The reverse TRBV30 gene of mammals: a defect or superiority in evolution?

At the 3' end of the C2 gene in the mammalian TRB locus, a distinct reverse TRBV30 gene (named TRBV31 in mice) has been conserved throughout evolution. In the fully annotated TRB locus of 14 mammals (including six orders), we observed noteworthy variations in the localization and quality of the reverse V30 genes and Recombination Signal Sequences (RSSs) in the gene trees of 13 mammals. Conversely, the forward V29 genes and RSSs were generally consistent with the species tree of their corresponding species. This finding suggested that the evolution of the reverse V30 gene was not synchronous and likely played a crucial role in regulating adaptive immune responses. To further investigate this possibility, we utilized single-cell TCR sequencing (scTCR-seq) and high-throughput sequencing (HTS) to analyze TCRß CDR3 repertoires from both central and peripheral tissues of Primates (Homo sapiens and Macaca mulatta), Rodentia (Mus musculus: BALB/c, C57BL/6, and Kunming mice), Artiodactyla (Bos taurus and Bubalus bubalis), and Chiroptera (Rhinolophus affinis and Hipposideros armige). Our investigation revealed several novel observations: (1) The reverse V30 gene exhibits classical rearrangement patterns adhering to the '12/23 rule' and the 'D-J rearrangement preceding the V-(D-J) rearrangement'. This results in the formation of rearranged V30-D2J2, V30-D1J1, and V30-D1J2. However, we also identified 'special rearrangement patterns' wherein V30-D rearrangement preceding D-J rearrangement, giving rise to rearranged V30-D2-J1 and forward Vx-D2-J. (2) Compared to the 'deletional rearrangement' (looping out) of forward V1-V29 genes, the reverse V30 gene exhibits preferential utilization with 'inversional rearrangement'. This may be attributed to the shorter distance between the V30 gene and D gene and the 'inversional rearrangement' modes. In summary, in the mammalian TRB locus, the reverse V30 gene has been uniquely preserved throughout evolution and preferentially utilized in V(D)J recombination, potentially serving a significant role in adaptive immunity. These results will pave the way for novel and specialized research into the mechanisms, efficiency, and function of V(D)J recombination in mammals.

Mitophagy in plants: Emerging regulators of mitochondrial targeting for selective autophagy.

The degradation and turnover of mitochondria is fundamental to Eukaryotes and is a key homeostatic mechanism for maintaining functional mitochondrial populations. Autophagy is an important pathway by which mitochondria are degraded, involving their sequestration into membrane-bound autophagosomes and targeting to lytic endosomal compartments (the lysosome in animals, the vacuole in plants and yeast). Selective targeting of mitochondria for autophagy, also known as mitophagy, distinguishes mitochondria from other cell components for degradation and is necessary for the regulation of mitochondria-specific cell processes. In mammals and yeast, mitophagy has been well characterised and is regulated by numerous pathways with diverse and important functions in the regulation of cell homeostasis, metabolism and responses to specific stresses. In contrast, we are only just beginning to understand the importance and functions of mitophagy in plants, chiefly as the proteins that target mitochondria for autophagy in plants are only recently emerging. Here, we discuss the current progress of our understanding of mitophagy in plants, the importance of mitophagy for plant life and the regulatory autophagy proteins involved in mitochondrial degradation. In particular, we will discuss the recent emergence of mitophagy receptor proteins that selectively target mitochondria for autophagy, and discuss the missing links in our knowledge of mitophagy-regulatory proteins in plants compared to animals and yeast.

Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions.

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2.

Poor T-cell receptor ß repertoire diversity early posttransplant for severe combined immunodeficiency predicts failure of immune reconstitution.

BACKGROUND: Development of a diverse T-cell receptor ß (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution. OBJECTIVES: We investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID. METHODS: We used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements. RESULTS: TRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention. CONCLUSIONS: TRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

Circulating TRB3 and GRP78 levels in type 2 diabetes patients: crosstalk between glucose homeostasis and endoplasmic reticulum stress.

PURPOSE: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus (T2DM) and insulin resistance. Tribbles homolog 3 (TRB3) is a pseudokinase upregulated by ER stress and hyperglycemia. Glucose-regulated protein 78 (GRP78) is an ER stress protein that is overexpressed under ER stress conditions. The current study aimed to investigate serum levels of TRB3 and GRP78, as an ER stress marker, in T2DM patients and their correlations with the metabolic profile. METHODS: Fifty-seven patients with type 2 diabetes and 23 healthy control subjects were evaluated for serum concentrations of TRB3, GRP78, and AGEs by enzyme-linked immunosorbent assay (ELISA). Fasting plasma glucose (FPG), HbA1c, lipid profile, TNF-α and insulin were also measured, and insulin resistance was calculated using a homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: Serum concentrations of TRB3, GRP78, AGEs, and TNF-α were significantly higher in T2DM patients compared to the healthy controls. Moreover, a statistically significant positive correlation was observed between plasma concentrations of TRB3 and FPG, HbA1c, HOMA-IR, and AGE. GRP78 levels were positively correlated with HbA1c and AGEs. There was also a positive correlation between GRP78 and TRB3. AGEs levels were positively correlated with the levels of FPG, HbA1c, HOMA-IR, and TNF-α. CONCLUSION: The current findings suggest that TRB3 and GRP78 may contribute to the pathogenesis of T2DM and might be considered as a therapeutic targets for the treatment of this disease.

Healthy-like CD4+ Regulatory and CD4+ Conventional T-Cell Receptor Repertoires Predict Protection from GVHD Following Donor Lymphocyte Infusion.

Donor lymphocyte infusion (DLI) can (re-)induce durable remission in relapsing patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). However, DLI harbors the risk of increased non-relapse mortality due to the co-occurrence of graft-versus-host disease (GVHD). GVHD onset may be caused or accompanied by changes in the clonal T-cell receptor (TCR) repertoire. To investigate this, we analyzed T cells in a cohort of 21 patients receiving DLI after alloHSCT. We performed deep T-cell receptor ß (TRB) sequencing of sorted CD4+CD25+CD127low regulatory T cells (Treg cells) and CD4+ conventional T cells (Tcon cells) in order to track longitudinal changes in the TCR repertoire. GVHD following DLI was associated with less diverse but clonally expanded CD4+CD25+CD127low Treg and CD4+ Tcon TCR repertoires, while patients without GVHD exhibited healthy-like repertoire properties. Moreover, the diversification of the repertoires upon GVHD treatment was linked to steroid-sensitive GVHD, whereas decreased diversity was observed in steroid-refractory GVHD. Finally, the unbiased sample analysis revealed that the healthy-like attributes of the CD4+CD25+CD127low Treg TCR repertoire were associated with reduced GVHD incidence. In conclusion, CD4+CD25+CD127low Treg and CD4+ Tcon TRB repertoire dynamics may provide a helpful real-time tool to improve the diagnosis and monitoring of treatment in GVHD following DLI.

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in ~ 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.

Obesity-induced TRB3 negatively regulates Brown adipose tissue function in mice.

Brown adipose tissue (BAT) and stimulating adaptive thermogenesis have been implicated as anti-obese and anti-diabetic tissues due to their ability to dissipate energy as heat by the expression of UCP1. We have recently demonstrated that TRB3 impairs differentiation of brown preadipocytes via inhibiting insulin signaling. However, the roles of the protein in BAT function and thermogenesis in vivo have not yet been established. For this study we tested the hypothesis that TRB3 mediates obesity- and diabetes-induced impairments in BAT differentiation and function, and that inhibition of TRB3 improves BAT function. TRB3 expression was increased in BAT from high-fat fed mice and ob/ob mice, which was associated with decreased UCP1 expression. Incubation of brown adipocytes with palmitate increased TRB3 expression and decreased UCP1. Knockout of TRB3 in mice displayed higher UCP1 expression in BAT and cold resistance. Incubation of brown adipocytes with ER stressors increased TRB3 but decreased UCP1 and ER stress markers were elevated in BAT from high-fat fed mice and ob/ob mice. Finally, high-fat feeding in TRB3KO mice were protected from obesity-induced glucose intolerance and displayed cold resistance and higher expression of BAT-specific markers. These data demonstrate that high-fat feeding and obesity increase TRB3 in BAT, resulting in impaired tissue function.

TRB3 mediates vascular remodeling by activating the MAPK signaling pathway in hypoxic pulmonary hypertension.

BACKGROUND: Hypoxic pulmonary hypertension (PH) is a refractory pulmonary vascular remodeling disease, and the efficiency of current PH treatment strategies is unsatisfactory. Tribbles homolog 3 (TRB3), a member of the pseudokinase family, is upregulated in diverse types of cellular stresses and functions as either a pro-proliferative or pro-apoptotic factor depending on the specific microenvironment. The regulatory mechanisms of TRB3 in hypoxic PH are poorly understood. METHODS: We performed studies using TRB3-specific silencing and overexpressing lentiviral vectors to investigate the potential roles of TRB3 on hypoxic pulmonary artery smooth muscle cells (PASMCs). Adeno-associated virus type 1(AVV1) vectors encoding short-hairpin RNAs against rat TRB3 were used to assess the role of TRB3 on hypoxic PH. TRB3 protein expression in PH patients was explored in clinical samples by western blot analysis. RESULTS: The results of whole-rat genome oligo microarrays showed that the expression of TRB3 and endoplasmic reticulum stress (ERS)-related genes was upregulated in hypoxic PASMCs. TRB3 protein expression was significantly upregulated by hypoxia and thapsigargin. In addition, 4-PBA and 4µ8C, both inhibitors of ERS, decreased the expression of TRB3. TRB3 knockdown promoted apoptosis and damaged the proliferative and migratory abilities of hypoxic PASMCs as well as inhibited activation of the MAPK signaling pathway. TRB3 overexpression stimulated the proliferation and migration of PASMCs but decreased the apoptosis of PASMCs, which was partly reversed by specific inhibitors of ERK, JNK and p38 MAPK. The Co-IP results revealed that TRB3 directly interacts with ERK, JNK, and p38 MAPK. Knockdown of TRB3 in rat lung tissue reduced the right ventricular systolic pressure and decreased pulmonary medial wall thickness in hypoxic PH model rats. Further, the expression of TRB3 in lung tissues was higher in patients with PH compared with those who have normal pulmonary artery pressure. CONCLUSIONS: TRB3 was upregulated in hypoxic PASMCs and was affected by ERS. TRB3 plays a key role in the pathogenesis of hypoxia-induced PH by binding and activating the ERK, JNK, and p38 MAPK pathways. Thus, TRB3 might be a promising target for the treatment of hypoxic PH.

Notch Signaling and the Breast Cancer Microenvironment.

Notch promotes breast cancer progression through tumor initiating cell maintenance, tumor cell fate specification, proliferation, survival, and motility. In addition, Notch is recognized as a decisive mechanism in regulating various juxtacrine and paracrine communications in the tumor microenvironment (TME). In this chapter, we review recent studies on stress-mediated Notch activation within the TME and sequelae such as angiogenesis, extracellular matrix remodeling, changes in the innate and adaptive immunophenotype, and therapeutic perspectives.

TRB3 interacts with ERK and JNK and contributes to the proliferation, apoptosis, and migration of lung adenocarcinoma cells.

Tribbles homolog 3 (TRB3) has been accounted for regulation of a few cell processes through interaction with other significant proteins. The molecular mechanisms underlying TRB3 in tumorigenesis in lung adenocarcinoma have not been entirely elucidated. The present study is aimed at determining the function and fundamental mechanisms of TRB3 in lung adenocarcinoma progression. TRB3 was highly expressed in A549 and H1299 cells and lung adenocarcinoma tissues compared with human bronchial epithelial cells (HBEpC) and adjacent normal lung tissues. Hypoxia significantly upregulated the expression of TRB3 protein in A549 and H1299 cells in a time-dependent way. Gene expression profiling interactive analysis data analysis indicated that patients with lung adenocarcinoma with excessive expression of TRB3 mRNA had fundamentally shorter survival time. TRB3 knockdown in A549 cells can inhibit cell proliferation and migration, and promote cell apoptosis. TRB3 knockdown reduced the expression of p-ERK and p-JNK, but did not affect the expression of p-P38 MAPK. TRB3 overexpression enhances the malignant transformation abilities of HBEpC such as cell proliferation, migration and colony formation, which could be reversed by U0126 and SP600125. TRB3 overexpression promotes the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but was not affected by U0126 and SP600125. The results of coimmunoprecipitation experiments indicated that TRB3 binds directly to ERK and JNK. This study suggests that TRB3 has a potentially carcinogenic role in lung adenocarcinoma by binding to ERK and JNK and promoting the phosphorylation of ERK and JNK. TRB3 can be a possible therapeutic focus for lung adenocarcinoma.

The expansion of the TRB and TRG genes in domestic goats (Capra hircus) is characteristic of the ruminant species.

BACKGROUND: Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects their successful adaptation to diverse environmental conditions. Despite the relevance of this species, the genetic research on the goat traits is limited compared to other domestic species. Thanks to the latest goat reference genomic sequence (ARS1), which is considered to be one of the most continuous assemblies in livestock, we deduced the genomic structure of the T cell receptor beta (TRB) and gamma (TRG) loci in this ruminant species. RESULTS: Our analyses revealed that although the organization of the goat TRB locus is broadly similar to that of the other artiodactyl species, with three in-tandem D-J-C clusters located at the 3' end, a complex and extensive series of duplications have occurred in the V genes at the 5' end, leading to a marked expansion in the number of the TRBV genes. This phenomenon appears to be a feature of the ruminant lineage since similar gene expansions have also occurred in sheep and cattle. Likewise, the general organization of the goat TRG genes is typical of ruminant species studied so far, with two paralogous TRG loci, TRG1 and TRG2, located in two distinct and distant positions on the same chromosome as result of a split in the ancestral locus. Each TRG locus consists of reiterated V-J-J-C cassettes, with the goat TRG2 containing an additional cassette relative to the corresponding sheep and cattle loci. CONCLUSIONS: Taken together, these findings demonstrate that strong evolutionary pressures in the ruminant lineage have selected for the development of enlarged sets of TRB and TRG genes that contribute to a diverse T cell receptor repertoire. However, differences observed among the goat, sheep and cattle TRB and TRG genes indicate that distinct evolutionary histories, with independent expansions and/or contractions, have also affected each ruminant species.

Topology and expressed repertoire of the Felis catus T cell receptor loci.

BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.

Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis.

The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.

Transitional B cells involved in autoimmunity and their impact on neuroimmunological diseases.

Transitional B cells (TrB cells) represent a crucial link between immature B cells in the bone marrow and mature peripheral B cells. Although TrB cells represent one of the regulatory B cell subpopulations in healthy individuals, the frequency of CD24hiCD38hi TrB cells in circulation may be altered in individuals with autoimmune diseases, such as multiple sclerosis, neuromyelitisoptica spectrum disorders, systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, systemic sclerosis, and juvenile dermatomyositis. Although TrB cells play regulatory roles under inflammatory conditions, consequences of their functional impairment vary across autoimmune diseases. Since the origin, development, and function of TrB cells, especially in humans, remain unclear and controversial, this review aimed to discuss the characteristics of TrB cells at steady state and explore their role in various immune diseases, including autoimmune rheumatic diseases and neuroimmunological diseases.

Profiling the pattern of human TRB/IGH-CDR3 repertoire in liver transplantation patients via high-throughput sequencing analysis.

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell-mediated inflammatory disease accompanied by inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheral blood mononuclear cell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.

TRB3 stimulates SIRT1 degradation and induces insulin resistance by lipotoxicity via COP1.

Fatty acid-induced lipotoxicity plays an important role in the pathogenesis of diabetes mellitus. Our previous studies have documented that lipotoxicity contributes to the onset and development of diabetes via insulin resistance and/or compromised function of the pancreatic ß-cells. However, the underlying molecular mechanisms associating lipotoxicity with insulin resistance remain to be fully elucidated. In this study, we explored the role of TRB3-COP1-SIRT1 in lipotoxicity leading to insulin resistance in hepatocytes. High fat diet (HFD)-fed mice and hepG2 cells stimulated with palmitate were utilized as models of lipid metabolism disorders. We analyzed the interactions of SIRT1 and COP1 with each other and with TRB3 using co-immunoprecipitation, western blotting. SIRT1 ubiquitination was also explored. Animal and cell experiments showed that lipotoxicity induced SIRT1 down-regulation at the protein level without altering the mRNA level, whereas, lipotoxicity led to up-regulation of TRB3 and COP1 at both the gene and protein levels. Mechanistic analysis indicated that COP1 functioned as an E3 Ub-ligase of SIRT1, responsible for its proteasomal degradation under lipotoxic conditions. TRB3 recruited COP1 to SIRT1 to promote its ubiquitination. Our data indicated for the first time that TRB3-COP1-SIRT1 pathway played an important role in lipotoxicity leading to insulin resistance in hepatocytes, and suggested that COP1 could be a potential therapeutic choice for the treatment of diabetes mellitus, with lipotoxicity being the important pathomechanism.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-ß-induced Neuronal Death.

Amyloid-ß (Aß)-induced neuron death is considered central to the pathogenesis of Alzheimer's disease (AD). Among several death modalities, autophagy and apoptosis play important roles in Aß-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon Aß treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro Increased Trib3 levels inhibited the activity of the kinase Akt by interacting with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to Aß insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. We found that Trib3 indirectly activates unc-51-like autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from Aß insult. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt, and FoxO1 in Aß-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD.

Liver-specific Gene Inactivation of the Transcription Factor ATF4 Alleviates Alcoholic Liver Steatosis in Mice.

Although numerous biological functions of the activating transcription factor 4 (ATF4) have been identified, a direct effect of ATF4 on alcoholic liver steatosis has not been described previously. The aim of our current study is to investigate the role of ATF4 in alcoholic liver steatosis and elucidate the underlying mechanisms. Here, we showed that the expression of ATF4 is induced by ethanol in hepatocytes in vitro and in vivo, and liver-specific ATF4 knock-out mice are resistant to ethanol-induced liver steatosis, associated with stimulated hepatic AMP-activated protein kinase (AMPK) activity. Furthermore, adenovirus-mediated AMPK knockdown significantly reversed the suppressive effects of ATF4 deficiency on ethanol-induced liver steatosis in mice. In addition, ethanol-fed ATF4 knock-out mice exhibit AMPK-dependent inhibition of fatty acid synthase and stimulation of carnitine palmitoyltransferase 1 (CPT1) in the liver. Moreover, hepatic Tribbles homolog 3 (TRB3) expression was stimulated by ethanol in an ATF4-dependent manner, and adenovirus-mediated TRB3 knockdown blocked ATF4-dependent ethanol-induced AMPK inhibition and triglyceride accumulation in AML-12 cells. Finally, TRB3 directly interacted with AMPK to suppress its phosphorylation. Taken together, these results identify the ATF4-TRB3-AMPK axis as a novel pathway responsible for ethanol-induced liver steatosis.