Vertebrate lonesome kinase modulates the hepatocyte secretome to prevent perivascular liver fibrosis and inflammation

Vertebrate lonesome kinase (VLK) is the only known extracellular tyrosine kinase, but its physiological functions are largely unknown. We show that VLK is highly expressed in hepatocytes of neonatal mice, but downregulated during adulthood. To determine the role of VLK in liver homeostasis and regeneration, we generated mice with a hepatocyte-specific knockout of the VLK gene (Pkdcc). Cultured progenitor cells established from primary hepatocytes of Pkdcc knockout mice produced a secretome, which promoted their own proliferation in 3D spheroids and proliferation of cultured fibroblasts. In vivo, Pkdcc knockout mice developed liver steatosis with signs of inflammation and perivascular fibrosis upon aging, combined with expansion of liver progenitor cells. In response to chronic CCl4-induced liver injury, the pattern of deposited collagen was significantly altered in these mice. The liver injury marker alpha-fetoprotein (AFP) was increased in the secretome of VLK-deficient cultured progenitor cells and in liver tissues of aged or CCl4-treated knockout mice. These results support a key role for VLK and extracellular protein phosphorylation in liver homeostasis and repair through paracrine control of liver cell function and regulation of appropriate collagen deposition. This article has an associated First Person interview with the first author of the paper

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation

mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1

mTORC1 is a master regulator of cell growth with well-known functions in inhibiting autophagic vesicle formation. Here, the authors show that mTORC1 also affects Golgi architecture and vesicle secretion by phosphorylating the scaffold protein SCYL1. The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1

Protein and molecular characterization of a clinically compliant amniotic fluid stem cell derived extracellular vesicle fraction capable of accelerating muscle regeneration through the enhancement of angiogenesis

The secretome of human amniotic fluid stem cells (AFSC) has great potential as a therapeutic agent in regenerative medicine. However it must be produced in a clinically compliant manner before it can be used in humans. Here we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EV) that harbour much but not all the biological activity of the whole secretome. Proteomic characterisation of the EV and free secretome fraction show the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly we show that the EVs only contain miRNA and not mRNA. This suggests tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than the imposition of new, signalling pathways. The EVs harbour significant anti-inflammatory activity as well as promoting angiogenesis; the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury

Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions.

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation

Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins

Abstract Background The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair. Methods Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source. Results Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome. Conclusions Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation

Autophagy protein 5 controls flow-dependent endothelial functions

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing

Cardioprotection and lifespan extension by the natural polyamine spermidine

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease