mTORC1 restricts TFE3 activity by auto-regulating its presence on lysosomes.

To stimulate cell growth, the protein kinase complex mTORC1 requires intracellular amino acids for activation. Amino-acid sufficiency is relayed to mTORC1 by Rag GTPases on lysosomes, where growth factor signaling enhances mTORC1 activity via the GTPase Rheb. In the absence of amino acids, GATOR1 inactivates the Rags, resulting in lysosomal detachment and inactivation of mTORC1. We demonstrate that in human cells, the release of mTORC1 from lysosomes depends on its kinase activity. In accordance with a negative feedback mechanism, activated mTOR mutants display low lysosome occupancy, causing hypo-phosphorylation and nuclear localization of the lysosomal substrate TFE3. Surprisingly, mTORC1 activated by Rheb does not increase the cytoplasmic/lysosomal ratio of mTORC1, indicating the existence of mTORC1 pools with distinct substrate specificity. Dysregulation of either pool results in aberrant TFE3 activity and may explain nuclear accumulation of TFE3 in epileptogenic malformations in focal cortical dysplasia type II (FCD II) and tuberous sclerosis (TSC).

TRIB3 Modulates PPARγ-Mediated Growth Inhibition by Interfering with the MLL Complex in Breast Cancer Cells.

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.

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo.

OBJECTIVE: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression.

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.