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Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of liver steatosis, the most common liver disease, and substantially increases the mortality rate. However, limited therapies are currently available to prevent MASH development. Identifying potential pharmacological treatments for the condition has been hampered by its heterogeneous and complex nature. Here, we identified a hepatic nonneuronal cholinergic signaling pathway required for metabolic adaptation to caloric overload. We found that cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) is highly expressed in hepatocytes of mice and humans. Further, CHRNA2 is activated by a subpopulation of local acetylcholine-producing macrophages during MASH development. The activation of CHRNA2 coordinates defensive programs against a broad spectrum of MASH-related pathogenesis, including steatosis, inflammation, and fibrosis. Hepatocyte-specific loss of CHRNA2 signaling accelerates the disease onset in different MASH mouse models. Activation of this pathway via pharmacological inhibition of acetylcholine degradation protects against MASH development. Our study uncovers a hepatic nicotinic cholinergic receptor pathway that constitutes a cell-autonomous self-defense route against prolonged metabolic stress and holds therapeutic potential for combatting human MASH.
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Fígado Gorduroso , Hepatócitos , Fígado , Receptores Nicotínicos , Transdução de Sinais , Animais , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Macrófagos/metabolismo , Acetilcolina/metabolismo , Camundongos Knockout , Modelos Animais de DoençasRESUMO
While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.
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Escherichia coli , RNA de Transferência , Ribossomos , Saccharomyces cerevisiae , Uridina , RNA de Transferência/metabolismo , RNA de Transferência/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ribossomos/metabolismo , Uridina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Processamento Pós-Transcricional do RNA , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , tRNA Metiltransferases/metabolismo , tRNA Metiltransferases/genéticaRESUMO
Non-neuronal cholinergic signaling, mediated by acetylcholine, plays important roles in physiological processes including inflammation and immunity. Our group first discovered evidence of non-neuronal cholinergic circuitry in adipose tissue, whereby immune cells secrete acetylcholine to activate beige adipocytes during adaptive thermogenesis. Here, we reveal that macrophages are the cellular protagonists responsible for secreting acetylcholine to regulate thermogenic activation in subcutaneous fat, and we term these cells cholinergic adipose macrophages (ChAMs). An adaptive increase in ChAM abundance is evident following acute cold exposure, and macrophage-specific deletion of choline acetyltransferase (ChAT), the enzyme for acetylcholine biosynthesis, impairs the cold-induced thermogenic capacity of mice. Further, using pharmacological and genetic approaches, we show that ChAMs are regulated via adrenergic signaling, specifically through the ß2 adrenergic receptor. These findings demonstrate that macrophages are an essential adipose tissue source of acetylcholine for the regulation of adaptive thermogenesis, and may be useful for therapeutic targeting in metabolic diseases.
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Acetilcolina/metabolismo , Colina O-Acetiltransferase/genética , Macrófagos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Gordura Subcutânea/citologia , Animais , Células Cultivadas , Temperatura Baixa , Deleção de Genes , Técnicas de Inativação de Genes , Camundongos , Cultura Primária de Células , Gordura Subcutânea/metabolismo , TermogêneseRESUMO
Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant posttranscriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases, and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time, because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA-specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence Saccharomyces cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.
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Saccharomyces cerevisiae , Espectrometria de Massas em Tandem , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida , RNA de Transferência/química , Ribonucleases/metabolismoRESUMO
Change history: In this Article, an extraneous label appeared in Fig. 4b, and has been removed in the online version.
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The dopamine projection from ventral tegmental area (VTA) to nucleus accumbens (NAc) is critical for motivation to work for rewards and reward-driven learning. How dopamine supports both functions is unclear. Dopamine cell spiking can encode prediction errors, which are vital learning signals in computational theories of adaptive behaviour. By contrast, dopamine release ramps up as animals approach rewards, mirroring reward expectation. This mismatch might reflect differences in behavioural tasks, slower changes in dopamine cell spiking or spike-independent modulation of dopamine release. Here we compare spiking of identified VTA dopamine cells with NAc dopamine release in the same decision-making task. Cues that indicate an upcoming reward increased both spiking and release. However, NAc core dopamine release also covaried with dynamically evolving reward expectations, without corresponding changes in VTA dopamine cell spiking. Our results suggest a fundamental difference in how dopamine release is regulated to achieve distinct functions: broadcast burst signals promote learning, whereas local control drives motivation.
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Dopamina/metabolismo , Aprendizagem/fisiologia , Motivação/fisiologia , Recompensa , Animais , Sinais (Psicologia) , Tomada de Decisões/fisiologia , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Masculino , Núcleo Accumbens/citologia , Núcleo Accumbens/fisiologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Ratos , Ratos Long-Evans , Fatores de Tempo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologiaRESUMO
Biological interpretation of untargeted LC-MS-based metabolomics data depends on accurate compound identification, but current techniques fall short of identifying most features that can be detected. The human fecal metabolome is complex, variable, incompletely annotated, and serves as an ideal matrix to evaluate novel compound identification methods. We devised an experimental strategy for compound annotation using multidimensional chromatography and semiautomated feature alignment and applied these methods to study the fecal metabolome in the context of fecal microbiota transplantation (FMT) for recurrent C. difficile infection. Pooled fecal samples were fractionated using semipreparative liquid chromatography and analyzed by an orthogonal LC-MS/MS method. The resulting spectra were searched against commercial, public, and local spectral libraries, and annotations were vetted using retention time alignment and prediction. Multidimensional chromatography yielded more than a 2-fold improvement in identified compounds compared to conventional LC-MS/MS and successfully identified several rare and previously unreported compounds, including novel fatty-acid conjugated bile acid species. Using an automated software-based feature alignment strategy, most metabolites identified by the new approach could be matched to features that were detected but not identified in single-dimensional LC-MS/MS data. Overall, our approach represents a powerful strategy to enhance compound identification and biological insight from untargeted metabolomics data.
Assuntos
Transplante de Microbiota Fecal , Fezes , Metaboloma , Metabolômica , Espectrometria de Massas em Tandem , Humanos , Fezes/microbiologia , Fezes/química , Cromatografia Líquida/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Infecções por Clostridium/microbiologia , Infecções por Clostridium/metabolismo , Clostridioides difficile/metabolismo , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/análise , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Sensitive analytical techniques that are capable of detecting and quantifying disease-associated biomolecules are indispensable in our efforts to understand disease mechanisms and guide therapeutic intervention through early detection, accurate diagnosis, and effective monitoring of disease. Parkinson's Disease (PD), for example, is one of the most prominent neurodegenerative disorders in the world, but the diagnosis of PD has primarily been based on the observation of clinical symptoms. The protein α-synuclein (α-syn) has emerged as a promising biomarker candidate for PD, but a lack of analytical methods to measure complex disease-associated variants of α-syn has prevented its widespread use as a biomarker. Antibody-based methods such as immunoassays and mass spectrometry-based approaches have been used to measure a limited number of α-syn forms; however, these methods fail to differentiate variants of α-syn that display subtle differences in only the sequence and structure. In this work, we developed a cyclic ion mobility-mass spectrometry method that combines multiple stages of activation and timed ion selection to quantify α-syn variants using both mass- and structure-based measurements. This method can allow for the quantification of several α-syn variants present at physiological levels in biological fluid. Taken together, this approach can be used to galvanize future efforts aimed at understanding the underlying mechanisms of PD and serves as a starting point for the development of future protein-structure-based diagnostics and therapeutic interventions.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Humanos , alfa-Sinucleína/química , Doença de Parkinson/metabolismo , Biomarcadores/análise , Espectrometria de Massas , AnticorposRESUMO
The cycle time of a standard liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of ≥5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 µm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.
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Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.
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Microfluídica , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Microdiálise/métodos , Serotonina , Ácido Glutâmico , Neurotransmissores/análise , Soluções para DiáliseRESUMO
Obesity is one of the leading health concerns in the United States. Studies from human and rodent models suggest that inherent differences in the function of brain motivation centers, including the nucleus accumbens (NAc), contribute to overeating and thus obesity. For example, there are basal enhancements in the excitability of NAc GABAergic medium spiny neurons (MSN) and reductions in basal expression of AMPA-type glutamate receptors in obesity-prone vs obesity-resistant rats. However, very little is known about the regulation of extracellular glutamate and GABA within the NAc of these models. Here we gave obesity-prone and obesity-resistant rats stable isotope-labeled glucose (13 C6 -glucose) and used liquid chromatography mass spectrometry (LC-MS) analysis of NAc dialysate to examine the real-time incorporation of 13 C6 -glucose into glutamate, glutamine, and GABA. This novel approach allowed us to identify differences in glucose utilization for neurotransmitter production between these selectively bred lines. We found that voluntarily ingested or gastrically infused 13 C6 -glucose rapidly enters the NAc and is incorporated into 13 C2 -glutamine, 13 C2 -glutamate, and 13 C2 -GABA in both groups within minutes. However, the magnitude of increases in NAc 13 C2 -glutamine and 13 C2 -GABA were lower in obesity-prone than in obesity-resistant rats, while basal levels of glutamate were elevated. This suggested that there may be differences in the astrocytic regulation of these analytes. Thus, we next examined NAc glutamine synthetase, GAD67, and GLT-1 protein expression. Consistent with reduced 13 C2 -glutamine and 13 C2 -GABA, NAc glutamine synthetase and GLT-1 protein expression were reduced in obesity-prone vs obesity-resistant groups. Taken together, these data show that NAc glucose utilization differs dramatically between obesity-prone and obesity-resistant rats, favoring glutamate over GABA production in obesity-prone rats and that reductions in NAc astrocytic recycling of glutamate contribute to these differences. These data are discussed in light of established differences in NAc function between these models and the role of the NAc in feeding behavior.
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Ácido Glutâmico , Núcleo Accumbens , Humanos , Ratos , Animais , Ácido Glutâmico/metabolismo , Núcleo Accumbens/metabolismo , Glutamina/metabolismo , Glutamato-Amônia Ligase/metabolismo , Obesidade/metabolismo , Ácido gama-Aminobutírico/metabolismo , Glucose/metabolismoRESUMO
siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.
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Digestão , Espectrometria de Massas em Tandem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espectrometria de Massas em Tandem/métodos , Análise de Sequência de RNARESUMO
Synthetic biology relies on engineering cells to have desirable properties, such as the production of select chemicals. A bottleneck in engineering methods is often the need to screen and sort variant libraries for potential activity. Droplet microfluidics is a method for high-throughput sample preparation and analysis which has the potential to improve the engineering of cells, but a limitation has been the reliance on fluorescent analysis. Here, we show the ability to select cell variants grown in 20 nL droplets at 0.5 samples/s using mass-activated droplet sorting (MADS), a method for selecting droplets based on the signal intensity measured by electrospray ionization mass spectrometry (ESI-MS). Escherichia coli variants producing lysine were used to evaluate the applicability of MADS for synthetic biology. E. coli were shown to be effectively grown in droplets, and the lysine produced by these cells was detectable using ESI-MS. Sorting of lysine-producing cells based on the MS signal was shown, yielding 96-98% purity for high-producing variants in the selected pool. Using this technique, cells were recovered after screening, enabling downstream validation via phenotyping. The presented method is translatable to whole-cell engineering for biocatalyst production.
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Escherichia coli , Lisina , Engenharia Celular , Movimento Celular , CorantesRESUMO
Droplet microfluidics enables high-throughput experimentation and screening by encapsulating chemical and biochemical samples in aqueous droplets segmented by an immiscible fluid. In such experiments, it is critical that each droplet remains chemically distinct. A common approach is to use fluorinated oils with surfactants to stabilize droplets. However, some small molecules have been observed to transport between droplets under these conditions. Attempts to study and mitigate this effect have relied on evaluating crosstalk using fluorescent molecules, which inherently limits the analyte scope and conclusions drawn about the mechanism of the effect. In this work, transport of low molecular weight compounds between droplets was investigated using electrospray ionization mass spectrometry (ESI-MS) for measurement. The use of ESI-MS significantly expands the scope of analytes that can be tested. We tested 36 structurally diverse analytes that were found to exhibit crosstalk ranging from negligible to complete transfer using HFE 7500 as the carrier fluid and 008-fluorosurfactant as a surfactant. Using this data set, we developed a predictive tool showing that high log P and log D values correlate with high crosstalk, and high polar surface area and log S correlate with low crosstalk. We then investigated several carrier fluids, surfactants, and flow conditions. It was discovered that transport is strongly dependent on all of these factors and that experimental design and surfactant tailoring can reduce carryover. We present evidence for mixed crosstalk mechanisms including both micellar and oil partitioning transfer. By understanding the driving mechanisms, surfactant and oil compositions can be designed to better reduce chemical transport for screening workflows.
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High-throughput screening (HTS) workflows are revolutionizing many fields, including drug discovery, reaction discovery and optimization, diagnostics, sensing, and enzyme engineering. Liquid chromatography (LC) is commonly deployed during HTS to reduce matrix effects, distinguish isomers, and preconcentrate prior to detection, but LC separation time often limits throughput. Although subsecond LC separations have been demonstrated, they are rarely utilized during HTS due to limitations associated with the speed of common autosamplers. In this work, these limits are overcome by utilizing droplet microfluidics for sample introduction. In the method, a train of samples segmented by air are continuously pumped into the inlet of an LC injection valve that is actuated once each sample fills the sample loop. Coupled with 2.1 mm diameter × 5 mm long columns packed with 2.7 µm superficially porous C18 particles operated at 5 mL/min, the injector enabled separation of 3 components at 1 s/sample and analysis of a 96-well plate in 1.6 min with <2% peak area relative standard deviation. Analyte-dependent carryover was minimized by including wash droplets composed of organic solvent in between sample droplets. High-throughput LC coupled with mass spectrometric detection using the segmented flow injector was applied to a screen of inhibitors of a cytochrome P450-catalyzed hydroxylation reaction. Measurements of the reaction substrate and product concentrations made using fast LC with the segmented flow injector correlated well with measurements made using a more conventional, 3 min LC method. These results demonstrate the potential for droplet microfluidics to be used for sample introduction during high-throughput LC analysis.
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Microfluídica , Cromatografia Líquida/métodos , Espectrometria de Massas/métodosRESUMO
Tissue functions such as hormone secretion involve the interplay of multiple chemical signals and metabolic processes over time. Measuring the different components involved is useful in unraveling the interactions, but often requires use of multiple analytical techniques. The challenge of measuring the necessary components with temporal resolution is greater when tissue samples are limited. Here, an accessible microfluidic platform compatible with multiple measurement techniques to monitor cell secretions has been developed. The platform is applied to islets of Langerhans, micro-organs involved in glucose homeostasis and diabetes. The device houses 1 to 8 islets and the perfusion fluid can be controlled to change conditions, e.g., glucose concentration, in seconds. Samples are collected in fractions and split for offline analysis. The device is paired with a scaled-down immunoassay, AlphaLISA, for hormone quantification and liquid chromatography-mass spectrometry for small molecule quantification to study secretion dynamics. The combined system allows the first simultaneous measurement of insulin, glucagon, biogenic amines, and amino acids from islet secretions. The combined measurements revealed correlation in secretion events and differences in timing of release between hormones and biogenic amines and amino acids. These efforts decreased the number of islets required compared to standard approaches, thus decreasing necessary animal use, reagent use, and cost, while increasing information content achievable from one sample. The microfluidic device is a suitable platform for in-depth characterization of secretion from small tissue samples.
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Ilhotas Pancreáticas , Técnicas Analíticas Microfluídicas , Animais , Ilhotas Pancreáticas/metabolismo , Insulina/análise , Aminoácidos/análise , Glucose/análiseRESUMO
Within the realm of drug discovery, high-throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound libraries for biological and pharmacokinetic evaluation. Herein we report the development of a segmented flow mass spectrometry-based platform to enable the rapid exploration of photoredox reactions for early-stage drug discovery. Specifically, microwell plate-based photochemical reaction screens were reformatted to segmented flow format to enable delivery to nanoelectrospray ionization-mass spectrometry analysis. This approach was demonstrated for the late-stage modification of complex drug scaffolds, as well as the subsequent structure-activity relationship evaluation of synthesized analogs. This technology is anticipated to expand the robust capabilities of photoredox catalysis in drug discovery by enabling high-throughput library diversification.
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Descoberta de Drogas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas , Catálise , Espectrometria de Massas por Ionização por Electrospray/métodos , Ensaios de Triagem em Larga EscalaRESUMO
Dysregulated glucagon secretion deteriorates glycemic control in type 1 and type 2 diabetes. Although insulin is known to regulate glucagon secretion via its cognate receptor (insulin receptor, INSR) in pancreatic alpha cells, the role of downstream proteins and signaling pathways underlying insulin's activities are not fully defined. Using in vivo (knockout) and in vitro (knockdown) studies targeting insulin receptor substrate (IRS) proteins, we compared the relative roles of IRS1 and IRS2 in regulating alpha cell function. Alpha cell-specific IRS1-knockout mice exhibited glucose intolerance and inappropriate glucagon suppression during glucose tolerance tests. In contrast, alpha cell-specific IRS2-knockout animals manifested normal glucose tolerance and suppression of glucagon secretion after glucose administration. Alpha cell lines with stable IRS1 knockdown could not repress glucagon mRNA expression and exhibited a reduction in phosphorylation of AKT Ser/Thr kinase (AKT, at Ser-473 and Thr-308). AlphaIRS1KD cells also displayed suppressed global protein translation, including reduced glucagon expression, impaired cytoplasmic Ca2+ response, and mitochondrial dysfunction. This was supported by the identification of novel IRS1-specific downstream target genes, Trpc3 and Cartpt, that are associated with glucagon regulation in alpha cells. These results provide evidence that IRS1, rather than IRS2, is a dominant regulator of pancreatic alpha cell function.
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Células Secretoras de Glucagon/patologia , Glucagon/metabolismo , Intolerância à Glucose/patologia , Proteínas Substratos do Receptor de Insulina/fisiologia , Resistência à Insulina , Animais , Feminino , Células Secretoras de Glucagon/metabolismo , Intolerância à Glucose/etiologia , Intolerância à Glucose/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Transdução de SinaisRESUMO
Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.
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Antivirais/metabolismo , Colesterol/biossíntese , Proteínas/metabolismo , Vias Biossintéticas , Citidina Trifosfato/metabolismo , Células HEK293 , Humanos , Transferases Intramoleculares/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Ligação Proteica , Proteínas/química , Esqualeno Mono-Oxigenase/metabolismoRESUMO
Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding; however, larger scale screens have not been implemented due to limitations associated with throughput and sample introduction. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach using 40 nL droplet sample volumes and 2 s dwell times at each collision voltage. This workflow, which increased throughput by â¼16-fold over conventional nanospray CIU methods, was applied to a 96-compound screen against Sirtuin-5, a protein target of clinical interest. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis.