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Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype. [ABSTRACT FROM AUTHOR]

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer. [ABSTRACT FROM AUTHOR]

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. Here, we report that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions. [ABSTRACT FROM AUTHOR]

Limited oxygen availability impairs normal body growth, although the underlying mechanisms are not fully understood. In Drosophila, hypoxic responses in the larval fat body (FB) disturb the secretion of insulin‐like peptides from the brain, inhibiting body growth. However, the cell‐autonomous effects of hypoxia on the insulin‐signaling pathway in larval FB have been underexplored. In this study, we aimed to examine the effects of overexpression of Sima, a Drosophila hypoxia‐inducible factor‐1 (HIF‐1) α homolog and a key component of HIF‐1 transcription factor essential for hypoxic adaptation, on the insulin‐signaling pathway in larval FB. Forced expression of Sima in FB reduced the larval body growth with reduced Akt phosphorylation levels in FB cells and increased hemolymph sugar levels. Sima‐mediated growth inhibition was reversed by overexpression of TOR or suppression of FOXO. After Sima overexpression, larvae showed higher expression levels of Tribbles, a negative regulator of Akt activity, and a simultaneous knockdown of Tribbles completely abolished the effects of Sima on larval body growth. Furthermore, a reporter analysis revealed Tribbles as a direct target gene of Sima. These results suggest that Sima in FB evokes Tribbles‐mediated insulin resistance and consequently protects against aberrant insulin‐dependent larval body growth under hypoxia. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death due to cancer in the world. Therefore, the identification of novel druggable targets is urgently needed. Tribbles proteins belong to a pseudokinase family, previously recognized in CRC as oncogenes and potential therapeutic targets. Here, we analyzed the expression of TRIB1, TRIB2, and TRIB3 simultaneously in 33 data sets from CRC based on available GEO profiles. We show that all three Tribbles genes are overrepresented in CRC cell lines and primary tumors, though depending on specific features of the CRC samples. Higher expression of TRIB2 in the tumor microenvironment and TRIB3 overexpression in an early stage of CRC development, unveil a potential and unexplored role for these proteins in the context of CRC. Differential Tribbles expression was also explored in diverse cellular experimental conditions where either genetic or pharmacological approaches were used, providing novel hints for future research. This comprehensive bioinformatic analysis provides new insights into Tribbles gene expression and transcript regulation in CRC. [ABSTRACT FROM AUTHOR]

The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3'untranslated region (3'UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple "high confidence" miRNAs potentially binding to the 3'UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3'UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells. [ABSTRACT FROM AUTHOR]

Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the 'dark' pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin‐producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co‐evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis‐independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species‐level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome. [ABSTRACT FROM AUTHOR]

The basic leucine-zipper (bZIP) domain transcription factors CCAAT/enhancer-binding proteins (C/EBP) have a variety of roles in cell proliferation, differentiation, and stress response. In the nervous system, several isoforms of C/EBP function in learning and memory, neuronal plasticity, neuroinflammation, and axon regeneration. We previously reported that the Caenorhabditis elegans C/EBP homolog, CEBP-1, is essential for axon regeneration. CEBP-1 consists of 319 amino acids, with its bZIP domain at the C-terminus and a long N-terminal fragment with no known protein motifs. Here, using forward genetic screening with targeted genome editing, we have identified a unique domain in the N-terminus that is critical for its in vivo function. Additionally, we characterized three nuclear localization signals (NLS) in CEBP-1 that act together to mediate CEBP-1's nuclear import. Moreover, the Importin-α, IMA-3, can bind to CEBP-1 via one of the NLS. ima-3 is ubiquitously expressed in all somatic cells, and ima-3 null mutants are larval lethal. Using Cre-lox dependent neuron-specific deletion strategy, we show that ima-3 is not critical for axon development, but is required for axon regeneration in adults. Together, these data advance our understanding of CEBP-1's function, and suggest new regulators that remain to be identified to expand the CEBP-1 protein interactome. [ABSTRACT FROM AUTHOR]

COP1 is a highly conserved ubiquitin ligase that regulates diverse cellular processes in plants and metazoans. Tribbles pseudokinases, which only exist in metazoans, act as scaffolds that interact with COP1 and its substrates to facilitate ubiquitination. Here, we report that, in addition to this scaffolding role, TRIB1 promotes nuclear localization of COP1 by disrupting an intramolecular interaction between the WD40 domain and a previously uncharacterized regulatory site within COP1. This site, which we have termed the pseudosubstrate latch (PSL), resembles the consensus COP1‐binding motif present in known COP1 substrates. Our findings support a model in which binding of the PSL to the WD40 domain stabilizes a conformation of COP1 that is conducive to CRM1‐mediated nuclear export, and TRIB1 displaces this intramolecular interaction to induce nuclear retention of COP1. Coevolution of Tribbles and the PSL in metazoans further underscores the importance of this role of Tribbles in regulating COP1 function. Synopsis: COP1 is a highly conserved E3 ubiquitin ligase that shuttles between the nucleus and cytoplasm. In plants, COP1 localization is controlled by light, while in metazoans, the pseudokinase TRIB1 promotes COP1 nuclear localization by competing with an intramolecular site that regulates CRM1‐mediated nuclear export of COP1. Binding of the TRIB1 C‐terminal tail to the COP1 WD40 domain potentiates COP1 nuclear localization.COP1 nucleocytoplasmic shuttling is controlled by an intramolecular site, termed the pseudosubstrate latch (PSL), that competes with TRIB1 for binding to the WD40 domain.Disruption of the PSL/WD40 interaction through mutations or TRIB1 binding interferes with CRM1‐mediated nuclear export of COP1.Phylogenetic analysis indicates that the COP1‐binding motifs in the TRIB1 tail and COP1 PSL coevolved in metazoan species. Localization of the conserved ubiquitin ligase COP1, known to be light‐controlled in plants, depends on regulated disruption of an intramolecular pseudo‐substrate interaction in metazoans. [ABSTRACT FROM AUTHOR]

Background/Aims: Obesity is a serious health risk factor strongly associated with insulin resistance and type 2 diabetes; however, the underlying mechanisms associating obesity with insulin resistance remain unknown. In this study, we explored the physiological role of Trib3 in regulating glucose metabolism in skeletal muscle tissues in a Trib3 transgenic mice model. Methods: Glucose metabolism in transgenic mice overexpressing Trib3 specifically in the skeletal muscle was examined by glucose/insulin tolerance test, metabolic cage studies, and glucose uptake assay. The effect of Trib3 overexpression on AKT phosphorylation and AKT protein turnover were assessed by RT-PCR and immunoblot analysis. Subcellular distribution of Trib3 and AKT1/2 was determined by microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. Ubiquitin assay was performed and ATG7 deficient cell line was employed to address the mechanisms of Trib3-dependent AKT protein homeostasis. Results: We found that Trib3 expression in skeletal muscle is elevated in obese conditions, and transgenic mice that overexpressed Trib3, specifically in skeletal muscle tissues, displayed impaired glucose homeostasis by suppressing insulin-stimulated glucose uptake. Disruption of insulin signaling in skeletal muscle Trib3 transgenic mice may occur due to the specific downregulation of AKT2 but not AKT1. Autophagy regulated AKT2 protein turnover, and Trib3 overexpression stimulated autophagic degradation of AKT2 by promoting AKT2 ubiquitination. Conclusion: Because diet-induced obesity upregulates Trib3 and downregulates AKT2 in skeletal muscle tissues, Trib3 may play a key role in establishing an association between obesity and insulin resistance by regulating AKT2 protein homeostasis. [ABSTRACT FROM AUTHOR]

CD8+ T cell exhaustion (T EX) impairs the ability of T cells to clear chronic infection or cancer. While T EX are hypofunctional, some T EX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ T EX (T KLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell T cell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts T EX toward CX3CR1+ intermediates with robust enrichment of T KLR via clonal T cell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ T KLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ T cells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ T EX whose targeting enhances the T KLR effector state and improves checkpoint inhibitor therapy. [Display omitted] • Trib1 deficiency promotes effector gene expression during T cell exhaustion (T EX) • Trib1 deletion in T EX drives clonal expansion of effector-like CD8+ T INT and T KLR • Promotion of T KLR in Trib1 KO is CD8 intrinsic, but viral control requires CD4 cells • Trib1 deletion improves viral control during PD-L1 blockade McClory et al. demonstrate that deletion of Trib1 in T cells promotes clonal expansion of exhausted CD8+ T INT and T KLR cells with an effector-like transcriptional program during chronic LCMV infection. Trib1 deletion improves viral control during PD-L1 blockade, suggesting that targeting Trib1 may improve outcomes during chronic infection and cancer. [ABSTRACT FROM AUTHOR]

Background: Tribbles proteins are conserved pseudokinases that function to control kinase signalling and transcription in diverse biological processes. Abnormal function in human Tribbles has been implicated in a number of diseases including leukaemia, metabolic syndromes and cardiovascular diseases. Caenorhabditis elegans Tribbles NIPI-3 was previously shown to activate host defense upon infection by promoting the conserved PMK-1/p38 mitogen-activated protein kinase (MAPK) signalling pathway. Despite the prominent role of Tribbles proteins in many species, our knowledge of their mechanism of action is fragmented, and the in vivo functional relevance of their interactions with other proteins remains largely unknown. Results: Here, by characterizing nipi-3 null mutants, we show that nipi-3 is essential for larval development and viability. Through analyses of genetic suppressors of nipi-3 null mutant lethality, we show that NIPI-3 negatively controls PMK-1/p38 signalling via transcriptional repression of the C/EBP transcription factor CEBP-1. We identified CEBP-1's transcriptional targets by ChIP-seq analyses and found them to be enriched in genes involved in development and stress responses. Unlike its cell-autonomous role in innate immunity, NIPI-3 is required in multiple tissues to control organismal development. Conclusions: Together, our data uncover an unprecedented crosstalk involving multiple tissues, in which NIPI-3 acts as a master regulator to inhibit CEBP-1 and the PMK-1/p38 MAPK pathway. In doing so, it keeps innate immunity in check and ensures proper organismal development. [ABSTRACT FROM AUTHOR]

Tribbles (TRIB) proteins, a family of evolutionary conserved psuedokinase proteins, modulate various signalling pathways within the cell. The regulatory roles of TRIB make them an important part of a number of biological processes ranging from cell proliferation to metabolism, immunity, inflammation and carcinogenesis. Innate immune system plays a pivotal role during the regulation of reproductive processes that allows successful creation of an offspring. Its involvement initiates from fertilization of the oocyte by spermatozoon and lasts throughout early embryonic development, pregnancy and labour. Therefore, there is a close cooperation between the reproductive system and the innate immune system. Evidence from our lab has demonstrated that improper activation of the innate immune system can reduce embryo implantation, thus leading to infertility. Therefore, control mechanisms regulating the innate immune system function can be critical for successful reproductive events. [ABSTRACT FROM AUTHOR]

The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types. [ABSTRACT FROM AUTHOR]

Tribbles (TRIB) proteins are pseudokinase mediators of eukaryotic signalling that have evolved important roles in lipoprotein metabolism, immune function and cellular differentiation and proliferation. In addition, an evolutionary-conserved modulation of PI3K/AKT signalling pathways highlights them as novel and rather unusual pharmaceutical targets. The three human TRIB family members are uniquely defined by an acidic pseudokinase domain containing a 'broken' a C-helix and a MEK (MAPK/ERK)-binding site at the end of the putative C-lobe and a distinct C-terminal peptide motif that interacts directly with a small subset of cellular E3 ubiquitin ligases. This latter interaction drives proteasomal-dependent degradation of networks of transcription factors, whose rate of turnover determines the biological attributes of individual TRIB family members. Defining the function of individual Tribs has been made possible through evaluation of individual TRIB knockout mice, siRNA/overexpression approaches and genetic screening in flies, where the single TRIB gene was originally described 15 years ago. The rapidly maturing TRIB field is primed to exploit chemical biology approaches to evaluate endogenous TRIB signalling events in intact cells. This will help define how TRIB-driven protein-protein interactions and the atypical TRIB ATP-binding site, fit into cellular signalling modules in experimental scenarios where TRIB-signalling complexes remain unperturbed. In this minireview, we discuss how small molecules can reveal rate-limiting signalling outputs and functions of Tribs in cells and intact organisms, perhaps serving as guides for the development of new drugs. We predict that appropriate small molecule TRIB ligands will further accelerate the transition of TRIB pseudokinase analysis into the mainstream of cell signalling. [ABSTRACT FROM AUTHOR]

TRIB2 (tribbles homolog 2) encodes one of three members of the tribbles family in mammals. These members share a Trb (tribbles) domain, which is homologous to protein serine-threonine kinases, but lack the active site lysine. The tribbles proteins interact and modulate the activity of signal transduction pathways in a number of physiological and pathological processes. TRIB2 has been identified as an oncogene that inactivates the transcription factor CCAAT/enhancer-binding protein a (C/EBPa) and causes acute myelogenous leukaemia (AML). Recent research provided compelling evidence that TRIB2 can also act as oncogenic driver in solid tumours, such as lung and liver cancer. In particular, our recent work demonstrated that TRIB2 is dramatically overexpressed in malignant melanomas compared with normal skin and promotes the malignant phenotype of melanoma cells via the down-regulation of FOXO (forkhead box protein O) tumour suppressor activity in vitro and in vivo. TRIB2 was found to be expressed in normal skin, but its expression consistently increased in benign nevi, melanoma and was highest in samples from patients with malignant melanoma. The observation that TRIB2 strongly correlates with the progression of melanocytederived malignancies suggests TRIB2 as a meaningful biomarker to both diagnose and stage melanoma. In addition, interfering with TRIB2 activity might be a therapeutic strategy for the treatment of several different tumour types. [ABSTRACT FROM AUTHOR]

The protein tribbles-1, encoded by the gene TRIB1, is increasingly recognized as a major regulator of multiple cellular and physiological processes in humans. Recent human genetic studies, as well as molecular biological approaches, have implicated this intriguing protein in the aetiology of multiple human diseases, including myeloid leukaemia, Crohn's disease, non-alcoholic fatty liver disease (NAFLD), dyslipidaemia and coronary artery disease (CAD). Genome-wide association studies (GWAS) have repeatedly identified variants at the genomic TRIB1 locus as being significantly associated with multiple plasma lipid traits and cardiovascular disease (CVD) in humans. The involvement of TRIB1 in hepatic lipid metabolism has been validated through viral-mediated hepatic overexpression of the gene in mice; increasing levels of TRIB1 decreased plasma lipids in a dose-dependent manner. Additional studies have implicated TRIB1 in the regulation of hepatic lipogenesis and NAFLD. The exact mechanisms of TRIB1 regulation of both plasma lipids and hepatic lipogenesis remain undetermined, although multiple signalling pathways and transcription factors have been implicated in tribbles-1 function. Recent reports have been aimed at developing TRIB1- based lipid therapeutics. In summary, tribbles-1 is an important modulator of human energy metabolism and metabolic syndromes and worthy of future studies aimed at investigating its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

Inflammation is part of the physiological innate immune response to invading pathogens and tissue injury. However, unresolved inflammation leads to human disease. The tribbles (TRIB) family of pseudokinase proteins has been shown to modulate key inflammatory signalling pathways, including the MAPK (mitogenactivated protein kinase) and PI3K (phosphoinositide 3-kinase) networks. This review summarizes our current knowledge on TRIBs in the context of inflammation, both at the level of molecular mechanisms and in disease development. [ABSTRACT FROM AUTHOR]

The pseudo-kinase family of tribbles (TRIB) proteins has been linked to a variety of cell signalling pathways and appears to have functionally divergent roles with respect to intracellular protein degradation and the ability to regulate signal transduction pathways. In the arthritides, inflammation and a wide variety of proinflammatory pathways have been implicated to drive the cartilage destruction and consequent disability associated with both rheumatoid arthritis (RA) and osteoarthritis (OA). Despite burgeoning evidence linking the TRIB to inflammation-related pathologies such as diabetes, multiple sclerosis and cancer, very little is known about their roles in arthritis. The present review discusses current knowledge of the impact of TRIB on pro-inflammatory cellular mechanisms and pathways known to be important in the pathogenesis of RA and OA. [ABSTRACT FROM AUTHOR]

The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the 'pseudocatalytic' domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metalindependent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an 'analogue-sensitive' (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolopyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery. [ABSTRACT FROM AUTHOR]

Background In mammals, the Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that are involved in multiple biological processes including cell proliferation and fatty acid (FA) metabolism. Our recent studies highlighted the importance of FA metabolism in cumulus cells (CC) during oocyte maturation in vertebrates and reported a higher TRIB1 expression in CC surrounding in vivo mature oocytes as compared to immature ooocytes in mice and cows. The objective was to investigate Tribbles expression patterns in bovine CC during in vitro maturation (IVM) and to examine their roles in the cumulus-oocyte complex. Methods Tribbles gene expression was analyzed in bovine and murine CC using quantitative RT-PCR. Proteins were detected using Western blot and intracellular localization was assessed by immunofluorescence. Bovine COCs were treated with etomoxir, an inhibitor of FA oxidation (FAO) which blocks CPT1 activity, during 6 h and 18 h IVM. Oocyte meiotic stage was assessed and expression of Tribbles and lipid metabolism genes was quantified in CC. Results and discussion TRIB1 and TRIB3 were more strongly expressed whereas TRIB2 was less expressed in CC surrounding the oocytes from preovulatory follicles than in CC of immature ones. In CC, Tribbles were located in the cytoplasm and nucleus; in mitotic cells TRIB2 and TRIB3 were detected in the spindle. In the oocyte, Tribbles proteins were detected in the ooplasm; also TRIB2 and TRIB3 were more accumulated in the germinal vesicle. In bovine CC, expression of TRIB1 and TRIB3 was transiently increased at a time preceding oocyte meiosis resumption in vitro. Treatment with etomoxir (150 μM) during IVM resulted in a significant reduction of oocyte maturation rate and decreased MAPK3/1 phosphorylation in the oocytes. In CC, 18 h IVM of etomoxir treatment significantly increased expression of TRIB1, TRIB3, CPTA1 (enzyme regulating FA entry in mitochondria for FAO) and CD36 (thrombospondin receptor involved in FA transport). Under the same conditions, expression of TRIB2 and ACACA (Acetyl coenzyme A carboxylase involved in FA synthesis) decreased in CC. All considered, Tribbles family members may be involved in FAO signaling in CC and participate in oocyte meiotic resumption regulation. [ABSTRACT FROM AUTHOR]

Narcolepsy is characterized by excessive daytime sleepiness, with or without cataplexy. Associated features include sleep paralysis, hypnagogic or hypnopompic hallucinations, and disturbed nocturnal sleep. Narcolepsy is strongly associated with the HLA DQB1*0602 allele, and its symptoms stem from destruction of hypocretin-secreting neurons in the hypothalamus. Recently identified autoantibodies to Tribbles homologue 2 in some patients, as well as cases associated with H1N1 vaccination, support an autoimmune mechanism. There are many challenges in diagnosing and treating pediatric narcolepsy. Caution must also be used in interpreting polysomnography and multiple sleep latency test results in children. HLA testing is nonspecific, and no commercial test exists to measure cerebrospinal fluid hypocretin levels. Neuroimaging has not yet proven useful in primary narcolepsy. Treatment of sleepiness and cataplexy in children requires extrapolating from adult studies. Hopefully, further insights into the pathophysiology of narcolepsy will allow for new therapeutics to manage the symptoms and modify the course of the disease. [ABSTRACT FROM AUTHOR]

Sensing and interpreting extracellular signals in response to changes in the environment has been a fundamental feature of all life forms from the very beginning of evolution. To fulfil this function, networks of proteins have evolved, forming the intracellular signal transduction machinery. Whereas the appropriate control of these signal transduction systems is essential to homoeostasis, dysregulation of signalling leads to disease and often the death of the organism. The tribbles family of pseudokinases have emerged in recent years as key controllers of signal transduction via their interactions with several key kinases, ubiquitin ligases and transcription factors. In line with their role in regulating fundamentally important signalling pathways, members of the tribbles family have been implicated in the development of a range of human diseases. Whereas our mechanistic understanding of how these proteins contribute to disease is far from complete, the present paper attempts to summarize some of the most important recent developments in this field of research. [ABSTRACT FROM AUTHOR]

Identification of rate-limiting steps or components of intracellular second messenger systems holds promise to effectively interfere with these pathways under pathological conditions. The emerging literature on a recently identified family of signalling regulator proteins, called tribbles gives interesting clues for how these proteins seem to link several ‘independent’ signal processing systems together. Via their unique way of action, tribbles co-ordinate the activation and suppression of the various interacting signalling pathways and therefore appear to be key in determining cell fate while responding to environmental challenges. This review summarises our current understanding of tribbles function and also provides an evolutionary perspective on the various tribbles genes. [ABSTRACT FROM AUTHOR]

Simple Summary: Tribbles proteins play various roles in cancer initiation and progression. However, still little is known about their molecular actions. Here we developed a mass spectrometry-based approach to study the Tribbles interactomes, allowing us to discover new interactors and functions that might help to understand their behavior better. Our proteomics data highlight the ability of TRIB3 to interact with transcription regulatory proteins and point to a new role in gene repression. Systematic analyses like these will help to evaluate the potential of Tribbles proteins as biomarkers for disease diagnosis and prognosis. The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles' interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology. [ABSTRACT FROM AUTHOR]

Background TRIB1, TRIB2 and TRIB3 belong to the mammalian Tribbles family of pseudokinases proteins. Several studies reported Tribbles oncogenic role in different types of cancer, including colorectal cancer (CRC). Though current CRC treatment can be curative, patients are in risk of disease recurrence, meaning novel pharmacological targets and strategies are required. Our goal was to analyze Tribbles gene expression in CRC in response to different drugs. Methods Tribbles transcript levels were obtained from GEO profiles database (NCBI). Gene data sets (GDS) were selected based on experimental drug treatment description. Statistical analysis was performed at GraphPadPrism. Results Compared to non-treated control, TRIB2 expression was ∼2-fold increased in colorectal adenocarcinoma samples from patients treated with cyclooxygenase-2 inhibitor celecoxib (GDS3384), though not statistically significant (P < 0.1). TRIB1 was unaltered and data for TRIB3 was not available. By contrast, all Tribbles showed differential expression after treatment of SW620 colon cancer cells with supercritical rosemary extract in progressive increasing doses (0, 30, 60, 100 μg/mL) (P < 0.01 ;GDS5416). While both TRIB1 and TRIB3 were moderately increased in a dose-dependent manner (∼18% and 13%, respectively), TRIB2 was maximally down-regulated by ∼15% after 60 μg/mL. Conclusions Although celecoxib exhibits antiproliferative effects in different cancer cell types, TRIB2 gene expression showed a trend to be induced after treatment, in contrast to several genes involved in fatty acid oxidation that were down-regulated, which could result from a compensatory mechanism based on a metabolic shift. Since TRIB1 / TRIB3 and TRIB2 were oppositely modulated in response to rosemary extract, additional studies are needed to validate its specific pharmacological potential interest for CRC treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Tribbles proteins possess the structure of protein kinases but function by forming protein complexes rather than phosphorylating substrates. Here we review the structure–function relationship of TRIB1 in cancers. Of the Tribbles proteins, TRIB1 is currently the most well characterised structurally. TRIB1 has different states that could potentially be targeted by small-molecule inhibitors and well-established relevance in acute myeloid leukaemia through degradation of transcription factors. Less is understood about the role of TRIB1 in solid tumours. Further research is required to fully realise the potential of TRIB1 as either a direct target of small-molecule drugs or a biomarker of treatment response across diverse cancer types. The Tribbles family of proteins—comprising TRIB1, TRIB2, TRIB3 and more distantly related STK40—play important, but distinct, roles in differentiation, development and oncogenesis. Of the four Tribbles proteins, TRIB1 has been most well characterised structurally and plays roles in diverse cancer types. The most well-understood role of TRIB1 is in acute myeloid leukaemia, where it can regulate C/EBP transcription factors and kinase pathways. Structure–function studies have uncovered conformational switching of TRIB1 from an inactive to an active state when it binds to C/EBPα. This conformational switching is centred on the active site of TRIB1, which appears to be accessible to small-molecule inhibitors in spite of its inability to bind ATP. Beyond myeloid neoplasms, TRIB1 plays diverse roles in signalling pathways with well-established roles in tumour progression. Thus, TRIB1 can affect both development and chemoresistance in leukaemia; glioma; and breast, lung and prostate cancers. The pervasive roles of TRIB1 and other Tribbles proteins across breast, prostate, lung and other cancer types, combined with small-molecule susceptibility shown by mechanistic studies, suggests an exciting potential for Tribbles as direct targets of small molecules or biomarkers to predict treatment response. [ABSTRACT FROM AUTHOR]

Simple Summary: The Tribbles family of pseudokinases controls a wide number of processes during cancer on-set and progression. However, the exact contribution of each of the three family members is still to be defined. Their functions appear to be context-dependent as they can act as oncogenes or tumor suppressor genes. They act as scaffolds modulating the activity of several signaling pathways involved in different cellular processes. In this review, we discuss the state-of-knowledge for TRIB1, TRIB2 and TRIB3 in the development and progression of colorectal cancer. We take a perspective look at the role of Tribbles proteins as potential biomarkers and therapeutic targets. The Tribbles family of pseudokinases controls a wide number of processes during cancer on-set and progression. However, the exact contribution of each of the three family members is still to be defined. Their function appears to be context-dependent as they can act as oncogenes or tumor suppressor genes. They act as scaffolds modulating the activity of several signaling pathways involved in different cellular processes. In this review, we discuss the state-of-knowledge for TRIB1, TRIB2 and TRIB3 in the development and progression of colorectal cancer. We take a perspective look at the role of Tribbles proteins as potential biomarkers and therapeutic targets. Specifically, we chronologically systematized all available articles since 2003 until 2020, for which Tribbles were associated with colorectal cancer human samples or cell lines. Herein, we discuss: (1) Tribbles amplification and overexpression; (2) the clinical significance of Tribbles overexpression; (3) upstream Tribbles gene and protein expression regulation; (4) Tribbles pharmacological modulation; (5) genetic modulation of Tribbles; and (6) downstream mechanisms regulated by Tribbles; establishing a comprehensive timeline, essential to better consolidate the current knowledge of Tribbles' role in colorectal cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: The Tribbles proteins are members of CAMK Ser/Thr protein kinase family. They are evolutionary conserved pseudokinases found in most tissues of eukaryotic organisms. This ubiquitously expressed protein family is characterized by containing a catalytically deficient kinase domain which lacks amino acid residues required for the productive interaction with ATP and metal ions. Tribbles proteins exert their biological functions mainly through direct interaction with MAPKK and AKT proteins, therefore regulating important pathways involved in cell proliferation, apoptosis and differentiation. Due to the role of MAPKK and AKT signalling in the context of cancer development, Tribbles proteins have been recently considered as biomarkers of cancer progression. Furthermore, as the atypical pseudokinase domain retains a binding platform for substrates, Tribbles targeting provides an attractive opportunity for drug development. The Tribbles pseudokinases family consists of TRIB1, TRIB2, TRIB3 and STK40 and, although evolutionarily conserved, they have distinctive characteristics. Tribbles members are expressed in a context and cell compartment-dependent manner. For example, TRIB1 and TRIB2 have potent oncogenic activities in vertebrate cells. Since the identification of Tribbles proteins as modulators of multiple signalling pathways, recent studies have linked their expression with several pathologies, including cancer. Tribbles proteins act as protein adaptors involved in the ubiquitin-proteasome degradation system, as they bridge the gap between substrates and E3 ligases. Between TRIB family members, TRIB2 is the most ancestral member of the family. TRIB2 is involved in protein homeostasis regulation of C/EBPα, β-catenin and TCF4. On the other hand, TRIB2 interacts with MAPKK, AKT and NFkB proteins, involved in cell survival, proliferation and immune response. Here, we review the characteristic features of TRIB2 structure and signalling and its role in many cancer subtypes with an emphasis on TRIB2 function in therapy resistance in melanoma, leukemia and glioblastoma. The strong evidence between TRIB2 expression and chemoresistance provides an attractive opportunity for targeting TRIB2. [ABSTRACT FROM AUTHOR]

Simple Summary: Proteasome inhibitors are currently used in the treatment of certain blood cancers, and clinical trials to treat solid tumors, including liver cancer, have also been conducted. However, different malignancies are not equally susceptible to proteasome inhibitors, and resistance to the drug may develop during the therapy. Here, we characterize the molecular mechanisms underlying the resilience of liver cancer cells to the proteasome inhibitor bortezomib. The results demonstrate that the activity of the eIF2α–ATF4 stress response pathway affects the viability of cells treated with bortezomib. We found that the pseudokinase TRIB3, an endogenous regulator of ATF4 and a gene highly expressed in liver cancer, resides predominantly at the same chromatin sites as ATF4 and constrains ATF4 activity. The survival of bortezomib-exposed hepatoma cells proved sensitive to TRIB3 overexpression and inactivation. Thus, TRIB3 is a novel factor contributing to bortezomib resistance of liver cancer cells. The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib. [ABSTRACT FROM AUTHOR]

The article offers information on the meeting of the Biochemical Society entitled "Tribbles pseudokinases at the crossroads of metabolism, cancer, immunity and development" held in Budapest, Hungary in April 2015. Topics discussed include oncogenic compared with tumour suppressor roles for Interest in the biology of tribbles (TRIB) family, interaction between TRIB and constitutive photomorphogenic 1 (COP1) E3 ubiquitin ligase and catalytic activity of TRIB.