Arginine Sensor Research Articles

CASTOR1 phosphorylation predicts poor survival in male patients with KRAS-mutated lung adenocarcinoma

BackgroundLung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. We have previously shown a tumor suppressive role of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), which is targeted for degradation upon phosphorylation at S14 (pCASTOR1) in multiple types of cancer. This study focuses on the predictive value of pCASTOR1 in lung adenocarcinoma (LUAD) patients with KRAS mutations.ResultsEmploying a newly developed pCASTOR1 specific antibody, we found that tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells (P < 0.05). Higher pCASTOR1 scores predicted poorer overall survival (OS) (HR = 3.3, P = 0.0008) and relapse-free survival (RFS) (HR = 3.0, P = 0.0035) in male patients with KRAS mutations. pCASTOR1 remained an independent predictor for OS (HR = 4.1, P = 0.0047) and RFS (HR = 3.5, P = 0.0342) after controlling for other factors. Notably, in early-stage LUAD, elevated pCASTOR1 scores were associated with significantly worse OS (HR = 3.3, P = 0.0176) and RFS (HR = 3.1, P = 0.0277) in male patients with KRAS mutations, akin to late-stage patients.ConclusionElevated pCASTOR1 scores serve as biomarkers predicting poorer OS and RFS in male LUAD patients with KRAS mutations, offering potential clinical utility in optimizing treatment strategies for this subgroup.

PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets

Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P &amp;lt; 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P &amp;lt; 0.05). The phosphorylation of Akt was increased (P &amp;lt; 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P &amp;lt; 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P &amp;lt; 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P &amp;lt; 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P &amp;lt; 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P &amp;lt; 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.

Dietary protein re-alimentation following restriction improves protein deposition via changing amino acid metabolism and transcriptional profiling of muscle tissue in growing beef bulls

This study aimed to develop a compensatory growth model using growing beef cattle by changing dietary protein and to investigate the underlying mechanisms of compensatory protein deposition in muscle tissue. Twelve Charolais bulls were randomly assigned to one of two groups with two periods: 1) a control group (CON) fed a 13% crude protein (CP) diet for 6 weeks; 2) a treatment group (REC) fed a 7% CP diet for 4 weeks (restriction period) and fed a 13% CP diet in the following 2 weeks (re-alimentation period). Growth performance, digestibility, nitrogen balance, targeted metabolomics of amino acids (AA) in plasma, and transcriptional profiling in muscle tissue were analyzed. Protein restriction decreased average daily gain (ADG; P < 0.05), while protein re-alimentation increased ADG relative to the CON (P < 0.05). Compared to the CON, REC reduced retained N (P < 0.05), and protein re-alimentation increased retained N and N utilization efficiency (P < 0.05), due to reduced urinary urea, hippuric acid, and creatinine excretions (P < 0.05). Ruminal NH3-N in the REC was lower than that in the CON in the protein re-alimentation period (P < 0.05). However, there was no difference in microbial protein and plasma urea nitrogen concentrations. Dietary protein restriction decreased plasma valine and aspartic acid concentrations relative to the CON (P < 0.05), and increased proline and 3-methyl-L-histidine concentrations (P < 0.05). After dietary protein re-alimentation, REC increased plasma citrulline concentrations (P < 0.05). The transcriptional profiling revealed that REC upregulated the AA transporter SLC3A1, and protein re-alimentation downregulated SLC7A8 in the muscle cell membrane. Within the muscle cell, upregulated cytosolic arginine sensor for mTORC1 subunit 2 (CASTOR2) inhibited protein synthesis by inhibiting the mammalian target of rapamycin complex 1 phosphorylation in the protein restriction period, while DNA-damage-inducible transcript 4 (DDIT4) activated the mTOR signaling pathway and promoted protein synthesis in the protein re-alimentation period. In summary, the targeted metabolomics and transcriptomics analyses demonstrated that protein re-alimentation following restriction can promote protein synthesis and reduce muscle breakdown by regulating AA metabolism and functional transcripts related to AA transporters and the mTOR signaling pathway.

CASTOR1 phosphorylation as a predictor of survival in male patients with KRAS-mutated lung adenocarcinoma.

e20015 Background: Lung cancer, a leading global cause of cancer-related mortality, necessitates enhanced prognostic markers for improved treatment outcomes. This study focuses on the predictive value of phosphorylated cytosolic arginine sensor for mTORC1 subunit 1 at S14 (pCASTOR1) in lung adenocarcinoma (LUAD) patients, specifically in males with KRAS mutations. Methods: This retrospective study, approved by University of Pittsburgh IRB, utilized formaline-fixed paraffin embedded primary tissues from two independent cohorts of patients who underwent thoracic surgical procedures at the University of Pittsburgh Medical Center (UPMC) from 2002 to 2011. Comprehensive clinical, demographical and pathological information was obtained from UPMC Cancer Registry, with cases lacking incomplete information excluded from analysis. Study endpoints included overall survival (OS) and relapse-free survival (RFS). An antibody targeting pCASTOR1 was employed to analyze LUAD tumor and adjacent non-tumor lung tissues from both cohorts. A pathologist, blinded to patient information and survival outcomes, conducted scoring in 5% increments. Scores were categorized as low or high pCASTOR1 based on second or third quartile. Correlations between pCASTOR1 scores and OS/RFS were examined, stratifying by clinical, pathological, demographic, or genotype data. Univariate OS and RFS analyses were conducted using the Kaplan-Meier method and log-rank test, while multivariate survival significance was assessed via Cox regression analysis. Additionally, unpaired T-tests were utilized to compare pCASTOR1 levels between non-tumor and tumor cells. A significance threshold of p &lt; 0.05 was applied for all analyses. Results: Tumor cells exhibited significantly elevated pCASTOR1 scores compared to non-tumor cells in both cohorts (P &lt; 0.05). High pCASTOR1 scores predicted poor OS (HR = 3.3, P = 0.0008) and RFS (HR = 3.0, P = 0.0035) in male patients with KRAS mutations in Cohort 1. pCASTOR1 remained an independent predictor for OS (HR = 4.1, P = 0.0047) and RFS (HR = 3.5, P = 0.0342) after controlling for other factors. Cohort 2, comprised solely of smokers with incomplete genotype information, validated these findings, with high pCASTOR1 scores correlating with worse OS (HR = 4.8, P = 0.0363). Notably, in early-stage LUAD, elevated pCASTOR1 scores were associated with significantly worse OS (HR = 3.3, P = 0.0176) and RFS (HR = 3.1, P = 0.0277) in male patients with KRAS mutations in Cohort 1, and worse OS (HR = 5.0, P = 0.0069) and RFS (HR = 5.0, P = 0.0069) in Cohort 2, akin to late-stage patients. Conclusions: Elevated pCASTOR1 score serves as a robust biomarker predicting poor OS and RFS in male LUAD patients with KRAS mutations, offering potential clinical utility in optimizing treatment strategies for this subgroup.

Unraveling CASTOR1's tumor suppressive role: Genetic ablation of CASTOR1 and KRAS-driven mouse model of non-small cell lung cancer.

8528 Background: Cytosolic Arginine Sensor for mTORC1 Subunit 1 (CASTOR1) has emerged as a potent suppressor of mTORC1 activity, playing a pivotal role in various cancer contexts. Previously implicated as a tumor suppressor in multiple cancers, CASTOR1 is tightly regulated in cancer, including inhibition by viral miRNAs in KSHV-induced cancer and degradation via AKT-phosphorylation and RNF167-mediated ubiquitination. Notably, CASTOR1's role extends beyond normal cellular contexts, as evidenced by its tumor-suppressive function in breast cancer xenograft models and its prognostic value in lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). This study aims to elucidate CASTOR1’s role in a transgenic Kras-driven LUAD mouse model, shedding light on its tumor-suppressive potential. Methods: Utilizing a novel CASTOR1 knockout (KO) mouse model crossed with the Kras (LSL-KrasG12D/+) NSCLC model, we established a new mouse model with simultaneous Kras activation and homozygous CASTOR1 ablation. Tumor incidence and growth were monitored in Kras mice harboring wild-type (WT) and KO CASTOR1. Further investigations examined the proliferative dynamics and activation status of various oncogenic pathways, and assessed mTORC1 inhibitor sensitivities in human LUAD cell lines with altered CASTOR1 expression levels. Results: CASTOR1 deficiency markedly heightened lung tumor incidence and size, concomitant with an elevated proliferative index. Intriguingly, a novel antibody identified robust pCASTOR1 levels in CASTOR1 WT mouse LUAD tumors, positively correlated with tumor stage and inversely related to total CASTOR1 abundance, hinting at an active degradation mechanism promoting tumor progression. Elevated mTORC1 signaling, evidenced by increased levels of p4EBP and pS6, underscored the pathway's activation consequent to CASTOR1 loss, likely driving tumor development. Furthermore, heightened pERK levels in CASTOR1 KO tumors unveiled a novel feedback loop intertwining CASTOR1/mTORC1 and the Kras/ERK pathway. Notably, treatment with mTORC1 inhibitors sensitized organoids of Kras tumors with mTORC1 hyperactivation to Kras G12D inhibitor. Conclusions: Our findings elucidate CASTOR1's tumor-suppressive role in Kras-dependent mouse LUAD tumors, suggesting the potential efficacy of combined Kras and mTORC1 inhibition in LUAD subsets characterized by hyperactivated Kras and diminished CASTOR1 levels or mTORC1 hyperactivity.

The DRAP1/DR1 Repressor Complex Increases mTOR Activity to Promote Progression and Confer Everolimus Sensitivity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Transcriptional dysregulation is a hallmark of cancer, and several transcriptional regulators have been demonstrated to contribute to cancer progression. In this study, we identified an upregulation of the transcriptional corepressor downregulator of transcription 1-associated protein 1 (DRAP1) in TNBC, which was closely associated with poor recurrence-free survival in patients with TNBC. DRAP1 promoted TNBC proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, the downregulator of transcription 1 (DR1)/DRAP1 heterodimer complex inhibited expression of the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1) and thereby increased activation of mTOR, which sensitized TNBC to treatment with the mTOR inhibitor everolimus. DRAP1 and DR1 also formed a positive feedback loop. DRAP1 enhanced the stability of DR1 by recruiting the deubiquitinase USP7 to inhibit its proteasomal degradation; in turn, DR1 directly promoted DRAP1 transcription. Collectively, this study uncovered a DRAP1-DR1 bidirectional regulatory pathway that promotes TNBC progression, suggesting that targeting the DRAP1/DR1 complex might be a potential therapeutic strategy to treat TNBC. Significance: DR1 and DRAP1 form a positive feedback loop and a repressor complex to cooperatively inhibit cytosolic arginine sensor for mTORC1 subunit 1 transcription and stimulate mTOR signaling, leading to progression and increased everolimus sensitivity in triple-negative breast cancer.

L-arginine alleviates heat stress-induced mammary gland injury through modulating CASTOR1-mTORC1 axis mediated mitochondrial homeostasis

As global warming intensifies, extreme heat is becoming increasingly frequent. These extreme heatwaves have decreased the milk production of dairy animals such as cows and goats and have caused significant damage to the entire dairy industry. It is known that heat stress (HS) can induce the apoptosis and autophagy of mammary epithelial cells (MECs), leading to a decrease in lactating MECs. L-arginine can effectively attenuate HS-induced decreases in milk yield, but the exact mechanisms are not fully understood. In this study, we found that HS upregulated the arginine sensor CASTOR1 in mouse MECs. Arginine activated mTORC1 activity through CASTOR1 and promoted mitochondrial biogenesis through the mTORC1/PGC-1α/NRF1 pathway. Moreover, arginine inhibited mitophagy through the CASTOR1/PINK1/Parkin pathway. Mitochondrial homeostasis ensures ATP synthesis and a stable cellular redox state for MECs under HS, further alleviating HS-induced damage and improving the lactation performance of MECs. In conclusion, these findings reveal the molecular mechanisms by which L-arginine relieves HS-induced mammary gland injury, and suggest that the intake of arginine-based feeds or feed additives is a promising method to increase the milk yield of dairy animals in extreme heat conditions.

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.

Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway.

Microglia are resident immune cells in the brain and are closely associated with central nervous system inflammation and neurodegenerative diseases. It is known that mammalian target of rapamycin (mTOR) pathway plays an important role in the polarization of microglia. Castor1 has been identified as the cytosolic arginine sensor for the mTOR complex 1 (mTORC1) pathway, but the role of Castor1 in microglial polarization is still unknown. The purpose of this study was to explore the regulatory effect of Castor1 on microglial polarization and the underlying mechanism. The results demonstrated that Castor1 expression was significantly decreased in lipopolysaccharides (LPS) and interferon (IFN)-γ treated microglia. Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers. However, the expression of M2-related genes was promoted when Castor1 was overexpressed in IL-4 treated microglia. Mechanistically, Castor1 overexpression inhibited the activation of mTOR signaling pathway. In addition, after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, indicating that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. We propose that Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization.

Structure of the nutrient-sensing hub GATOR2.

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 β-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through β-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 β-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

Stable Lanthanide Metal-Organic Frameworks with Ratiometric Fluorescence Sensing for Amino Acids and Tunable Proton Conduction and Magnetic Properties.

Four new isostructural lanthanide metal-organic frameworks (MOFs), namely {[Ln(DMTP-DC)1.5(H2O)3]·DMF}n [H2DMTP-DC = 2',5'-dimethoxytriphenyl-4,4″-dicarboxylic acid; LnIII = EuIII (1), GdIII (2), TbIII (3), and DyIII (4)], have been synthesized and characterized. Single-crystal structure analysis reveals that 1-4 are three-dimensional Ln-MOFs with rich H-bonding of coordinated H2O molecules in the network channels. The X-ray diffraction patterns indicate that Ln-MOF 1 displays good stabilities in organic solvents and aqueous solutions with distinct pH values. Both 1 and 3 show characteristic emission of LnIII ions. Ln-MOF 1 can be used as a ratiometric fluorescence sensor for arginine and lysine in aqueous solution, and the detection limits are 24.38 μM for arginine and 9.31 μM for lysine. All 1-4 show proton conductivity related to relative humidity (RH) and temperature, and the maximum conductivity values of 1-4 at 55 °C and 100% RH are 9.94 × 10-5, 1.62 × 10-4, 1.71 × 10-4, and 2.67 × 10-4 S·cm-1, respectively. The value of σ increases with the decrease in ionic radius, indicating that the radius of the LnIII ions can regulate the proton conductivity of these MOFs. Additionally, 2 exhibits a significant magnetocaloric effect (MCE) with a magnetic entropy change (-ΔSm) of 18.86 J kg-1 K-1 for ΔH = 7 T at 2 K, and 4 shows weak field-induced slow relaxation of magnetization. The coexistence of good fluorescence sensing capability, attractive proton conductivity, and relatively large MCE in Ln-MOFs is rare, and thus, 1-4 are potentially multifunctional MOF materials.

Downregulation of CASTOR1 Inhibits Heat-Stress-Induced Apoptosis and Promotes Casein and Lipid Synthesis in Mammary Epithelial Cells.

Heat stress is one of the most important factors limiting the milk yields of dairy animals. This decline can be attributed to the heat-stress-induced apoptosis of mammary epithelial cells (MECs). The cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1) is a crucial upstream regulator of the mechanistic target of rapamycin complex 1 (mTORC1) signaling, which has close connections with apoptosis. However, the specific roles of CASTOR1 in regulating the apoptosis and lactation of MECs are still obscure. In the present study, we found that heat stress promotes apoptosis and CASTOR1's expression in HC11 cells. Downregulation of CASTOR1 inhibits heat-stress-induced apoptosis through a ROS-independent pathway. In addition, silencing of CASTOR1 promotes cell proliferation, cell cycle progression, and milk component synthesis, and overexpressing of CASTOR1 reverses these observations. Furthermore, we found that silencing of CASTOR1 contributes to the nuclear transport of SREBP1 and promotes lipid synthesis. This study demonstrates the pivotal roles of CASTOR1 in heat-stress-induced apoptosis and milk component synthesis in MECs.

Effects of single ingestion of arginine on mTORC1 activation in rat fast- and slow-twitch muscles

This study aimed to examine whether a single ingestion of arginine activates the mammalian target of rapamycin complex 1 (mTORC1) in rat fast- and slow-twitch skeletal muscles. In the first experiment, the rats were orally administered arginine (3 or 10 mmol/kg body weight) in water. The plantaris, gastrocnemius, and soleus muscles were excised 1 h after the administration. Immunoblot analysis showed that the administration with a higher dose (10 mmol/kg) resulted in increased phosphorylation of ribosomal S6 kinase (S6K) and ribosomal protein S6 only in the soleus muscles. The amounts of cellular arginine sensor for mTORC1 subunit 1 (CASTOR1) expressed were similar in these three muscles. In the second experiment, the plantaris and soleus muscles were excised 1 h after the administration of 10 mmol/kg of arginine. The binding of CASTOR1 to the GATOR2 complex was not detected in either muscle in co-immunoprecipitation and immunoblot analyses, irrespective of arginine administration. In the third experiment, a role of nitric oxide (NO) was elucidated. Treatment with an inhibitor of NO synthase blocked the arginine-induced increase in S6K phosphorylation. These results indicate that a single ingestion of arginine is capable of activating mTORC1 only in slow-twitch muscles and suggest that the activation may be mediated via NO, but not via the CASTOR1-GATOR2 complex pathway.

Heat Stress: Effects on Rumen Microbes and Host Physiology, and Strategies to Alleviate the Negative Impacts on Lactating Dairy Cows.

Heat stress (HS) in dairy cows causes considerable losses in the dairy industry worldwide due to reduced animal performance, increased cases of metabolic disorders, altered rumen microbiome, and other health problems. Cows subjected to HS showed decreased ruminal pH and acetate concentration and an increased concentration of ruminal lactate. Heat-stressed cows have an increased abundance of lactate-producing bacteria such as Streptococcus and unclassified Enterobacteriaceae, and soluble carbohydrate utilizers such as Ruminobacter, Treponema, and unclassified Bacteroidaceae. Cellulolytic bacteria, especially Fibrobacteres, increase during HS due to a high heat resistance. Actinobacteria and Acetobacter, both acetate-producing bacteria, decreased under HS conditions. Rumen fermentation functions, blood parameters, and metabolites are also affected by the physiological responses of the animal during HS. Isoleucine, methionine, myo-inositol, lactate, tryptophan, tyrosine, 1,5-anhydro-D-sorbitol, 3-phenylpropionic acid, urea, and valine decreased under these conditions. These responses affect feed consumption and production efficiency in milk yield, growth rate, and reproduction. At the cellular level, activation of heat shock transcription factor (HSF) (located throughout the nucleus and the cytoplasm) and increased expression of heat shock proteins (HSPs) are the usual responses to cope with homeostasis. HSP70 is the most abundant HSP family responsible for the environmental stress response, while HSF1 is essential for increasing cell temperature. The expression of bovine lymphocyte antigen and histocompatibility complex class II (DRB3) is downregulated during HS, while HSP90 beta I and HSP70 1A are upregulated. HS increases the expression of the cytosolic arginine sensor for mTORC1 subunits 1 and 2, phosphorylation of mammalian target of rapamycin and decreases the phosphorylation of Janus kinase-2 (a signal transducer and activator of transcription factor-5). These changes in physiology, metabolism, and microbiomes in heat-stressed dairy cows require urgent alleviation strategies. Establishing control measures to combat HS can be facilitated by elucidating mechanisms, including proper HS assessment, access to cooling facilities, special feeding and care, efficient water systems, and supplementation with vitamins, minerals, plant extracts, and probiotics. Understanding the relationship between HS and the rumen microbiome could contribute to the development of manipulation strategies to alleviate the influence of HS. This review comprehensively elaborates on the impact of HS in dairy cows and introduces different alleviation strategies to minimize HS.

RNF167 activates mTORC1 and promotes tumorigenesis by targeting CASTOR1 for ubiquitination and degradation

mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.

Development of a tripeptide based arginine sensor via applying the concept of molecular engineering

In this present work, we have synthesized two tripeptides and one of them is the molecular engineered version of the other one. The study shows how a single nitro group can abruptly change the physicochemical properties of a tripeptide. Tripeptide 3 shows interesting self-assembled nanoscale supramolecular architectures. We experienced a gradual transition from toroid to sphere morphology. Contrary to this, 4, the molecular engineered version of 3, organizes in a completely different manner. Instead of producing aggregated spheres, it produces a structure similar to a chain of pearls. In the case of secondary structure, compound 3 shows beta sheet-like structure while compound 4 exhibits alpha helix-like assembly. Compound 4 acts as an arginine sensor with a detection limit of 1.38 μM in the water. Furthermore, using Density-functional theory, optimization of arginine bound 4, calculation of adsorption energies, and prediction of absorption spectra were obtained which support the experimental data.

A conformational change in the N terminus of SLC38A9 signals mTORC1 activation

mTORC1 is a central hub that integrates environmental cues, such as cellular stresses and nutrient availability to modulate metabolism and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, emerged as a sensor for luminal arginine and as an activator of mTORC1. The amino acid-mediated activation of mTORC1 is regulated by the N-terminal domain of SLC38A9. Here, we determined the crystal structure of zebrafish SLC38A9 (drSLC38A9) and found the N-terminal fragment inserted deep within the transporter, bound in the substrate-binding pocket where normally arginine would bind. This represents a significant conformational change of the N-terminal domain (N-plug) when compared with our recent arginine-bound structure of drSLC38A9. We propose a ball-and-chain model for mTORC1 activation, where N-plug insertion and Rag GTPase binding with SLC38A9 is regulated by luminal arginine levels. This work provides important insights into nutrient sensing by SLC38A9 to activate the mTORC1 pathways in response to dietary amino acids.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Anthracene-Tagged UiO-67-MOF as Highly Selective Aqueous Sensor for Nanoscale Detection of Arginine Amino Acid.

In the present paper, new functionalized UiO-67 metal-organic frameworks (MOF) which contain aromatic tagged groups such as phenyl, naphthyl, and anthracene have been synthesized, characterized, and used for sensing water-soluble amino acids. The results show that anthracene-tagged UiO-67-MOF is shown to act as a highly efficient and selective aqueous sensor for arginine over other water-soluble amino acids in nanoscale. Upon adding an increasing amount of arginine, PL bands of the anth-UiO-67 MOF quenched completely, while there is no perturbation in the PL bands for other amino acid observed. This MOF allows a selective ratiometric detection of arginine without any interference from other amino acids.

Transcriptome Functional Analysis of Mammary Gland of Cows in Heat Stress and Thermoneutral Condition.

Simple SummaryThe current study employed RNA-seq technology to analyze the impact of heat stress on the whole transcript sequencing profile in the mammary glands of lactating Holstein dairy cows. In the findings of the current study, heat stress downregulated the expression of casein genes, which resulted in a decrease in milk production. Moreover, heat stress upregulated the gene expression of HSPA1A and HSP90B1, while it downregulated the expression of immune response-related genes that resulted in a reduction in milk yield. Furthermore, there was an increased synthesis of heat shock proteins and unfolded proteins that could reduce the availability of circulating amino acids for milk protein synthesis. The findings of the current experiment may help to explore the impact of heat stress on immune function, milk production, and milk protein synthesis in cows.Heat stress (HS) exerts significant effects on the production of dairy animals through impairing health and biological functions. However, the molecular mechanisms related to the effect of HS on dairy cow milk production are still largely unknown. The present study employed an RNA-sequencing approach to explore the molecular mechanisms associated with a decline in milk production by the functional analysis of differentially expressed genes (DEGs) in mammary glands of cows exposed to HS and non-heat-stressed cows. The results of the current study reveal that HS increases the rectal temperature and respiratory rate. Cows under HS result in decreased bodyweight, dry matter intake (DMI), and milk yield. In the current study, a total of 213 genes in experimental cow mammary glands was identified as being differentially expressed by DEGs analysis. Among identified genes, 89 were upregulated, and 124 were downregulated. Gene Ontology functional analysis found that biological processes, such as immune response, chaperone-dependent refolding of protein, and heat shock protein binding activity, were notably affected by HS. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis found that almost all of the top-affected pathways were related to immune response. Under HS, the expression of heat shock protein 90 kDa beta I (HSP90B1) and heat shock 70 kDa protein 1A was upregulated, while the expression of bovine lymphocyte antigen (BoLA) and histocompatibility complex, class II, DRB3 (BoLA-DRB3) was downregulated. We further explored the effects of HS on lactation-related genes and pathways and found that HS significantly downregulated the casein genes. Furthermore, HS increased the expression of phosphorylation of mammalian target of rapamycin, cytosolic arginine sensor for mTORC1 subunit 2 (CASTOR2), and cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1), but decreased the phosphorylation of Janus kinase-2, a signal transducer and activator of transcription factor-5. Based on the findings of DMI, milk yield, casein gene expression, and the genes and pathways identified by functional annotation analysis, it is concluded that HS adversely affects the immune function of dairy cows. These results will be beneficial to understand the underlying mechanism of reduced milk yield in HS cows.