Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the ...Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.
Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The ...SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.
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•Small extracellular vesicles (sEVs) mediate paracrine senescence•Inhibition of sEV biogenesis prevents paracrine senescence•Multivesicular bodies and CD63 are increased during senescence in vivo•MS analysis identifies IFITM3 as partially responsible for sEV-paracrine senescence
Borghesan et al. show that the soluble fraction and small extracellular vesicles (sEVs) mediate paracrine senescence. RNA sequencing and loxP reporter systems confirm sEV-mediated paracrine senescence, while preventing sEV release averts senescence. Mass spectrometry and functional analysis show that the IFN protein, IFITM3, is partially responsible for this phenotype.
The AKT‐mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how ...cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K‐AKT activity by sustaining mTORC1‐ and S6K1‐dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55‐dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL‐PP2A/B55 kinase‐phosphatase module in controlling AKT and maintaining metabolic homeostasis.
Synopsis
In the presence of excessive signaling, the AKT‐mTOR pathway limits its activity by phosphorylating upstream regulators. When phosphorylated by mTOR, the MASTL/Greatwall kinase contributes to this negative feedback loop by inhibiting the phosphatase PP2A to prevent excessive AKT activity.
MASTL is a new component of the feedback loop that restricts mTOR activity.
MASTL inhibits dephosphorylation of IRS1 and GRB10 by PP2A/B55.
mTOR phosphorylates and activates MASTL to inhibit PP2A/B55.
MASTL inhibition results in improved glucose homeostasis in vivo.
The MASTL/Greatwall kinase is a new component of the feedback loop that restricts mTOR pathway activity in the presence of excessive signaling.
ABSTRACT
Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and ...extracellular vesicles. During this process, known as pre‐metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow‐derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell‐deficient mouse models (Wsh and MCTP5‐Cre‐RDTR), supporting a pro‐metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co‐culture of bone‐marrow‐derived mast cells with melanoma cells showed that they have an intrinsic anti‐metastatic activity. Mass spectrometry analysis of melanoma‐mast cell co‐cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16‐F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease‐free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti‐metastatic target in melanoma as previously suggested in other cancer types.
Melanoma lung metastasis is reduced in mast cell‐deficient mouse models. HMGA1 from melanoma cells promotes metastatic behaviour. Targeting HMGA1 expression by mast cells actions reduce melanoma metastasis.
Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional ...prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD+) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD+ pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD+ can be prophylactic or therapeutic in HCC.
•URI causes NAD+ depletion-dependent DNA damage leading to HCC development•Restoring NAD+ pools in vivo protects from DNA damage and HCC•URI inhibits AhR/ER transcriptional activity-mediated de novo NAD+ synthesis•URI-mediated de novo NAD+ synthesis inhibition may occur in human HCC
Tummala et al. show that overexpression of URI in mouse liver inhibits NAD+ metabolism, thereby causing DNA damage and tumorigenesis. Importantly, restoring NAD+ pools prevents DNA damage and tumor formation. URI expression in human HCC correlates negatively with L-tryptophan catabolism and patient survival.
Mouse embryonic stem cells (mESCs) in the primed pluripotency state, which resembles the post-implantation epiblast, can be de-differentiated in culture to a naive state that resembles the ...pre-implantation inner cell mass. We report that primed-to-naive mESC transition entails a significant slowdown of DNA replication forks and the compensatory activation of dormant origins. Using isolation of proteins on nascent DNA coupled to mass spectrometry, we identify key changes in replisome composition that are responsible for these effects. Naive mESC forks are enriched in MRE11 nuclease and other DNA repair proteins. MRE11 is recruited to newly synthesized DNA in response to transcription-replication conflicts, and its inhibition or genetic downregulation in naive mESCs is sufficient to restore the fork rate of primed cells. Transcriptomic analyses indicate that MRE11 exonuclease activity is required for the complete primed-to-naive mESC transition, demonstrating a direct link between DNA replication dynamics and the mESC de-differentiation process.
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•Primed-to-naive mESC transition triggers replication fork slowdown•iPOND-MS reveals differences in replisome composition of naive and primed mESCs•MRE11 exonuclease mediates fork slowdown in the primed-to-naive mESC transition•Inhibition of MRE11 exonuclease prevents complete primed-to-naive mESC transition
Ubieto-Capella et al. report that MRE11 nuclease is enriched at replication forks during primed-to-naive mESC transition and is responsible for replication fork slowdown. Inhibition of MRE11 exonuclease activity prevents the decrease in fork rate and interferes with complete primed-to-naive transition. This study links DNA replication dynamics with mESC reprogramming capacity.
The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an ...evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.
Protein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses ...have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.
We used a brief trypsin treatment followed by peptide separation and identification using nano‐LC followed by off‐line MS/MS to identify the surface proteins on live Candida albicans organisms ...growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty‐one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.
Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found ...S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin. S100A9 is shown here to function as a chromatin component modulating C3 expression in mouse and human cells by binding to a region upstream of the C3 start site. When S100A9 was genetically deleted in mouse models of skin inflammation, the psoriasis-like skin disease and inflammation were strongly attenuated, with a mild immune infiltrate and decreased amounts of C3. In addition, inhibition of C3 in the mouse model strongly reduced the inflammatory skin disease. Thus, S100A8-S100A9 can regulate C3 at the nuclear level and present potential new therapeutic targets for psoriasis.
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•S100A8-S100A9 regulate complement C3 expression•S100A8-S100A9 binds to chromatin at the C3 promoter•Loss of S100A8-S100A9 and/or C3 improves psoriasis-like symptoms•S100A8-S100A9 and C3 are specifically expressed in psoriasis
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