Amino Acid Sensor Research Articles

Excited-state intramolecular proton transfer (ESIPT) salicylaldehyde Schiff bases: ratiometric sensing of ammonia and biologically relevant ions in solution and solid state

ABSTRACT The intricate molecular architecture of ESIPT salicylaldehyde Schiff bases facilitates dynamic processes, inducing tunable photoluminescent properties. Notably, their halochromic nature, exhibiting colour changes in response to external stimuli, adds a vibrant dimension to their molecular repertoire. This sensitivity extends to environmental factors, making them valuable indicators for alterations in surroundings. The compound (E)-N’-(3,5-dibromo-2-hydroxybenzylidene)-4-methylbenzohydrazide (PTBH) demonstrates exceptional sensitivity to ammonia, enabling real-time detection (LOD = 0.14 nM) in both solution (ratiometric) and the solid state. Moreover, their metal chelation capability allows simultaneous sensing of Mg2+ and Fe2+ ions, addressing environmental hazards. Exploiting molecular recognition, the fluorescent probe serves as sensors for amino acids, opening new avenues in biomedical diagnostics. The study introduces a novel solid-state emissive Schiff base, highlighting its stimuli-responsive photoluminescent properties and diverse applications, emphasising its potential in intelligent fluorescent materials for analytical and sensing technologies.

Animal amino acid sensor.

Cell growth and metabolism necessitate the involvement of amino acids, which are sensed and integrated by the mammalian target of rapamycin complex 1 (mTORC1). However, the molecular mechanisms underlying amino acid sensing remain poorly understood. Research indicates that amino acids are detected by specific sensors, with the signals being relayed to mTORC1 indirectly. This paper reviews the structures and biological functions of the amino acid sensors identified thus far. Additionally, it evaluates the potential role these sensors play in the developmental changes of the livestock production.

Label-Free Mode Based on Ferrocene/PEDOT:PSS-PPy for Molecularly Imprinted Electrochemically Ultrasensitive Detection of Amino Acids.

Generally, molecularly imprinted (MIP) electrochemical sensors for amino acids operate in a "label-like" mode. That is, after an amino acid is specifically recognized by an imprinted cavity at the sensing interface, the amino acid itself provides the sensing signal for quantitative detection. However, poorly electroactive amino acids impede electron transfer at the sensing interface and require high potentials to drive the reaction; thus, more interfering reactions tend to be triggered in practical applications, causing enhanced background noise in the detection. To address these issues, a "label-free" mode of the MIP sensor based on the ferrocene (Fc)/PEDOT:PSS-polypyrrole (PPy) composite was designed for the first time. The Fc/PEDOT:PSS-PPy is drop coated on the electrode surface as a substrate, and MIP polymers with specific recognition ability are immobilized on the substrate via electrostatic adsorption. As a proof of concept, l-tyrosine (l-Tyr) was selected as a model analyte and the "label-free" mode MIP/Fc/PEDOT:PSS-PPy sensor was constructed. The limit of detection (LOD) and linearity range of the MIP/Fc/PEDOT:PSS-PPy sensor were 2.31 × 10-11 M and from 100 pM to 5 mM, respectively. Compared with the label-like mode, the LOD was three orders of magnitude lower, the linear range was increased by three orders of magnitude, and the sensitivity was improved by more than four times. This work provides a universal and effective concept for MIP electrochemical sensing of amino acids.

Role of L-glutamine in the regulation of rat Sertoli cell proliferation by FSH.

FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that result from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing glutaminase isoform 2 and reducing glutamine synthetaseexpression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signaling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.

The amino acid sensor methionyl-tRNA synthetase is required for methionine-induced milk protein synthesis in a domestic pigeon model.

This study was conducted to investigate whether methionyl-tRNA synthetase (MetRS) is a mediator of methionine (Met)-induced crop milk protein synthesis via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signalling pathway in breeding pigeons. In Experiment 1, a total of 216 pairs of breeding pigeons were divided into three groups (control, Met-deficient, and Met-rescue groups). In Experiments 2 and 3, forty pairs of breeding pigeons from each experiment were allocated into four groups. The second experiment included a control group and three MetRS inhibitor (REP8839) groups. The third experiment included a Met-deficient group, Met-sufficient group, REP8839 + Met-deficient group and REP8839 + Met-sufficient group. Experiment 1 showed that Met supplementation increased crop development, crop milk protein synthesis, the protein expression of MetRS and JAK2/STAT5 signalling pathway, and improved squab growth. Experiment 2 showed that crop development, crop milk protein synthesis and the protein expression of MetRS and the JAK2/STAT5 signalling pathway were decreased, and squab growth was inhibited by the injection of 1·0 mg/kg body weight REP8839, which was the selected dose for the third experiment. Experiment 3 showed that Met supplementation increased crop development, crop milk protein synthesis and the expression of MetRS and JAK2/STAT5 signalling pathway and rescued squab growth after the injection of REP8839. Moreover, the co-immunoprecipitation results showed that there was an interaction between MetRS and JAK2. Taken together, these findings indicate that MetRS mediates Met-induced crop milk protein synthesis via the JAK2/STAT5 signalling pathway, resulting in improved squab growth in breeding pigeons.

Framework for exploring the sensory repertoire of the human gut microbiota.

Bacteria sense changes in their environment and transduce signals to adjust their cellular functions accordingly. For this purpose, bacteria employ various sensors feeding into multiple signal transduction pathways. Signal recognition by bacterial sensors is studied mainly in a few model organisms, but advances in genome sequencing and analysis offer new ways of exploring the sensory repertoire of many understudied organisms. The human gut is a natural target of this line of study: it is a nutrient-rich and dynamic environment and is home to thousands of bacterial species whose activities impact human health. Many gut commensals are also poorly studied compared to model organisms and are mainly known through their genome sequences. To begin exploring the signals human gut commensals sense and respond to, we have designed a framework that enables the identification of sensory domains, prediction of signals that they recognize, and experimental verification of these predictions. We validate this framework's functionality by systematically identifying amino acid sensors in selected bacterial genomes and metagenomes, characterizing their amino acid binding properties, and demonstrating their signal transduction potential.IMPORTANCESignal transduction is a central process governing how bacteria sense and respond to their environment. The human gut is a complex environment with many living organisms and fluctuating streams of nutrients. One gut inhabitant, Escherichia coli, is a model organism for studying signal transduction. However, E. coli is not representative of most gut microbes, and signaling pathways in the thousands of other organisms comprising the human gut microbiota remain poorly understood. This work provides a foundation for how to explore signals recognized by these organisms.

Dietary L-Glu sensing by enteroendocrine cells adjusts food intake via modulating gut PYY/NPF secretion

Amino acid availability is monitored by animals to adapt to their nutritional environment. Beyond gustatory receptors and systemic amino acid sensors, enteroendocrine cells (EECs) are believed to directly percept dietary amino acids and secrete regulatory peptides. However, the cellular machinery underlying amino acid-sensing by EECs and how EEC-derived hormones modulate feeding behavior remain elusive. Here, by developing tools to specifically manipulate EECs, we find that Drosophila neuropeptide F (NPF) from mated female EECs inhibits feeding, similar to human PYY. Mechanistically, dietary L-Glutamate acts through the metabotropic glutamate receptor mGluR to decelerate calcium oscillations in EECs, thereby causing reduced NPF secretion via dense-core vesicles. Furthermore, two dopaminergic enteric neurons expressing NPFR perceive EEC-derived NPF and relay an anorexigenic signal to the brain. Thus, our findings provide mechanistic insights into how EECs assess food quality and identify a conserved mode of action that explains how gut NPF/PYY modulates food intake.

New insights into GATOR2-dependent interactions and its conformational changes in amino acid sensing.

Eukaryotic cells coordinate growth under different environmental conditions via mechanistic target of rapamycin complex 1 (mTORC1). In the amino-acid-sensing signalling pathway, the GATOR2 complex, containing five evolutionarily conserved subunits (WDR59, Mios, WDR24, Seh1L and Sec13), is required to regulate mTORC1 activity by interacting with upstream CASTOR1 (arginine sensor) and Sestrin2 (leucine sensor and downstream GATOR1 complex). GATOR2 complex utilizes β-propellers to engage with CASTOR1, Sestrin2 and GATOR1, removal of these β-propellers results in substantial loss of mTORC1 capacity. However, structural information regarding the interface between amino acid sensors and GATOR2 remains elusive. With the recent progress of the AI-based tool AlphaFold2 (AF2) for protein structure prediction, structural models were predicted for Sentrin2-WDR24-Seh1L and CASTOR1-Mios β-propeller. Furthermore, the effectiveness of relevant residues within the interface was examined using biochemical experiments combined with molecular dynamics (MD) simulations. Notably, fluorescence resonance energy transfer (FRET) analysis detected the structural transition of GATOR2 in response to amino acid signals, and the deletion of Mios β-propeller severely impeded that change at distinct arginine levels. These findings provide structural perspectives on the association between GATOR2 and amino acid sensors and can facilitate future research on structure determination and function.

IR76b-expressing neurons in Drosophila melanogaster are necessary for associative reward learning of an amino acid mixture.

Learning where to find nutrients while at the same time avoiding toxic food is essential for survival of any animal. Using Drosophila melanogaster larvae as a study case, we investigate the role of gustatory sensory neurons expressing IR76b for associative learning of amino acids, the building blocks of proteins. We found surprising complexity in the neuronal underpinnings of sensing amino acids, and a functional division of sensory neurons. We found that the IR76b receptor is dispensable for amino acid learning, whereas the neurons expressing IR76b are specifically required for the rewarding but not the punishing effect of amino acids. This unexpected dissociation in neuronal processing of amino acids for different behavioural functions provides a study case for functional divisions of labour in gustatory systems.

Beyond antibiotic resistance: The whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. The conserved whiB7 stress response reduces the effectiveness of antibiotic therapy by activating several intrinsic antibiotic resistance mechanisms. Despite our comprehensive biochemical understanding of WhiB7, the complex set of signals that induce whiB7 expression remain less clear. We employed a reporter-based, genome-wide CRISPRi epistasis screen to identify a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 expression. We show that whiB7 expression is determined by the amino acid composition of the 5' regulatory uORF, thereby allowing whiB7 to sense amino acid starvation. Although deprivation of many amino acids can induce whiB7, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. These findings describe a metabolic function for whiB7 and help explain its evolutionary conservation across mycobacterial species occupying diverse ecological niches.

The role of Phafin proteins in cell signaling pathways and diseases.

Membrane-associated proteins are important membrane readers that mediate and facilitate the signaling and trafficking pathways in eukaryotic membrane-bound compartments. The protein members in the Phafin family are membrane readers containing two phosphoinositide recognition domains: the Pleckstrin Homology domain and the FYVE (Fab1, YOTB, Vac1, and early endosome antigen 1) domain. Phafin proteins, categorized into two subfamilies, Phafin1 and Phafin2, associate with cellular membranes through interactions involving membrane-embedded phosphoinositides and phosphoinositide-binding domains. These membrane-associated Phafin proteins play pivotal roles by recruiting binding partners and forming complexes, which contribute significantly to apoptotic, autophagic, and macropinocytotic pathways. Elevated expression levels of Phafin1 and Phafin2 are observed in various cancers. A recent study highlights a significant increase in Phafin1 protein levels in the lungs of idiopathic pulmonary fibrosis patients compared to normal subjects, suggesting a crucial role for Phafin1 in the pathogenesis of pulmonary fibrosis. Additionally, phosphatidylinositol-3-phosphate-binding 2 (Pib2), a close relative of the Phafin1 protein, functions as an amino acid sensor activating the TOCR1 pathway in yeasts. This review focuses on delineating the involvement of Phafin proteins in cellular signaling and their implications in diseases and briefly discusses the latest research findings concerning Pib2.

Branched-chain amino acid catabolic defect promotes α-cell proliferation via activating mTOR signaling

Elevated circulating level of branched-chain amino acids (BCAAs) is closely related to the development of type 2 diabetes. However, the role of BCAA catabolism in various tissues in maintaining glucose homeostasis remains largely unknown. Pancreatic α-cells have been regarded as amino acid sensors in recent years. Therefore, we generated α-cell specific branched-chain alpha-ketoacid dehydrogenase E1α subunit (BCKDHA) knockout (BCKDHA-αKO) mice to decipher the effects of BCAA catabolism in α-cells on whole-body energy metabolism. BCKDHA-αKO mice showed normal body weight, body fat, and energy expenditure. Plasma glucagon level and glucose metabolism also remained unchanged in BCKDHA-αKO mice. Whereas, the deletion of BCKDHA led to increased α-cell number due to elevated cell proliferation in neonatal mice. In vitro, only leucine among BCAAs promoted aTC1-6 cell proliferation, which was blocked by the agonist of BCAA catabolism BT2 and the inhibitor of mTOR Rapamycin. Like Rapamycin, BT2 attenuated leucine-stimulated phosphorylation of S6 in αTC1-6 cells. Elevated phosphorylation level of S6 protein in pancreatic α-cells was also observed in BCKDHA-αKO mice. These results suggest that local accumulated leucine due to defective BCAA catabolism promotes α-cell proliferation through mTOR signaling, which is insufficient to affect glucagon secretion and whole-body glucose homeostasis.

Therapeutic potential of X12Y12 (X=Al, B, and Y=N, P) nanocages as effective sensors for Ala, Val, Arg, and Asp amino acids

In view of the intensifying research to study the comparative proficiency of innocuous and bioavailable nanomaterials concordance with human systems for drug conveyance, this study was aimed to perusal the comparative efficiency of X12Y12 (X=B, Al, and Y= N, P) nanocages for the adsorption of four amino acids including alanine (Ala), valine (Val), arginine (Arg), and aspartic acid (Asp). Four different nanocages interacted with amino acids at the nitrogen (-NH2), oxygen (-CO), and oxygen (-OH) sites, ensuing in forty eight adsorbate-adsorbent complexes whose sensitivity and reactivity were examined via density functional theory (DFT) calculations at ωB97XD/6-311G+(d,p) level of theory with distinct prominence on the geometry, electronic, and topological scrutiny. The adsorption energy (Eads) for adsorbate-adsorbent complexes found to be highly exothermic and in the range of -15.02 to -58.35 kcal/mol. The consequences of thermodynamic calculations show that adsorption of considered amino acids over the surface of nanocages are thermodynamically favorable. The estimated value of energy gap (Egap) associated with X12Y12 nanocages found to be decreased after forming complex with the amino acid molecules. Therefore, these nanocages act as appropriate sensing material for the development of sensors for the determination of amino acids. To attain the insight into the interactions of amino acids with the nanocages; density of states (DOS), UV–visible spectrum and dipole moment are studied. The QTAIM examination is also accomplished to recognize the interaction in adsorbate-adsorbent complexes. Additionally, ETS-NOCV examination apprise that charge flow from amino acids to the nanocages in all the complexes. Consequently, the study demonstrated that nanocages act as perfect candidates for the adsorption of amino acids.

Amino acid and acetone fluorescence sensors based on a stable light-emitting Cd(II) metal-organic framework with derivatives of isonicotinic acid

As one of the most promising category of functional materials, luminescent metal–organic frameworks (LMOFs) have been widely investigated as fluorescence chemical sensors which can offer a reliable diagnostic tools in biomedical research. Herein, a luminescent Cd-MOF [Cd2(L)2(H2O)]n (Cd-MOF) (H2L = 3-(3-carboxylphenyl) isonicotinic acid), was synthesized by and characterized in detail. Single-crystal structure determination reveals that Cd-MOF crystalizes in the monoclinic crystal system with C2/c space group. Experimental results show that Cd-MOF is a highly selective and sensitive luminescent sensor for rapidly detecting acetone, l-Tryptophane (L-Trp) and l- Aspartic acid (L-Asp) in water. It can significantly test acetone in common solvents, selectively identify l-Trp-and l-Asp-in 12 amino acid aqueous solutions, and enhance the fluorescence signal of Tryptophan and quench l-Asp-with the detection limit of 17.6 μM and 68.8 μM respectively. However, upon the interaction with electron rich amino acid (L-Trp), the Cd-MOF shows the selective “Turn-On” behavior, which may also be supported by the DFT studies.

Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gβγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

Bifunctional metal-organic frameworks as selective turn-on fluorescence sensors for tryptophan and heterogeneous catalysts for Knoevenagel condensation reaction

Two new doubly interpenetrated metal-organic frameworks (MOFs) with formulas [{Zn(L1)(5-nipa)}·DMA]α (1a) and [{Cd(L2)(5-nipa)(H2O)}]α (1b) have been discussed herein, where [L1 = N,N'-(oxybis(4,1-phenylene))diisonicotinamide; L2 = N,N'-(methylenebis(4,1-phenylene)) diisonicotinamide; 5-H2nipa = 5-nitroisophthalic acid; DMA = dimethylacetamide]. A single crystal X-ray analysis reveals that 1a exhibits a 2D-interpenetrated interwoven sheet and finally adapts a 3D framework structure with the help of π···π stacking interactions. Similarly, 1b displays a two-fold interpenetrated 2D sheet-like architecture and is finally adjusted to a 3D structure. Both 1a and 1b feature channels with a cross-section of 25.06 × 25.06 Å2 and 24.01 × 24.01 Å2, respectively, running along the b-axis. Topological analysis of 1a and 1b by ToposPro suggests a (4)-connected uninodal sql topology with point symbol {44.62}. Further, 1a and 1b were exploited as molecular sensors for several amino acids exposing different shapes and sizes and exhibiting selective turn-on fluorescence sensing towards biologically important D/L-tryptophan with the notable limit of detection values of 0.084/0.110 μM and 0.143/0.141 μM, respectively, in aqueous medium. In addition, 1a and 1b were also exploited as heterogeneous catalysts towards several assorted aldehydes having different electronic environments, including sterically demanding aldehydes, under mild reaction conditions and obtained up to 97% of the respective Knoevenagel condensation product. Importantly, sterically hindered aldehydes also worked well with the MOF catalysts 1a and 1b, but showed comparatively low conversion. It is important to mention that in both cases, these MOF materials are recyclable for up to five consecutive runs without displaying any substantial loss in their sensing or catalytic activities. Moreover, the present investigation cultivates a new vision to design novel luminescent MOFs that can be used as multifunctional materials for the turn-on sensing of small organic molecules as well as instigated as heterogeneous catalysts in diverse organic transformation reactions.

Redundant electrostatic interactions between GATOR1 and the Rag GTPase heterodimer drive efficient amino acid sensing in human cells

Cells need to coordinate nutrient availability with their growth and proliferation. In eukaryotic cells, this coordination is mediated by the mechanistic target of the rapamycin complex 1 (mTORC1) pathway. mTORC1 activation is regulated by two GTPase units, the Rag GTPase heterodimer and the Rheb GTPase. The RagA-RagC heterodimer controls the subcellular localization of mTORC1, and its nucleotide loading states are strictly controlled by upstream regulators including amino acid sensors. A critical negative regulator of the Rag GTPase heterodimer is GATOR1. In the absence of amino acids, GATOR1 stimulates GTP hydrolysis by the RagA subunit to turn off mTORC1 signaling. Despite the enzymatic specificity of GATOR1 to RagA, a recent cryo-EM structural model of the human GATOR1-Rag-Ragulator complex reveals an unexpected interface between Depdc5, a subunit of GATOR1, and RagC. Currently, there is no functional characterization of this interface, nor do we know its biological relevance. Here, combining structure-function analysis, enzymatic kinetic measurements, and cell-based signaling assays, we identified a critical electrostatic interaction between Depdc5 and RagC. This interaction is mediated by the positively charged Arg-1407 residue on Depdc5 and a patch of negatively charged residues on the lateral side of RagC. Abrogating this interaction impairs the GAP activity of GATOR1 and cellular response to amino acid withdrawal. Our results reveal how GATOR1 coordinates the nucleotide loading states of the Rag GTPase heterodimer, and thus precisely controls cellular behavior in the absence of amino acids.

Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P<0.05) and muscle (P<0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.