SLC1A5 provides glutamine and asparagine necessary for bone development in mice

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.

OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis

O-GlcNAc transferase (OGT) is an essential X-chromosome-encoded enzyme that catalyzes the addition of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of serine and threonine residues on many nuclear and cytosolic proteins. This posttranslational modification is reversible and is actively removed by the O-GlcNAc’ase OGA. It was shown more than two decades ago that OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. Given the close association between OGT and human diseases, such as cancer, diabetes and cardiovascular disease, identification of the mechanisms by which OGT controls cell viability will facilitate the development of therapeutic strategies to manipulate OGT activity. Here, we employ a genome-wide CRISPR-Cas9 viability screen in mouse embryonic stem cells (mESCs) with inducible Ogt deletion to show that the block in cell viability induced by Ogt-deficiency stems from a deleterious increase in mitochondrial oxidative phosphorylation (OXPHOS). Mechanistically, we demonstrate that OGT safeguards mTOR (mechanistic target of rapamycin) activity to maintain mitochondrial fitness through modulation of proteasome activity and intracellular amino acid homeostasis. In the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR translocation and activation and increased oxidative phosphorylation. This mechanism also operates in CD8+ T cells, indicating its generality across mammalian cell types. Genome-wide proteomic and phosphoproteomic analyses show extensive changes in global signaling and confirm our finding of mTOR hyperactivation in OGT-deficient cells. In sum, our study highlights a novel function for OGT in regulating the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness and cell viability. Since many of the proteins involved in proteasome, mTOR and mitochondrial activity are aberrantly expressed in cancer, and since inhibitors for proteasome and mTOR have been used in cancer therapy, manipulating OGT activity may have therapeutic potential in diseases in which this signaling axis is impaired.

USP21 deubiquitinase elevates macropinocytosis to enable oncogenic KRAS bypass in pancreatic cancer

Activating mutations in KRAS (KRAS*) are present in nearly all pancreatic ductal adenocarcinoma (PDAC) cases and critical for tumor maintenance. By using an inducible KRAS* PDAC mouse model, we identified a deubiquitinase USP21-driven resistance mechanism to anti-KRAS* therapy. USP21 promotes KRAS*-independent tumor growth via its regulation of MARK3-induced macropinocytosis, which serves to maintain intracellular amino acid levels for anabolic growth. The USP21-mediated KRAS* bypass, coupled with the frequent amplification of USP21 in human PDAC tumors, encourages the assessment of USP21 as a novel drug target as well as a potential parameter that may affect responsiveness to emergent anti-KRAS* therapy.

SLC1A5 provides glutamine and asparagine necessary for bone development in mice

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here we identify Slc1a5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show Slc1a5 acts cell autonomously in osteoblasts to import glutamine and asparagine. Deleting Slc1a5 or reducing either glutamine or asparagine availability prevents protein synthesis and osteoblast differentiation. Mechanistically, glutamine and asparagine metabolism support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.

Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin β1

Although numerous recent studies have shown the importance of polymeric microfibrous extracellular matrices (ECMs) in maintaining cell behaviors and functions, the mechanistic nexus between ECMs and intracellular activities is largely unknown. Nevertheless, this knowledge will be critical in understanding and treating diseases with ECM remodeling. Therefore, we present our findings that ECM microstructures could regulate intracellular amino acid levels in liver cells mechanistically through integrin β1. Amino acids were studied because they are the fundamental blocks for protein synthesis and metabolism, two vital functions of liver cells. Two ECM conditions, flat and microfibrous, were prepared and studied. In addition to characterizing cell growth, albumin production, urea synthesis, and cytochrome p450 activity, we found that the microfibrous ECM generally upregulated the intracellular amino acid levels. Further explorations showed that cells on the flat substrate expressed more integrin β1 than cells on the microfibers. Moreover, after partially blocking integrin β1 in cells on the flat substrate, the intracellular amino acid levels were restored, strongly supporting integrin β1 as the linking mechanism. This is the first study to report that a non-biological polymer matrix could regulate intracellular amino acid patterns through integrin. The results will help with future therapy development for liver diseases with ECM changes (e.g., fibrosis).

A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence

ABSTRACT Bacterial cells utilize toxin-antitoxin systems to inhibit self-reproduction, while maintaining viability, when faced with environmental challenges. The activation of the toxin is often coupled to the induction of cellular response pathways, such as the stringent response, in response to multiple stress conditions. Under these conditions, the cell enters a quiescent state referred to as dormancy or persistence. How toxin activation triggers persistence and induces a systemic stress response in the alphaproteobacteria remains unclear. Here, we report that in Caulobacter, a hipA2-encoded bacterial toxin contributes to bacterial persistence by manipulating intracellular amino acid balance. HipA2 is a serine/threonine kinase that deactivates tryptophanyl-tRNA synthetase by phosphorylation, leading to stalled protein synthesis and the accumulation of free tryptophan. An increased level of tryptophan allosterically activates the adenylyltransferase activity of GlnE that, in turn, deactivates glutamine synthetase GlnA by adenylylation. The inactivation of GlnA promotes the deprivation of glutamine in the cell, which triggers a stringent response. By screening 69 stress conditions, we find that HipBA2 responds to multiple stress signals through the proteolysis of HipB2 antitoxin by the Lon protease and the release of active HipA2 kinase, revealing a molecular mechanism that allows disparate stress conditions to be sensed and funneled into a single response pathway. IMPORTANCE To overcome various environmental challenges, bacterial cells can enter a physiologically quiescent state, known as dormancy or persistence, which balances growth and viability. In this study, we report a new mechanism by which a toxin-antitoxin system responds to harsh environmental conditions or nutrient deprivation by orchestrating a dormant state while preserving viability. The hipA2-encoded kinase functions as a toxin in Caulobacter, inducing bacterial persistence by disturbing the intracellular tryptophan-glutamine balance. A nitrogen regulatory circuit can be regulated by the intracellular level of tryptophan, which mimics the allosteric role of glutamine in this feedback loop. The HipBA2 module senses different types of stress conditions by increasing the intracellular level of tryptophan, which in turn breaks the tryptophan-glutamine balance and induces glutamine deprivation. Our results reveal a molecular mechanism that allows disparate environmental challenges to converge on a common pathway that results in a dormant state.

Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure

ABSTRACTMechanical stresses, including high hydrostatic pressure, elicit diverse physiological effects on organisms. Gtr1, Gtr2, Ego1 (also known as Meh1) and Ego3 (also known as Slm4), central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa (∼250 kg/cm2) stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1 and PIB2 with GLN3. These results suggest that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC–TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrate the regulatory circuit that maintains intracellular amino acid homeostasis and suggest critical roles for the EGOC–TORC1 and Pib2–TORC1 complexes in the growth of yeast under high hydrostatic pressure.

Extracellular acidity and oxygen availability conjointly control eukaryotic cell growth via modulation of cytoplasmic translation

BackgroundOxygen availability and extracellular acidity both have a strong impact on growth and cultivation characteristics of eukaryotes, however they are often considered in isolation, whereby a single parameter is varied at a time to identify its impact, rendering the investigation of synergistic effects created by two or more factors non-achievable. This study identified the synergistic effect between environmental pH and oxygen levels on the physiological and cellular characteristics of the simplest eukaryote, Saccharomyces cerevisiae.Materials and methodsThe physiological, transcriptomic, and metabolic responses of yeast were investigated during batch growth in a 2 × 2 factorial design setting; environmental pH and oxygen availability were either controlled at their optimal settings, or allowed to follow their own course during cultivation.ResultsSynergistic effects had a significant impact on yeast physiology, which was provoked further by both the modulation of gene expression by transcription, and the modification of metabolite pools. Genes involved in cytoplasmic translation, the extracellular and intracellular amino acid and their precursor metabolite pools were significantly responsive to concurrent variations in these two factors.ConclusionThe synergistic effect of extracellular acidity and oxygenation on eukaryotic landscape of growth-associated events was significantly more pronounced than their individual effects.