasion in cutaneous squamous cell carcinoma via impaired keratin distribution.

BACKGROUNDS: FAM83H is essential for amelogenesis, but recent reports implicate that FAM83H is involved in the tumorigenesis. We previously clarified that TRIM29 binds to FAM83H to regulate keratin distribution and squamous cell migration. However, little is known about FAM83H in normal/malignant skin keratinocytes. OBJECTIVE: To investigate the expression of FAM83H in cutaneous squamous cell carcinoma (SCC) and its physiological function. METHODS: Immunohistochemical analysis and RT-PCR of human SCC tissues were performed. Next, we examined the effect of FAM83H knockdown/overexpression in SCC cell lines using cell proliferation, migration, and invasion assay. To investigate the molecular mechanism, immunoprecipitation of FAM83H was examined. Further, Immunofluorescence staining was performed. Finally, we examined the correlation between the expressions of FAM83H and the keratin distribution. RESULTS: FAM83H expression was lower in SCC lesions than in normal epidermis and correlated with differentiation grade. The mRNA expression levels of FAM83H in SCC tumors were also lower than in normal epidermis. The knockdown of FAM83H enhanced SCC cell migration and invasion, whereas the overexpression of FAM83H led to decreases in both. Furthermore, the knockdown of FAM83H enhanced the cancer cell metastasis in vivo. FAM83H formed a complex with TRIM29 and keratins. The knockdown of FAM83H altered keratin distribution and solubility. Clinically, the loss of FAM83H correlates with an altered keratin distribution. CONCLUSION: Our findings reveal a critical function for FAM83H in regulating keratin distribution, as well as in the migration/invasion of cutaneous SCC, suggesting that FAM83H could be a crucial molecule in the tumorigenesis of cutaneous SCC.

Fluticasone Propionate Suppresses Poly(I:C)-Induced ACE2 in Primary Human Nasal Epithelial Cells.

Background: From the first detection in 2019, SARS-CoV-2 infections have spread rapidly worldwide and have been proven to cause an urgent and important health problem. SARS-CoV-2 cell entry depends on two proteins present on the surface of host cells, angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). The nasal cavity is thought to be one of the initial sites of infection and a possible reservoir for dissemination within and between individuals. However, it is not known how the expression of these genes is regulated in the nasal mucosa. Objective: In this study, we examined whether the expression of ACE2 and TMPRSS2 is affected by innate immune signals in the nasal mucosa. We also investigated how fluticasone propionate (FP), a corticosteroid used as an intranasal steroid spray, affects the gene expression. Methods: Primary human nasal epithelial cells (HNECs) were collected from the nasal mucosa and incubated with Toll-like receptor (TLR) agonists and/or fluticasone propionate (FP), followed by quantitative PCR, immunofluorescence, and immunoblot analyses. Results: Among the TLR agonists, the TLR3 agonist Poly(I:C) significantly increased ACE2 and TMPRSS2 mRNA expression in HNECs (ACE2 36.212±11.600-fold change, p<0.0001; TMPRSS2 5.598±2.434-fold change, p=0.031). The ACE2 protein level was also increased with Poly(I:C) stimulation (2.884±0.505-fold change, p=0.003). The Poly(I:C)-induced ACE2 expression was suppressed by co-incubation with FP (0.405±0.312-fold change, p=0.044). Conclusion: The activation of innate immune signals via TLR3 promotes the expression of genes related to SARS-CoV2 cell entry in the nasal mucosa, although this expression is suppressed in the presence of FP. Further studies are required to evaluate whether FP suppresses SARS-CoV-2 viral cell entry.

Post-translational Wnt receptor regulation: Is the fog slowly clearing?: The molecular mechanism of RNF43/ZNRF3 ubiquitin ligases is not yet fully elucidated and still controversial.

Wnt signaling plays pivotal roles during our entire lives, from conception to death, through the regulation of morphogenesis in developing embryos and the maintenance of tissue homeostasis in adults. The regulation of Wnt signaling occurs on several levels: at the receptor level on the plasma membrane, at the ß-catenin protein level in the cytoplasm, and through transcriptional regulation in the nucleus. Several recent studies have focused on the mechanisms of Wnt receptor regulation, following the discovery that the Wnt receptor frizzled (Fzd) is a target of the ubiquitin ligases, RNF43 and ZNRF3. RNF43 and ZNRF3 are homologous genes that are mutated in several cancers. The details underlying their mechanism of action continue to unfold, while at the same time raising many new questions. In this review, we discuss advances and controversies in our understanding of Wnt receptor regulation.

Role of intracellular zinc in molecular and cellular function in allergic inflammatory diseases.

Zinc is an essential micronutrient in human body and a vital cofactor for the function of numerous proteins encoded by the human genome. Zinc has a critical role in maintaining many biochemical and physiological processes at the molecular, cellular, and multiple organ and systemic levels. The alteration of zinc homeostasis causes dysfunction of many organs and systems. In the immune system, zinc regulates the differentiation, proliferation and function of inflammatory cells, including T cells, eosinophils, and B cells, by modifying several signaling pathways such as NFκB signaling pathways and TCR signals. An adequate zinc level is essential for proper immune responses and decreased zinc levels were reported in many allergic inflammatory diseases, including atopic dermatitis, bronchial asthma, and chronic rhinosinusitis. Decreased zinc levels often enhance inflammatory activation. On the other hand, the inflammatory conditions alter the intracellular homeostasis of zinc, often decreasing zinc levels. These findings implied that there could be a vicious cycle between zinc deficiency and inflammatory conditions. In this review, we present recent evidence on the involvement of zinc in atopic dermatitis, bronchial asthma, and chronic rhinosinusitis, with insights into the involvement of zinc in the underlying molecular and cellular mechanisms related to these allergic inflammatory diseases.

A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis.

Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.

NONO Is a Negative Regulator of SOX2 Promoter.

BACKGROUND/AIM: Sex determining region Y (SRY)-box 2 (SOX2) is a transcription factor essential for the maintenance of proliferation and self-renewal of cancer stem cells and is associated with breast cancer initiation. Regulation of cancer stem cell plasticity by SOX2 requires both positive and negative SOX2 transcription factors, but the negative regulator is still largely unknown. MATERIALS AND METHODS: SOX2 promoter-binding proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry, luciferase assay, and chromatin immunoprecipitation. The effects of one such transcription factor on SOX2 expression was investigated by knockdown and overexpression experiments. RESULTS: Non-POU domain-containing octamer-binding protein (NONO) (also known as 54-kDa nuclear RNA-binding protein, P54NRB) was identified as a SOX2 promoter-binding protein and a negative regulator of SOX2 expression. Its activity was controlled by its coiled-coil domain and the C-terminal domain. CONCLUSION: These results suggest that NONO acts as a key regulator of SOX2 transcription through the repression of SOX2 promoter activity in breast cancer cells.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts.

We aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-γ (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy.Abbreviations: 3-MA: 3-methyladenine; ACPA: anti-citrullinated peptide antibodies; anti-CCP: anti-cyclic citrullinated peptide antibody; cVIM: citrullinated VIM; BECN1: beclin1; DAPI: 4',6-diamidino-2-phenylindole; FBS: fetal bovine serum; HLA: human leukocyte antigen; IFNG/IFN-γ: interferon gamma; IL6: interleukin 6; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence index; MHC: major histocompatibility complex; OA: osteoarthritis; PADI: peptidyl arginine deiminase; PepA: pepstatin A; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RA: rheumatoid arthritis; SFs: synovial fibroblasts; siRNA: small interfering RNA; VIM: vimentin.

Loss of TRIM29 Alters Keratin Distribution to Promote Cell Invasion in Squamous Cell Carcinoma.

: TRIM29 (tripartite motif-containing protein 29) is a TRIM family protein that has been implicated in breast, colorectal, and pancreatic cancers. However, its role in stratified squamous epithelial cells and tumors has not been elucidated. Here, we investigate the expression of TRIM29 in cutaneous head and neck squamous cell carcinomas (SCC) and its functions in the tumorigenesis of such cancers. TRIM29 expression was lower in malignant SCC lesions than in adjacent normal epithelial tissue or benign tumors. Lower expression of TRIM29 was associated with higher SCC invasiveness. Primary tumors of cutaneous SCC showed aberrant hypermethylation of TRIM29. Depletion of TRIM29 increased cancer cell migration and invasion; conversely, overexpression of TRIM29 suppressed these. Comprehensive proteomics and immunoprecipitation analyses identified keratins and keratin-interacting protein FAM83H as TRIM29 interactors. Knockdown of TRIM29 led to ectopic keratin localization of keratinocytes. In primary tumors, lower TRIM29 expression correlated with the altered expression of keratins. Our findings reveal an unexpected role for TRIM29 in regulating the distribution of keratins, as well as in the migration and invasion of SCC. They also suggest that the TRIM29-keratin axis could serve as a diagnostic and prognostic marker in stratified epithelial tumors and may provide a target for SCC therapeutics. SIGNIFICANCE: These findings identify TRIM29 as a novel diagnostic and prognostic marker in stratified epithelial tissues.

TRIM59 Promotes Gliomagenesis by Inhibiting TC45 Dephosphorylation of STAT3.

Aberrant EGFR signaling is a common driver of glioblastoma (GBM) pathogenesis; however, the downstream effectors that sustain this oncogenic pathway remain unclarified. Here we demonstrate that tripartite motif-containing protein 59 (TRIM59) acts as a new downstream effector of EGFR signaling by regulating STAT3 activation in GBM. EGFR signaling led to TRIM59 upregulation through SOX9 and enhanced the interaction between TRIM59 and nuclear STAT3, which prevents STAT3 dephosphorylation by the nuclear form of T-cell protein tyrosine phosphatase (TC45), thereby maintaining transcriptional activation and promoting tumorigenesis. Silencing TRIM59 suppresses cell proliferation, migration, and orthotopic xenograft brain tumor formation of GBM cells and glioma stem cells. Evaluation of GBM patient samples revealed an association between EGFR activation, TRIM59 expression, STAT3 phosphorylation, and poor prognoses. Our study identifies TRIM59 as a new regulator of oncogenic EGFR/STAT3 signaling and as a potential therapeutic target for GBM patients with EGFR activation.Significance: These findings identify a novel component of the EGFR/STAT3 signaling axis in the regulation of glioma tumorigenesis. Cancer Res; 78(7); 1792-804. ©2018 AACR.

Mutations in bassoon in individuals with familial and sporadic progressive supranuclear palsy-like syndrome.

Clinical diagnosis of progressive supranuclear palsy (PSP) is sometimes difficult because various phenotypes have been identified. Here, we report a mutation in the bassoon (BSN) gene in a family with PSP-like syndrome. Their clinical features resembled not only those of PSP patients but also those of individuals with multiple system atrophy and Alzheimer's disease. The neuropathological findings showed a novel three + four repeat tauopathy with pallido-luysio-nigral degeneration and hippocampal sclerosis. Whole-exome analysis of this family identified a novel missense mutation in BSN. Within the pedigree, the detected BSN mutation was found only in affected individuals. Further genetic analyses were conducted in probands from four other pedigrees with PSP-like syndrome and in 41 sporadic cases. Three missense mutations in BSN that are very rarely listed in databases of healthy subjects were found in four sporadic cases. Western blot analysis of tau following the overexpression of wild-type or mutated BSN revealed the possibility that wild-type BSN reduced tau accumulation, while mutated BSN lost this function. An association between BSN and neurological diseases has not been previously reported. Our results revealed that the neurodegenerative disorder associated with the original proband's pedigree is a novel tauopathy, differing from known dementia and parkinsonism syndromes, including PSP.