Conditions Of Amino Acid Starvation Research Articles

Impact of Protein Coronas on Lipid Nanoparticle Uptake and Endocytic Pathways in Cells.

Lipid nanoparticles (LNPs), widely used in disease diagnosis and drug delivery, face the challenge of being surrounded by biological macromolecules such as proteins upon entering the human body. These molecules compete for binding sites on the nanoparticle surfaces, forming a protein corona. The impact of different types of protein coronas on LNP delivery remains unclear. In this study, we employed a newly developed, highly sensitive LNP labeling platform and analyzed the endocytosis of HeLa cells under different nutritional conditions using proteomics to address this critical issue. Our research found that under conditions of complete medium and amino acid starvation, most DNA-FITC vesicles in HeLa cells were located in the perinuclear region 4 h after transfection. In contrast, under serum starvation conditions, only a small portion of DNA-FITC vesicles were in the perinuclear region. On the other hand, through proteomics, we discovered that cells that were enriched in amino acids and complete medium contained more proteins, whereas those under serum starvation had relatively fewer enriched proteins. Through KEGG pathway enrichment analysis, we identified the phagosome and endocytosis pathways as particularly important. Lastly, differential analysis of proteins in these pathways revealed that proteins such as F-actin, Coronin, vATPase, TUBA, TUBB, MHCII, and TSP may have significant impacts on cellular endocytosis. Our research findings indicate that it is necessary to regulate cellular endocytosis based on different protein coronas to achieve optimal cytoplasmic release.

Abstract 1873: AST-05X: A novel GCN2 inhibitor as a promising anti-cancer approach for sarcoma

Abstract Background Integrated stress response (ISR) is an adaptive signaling pathway that responds to stress and plays an important role in cell fate determination. This pathway enables cellular adaptation and survival under stress conditions by activating specific genes that reduce protein synthesis. Tumors exist in a state characterized by heightened intrinsic and extrinsic stress, heavily relying on a balanced ISR to manage the metabolic demands for rapid growth. ISR (Integrated Stress Response) consists of four major kinases: GCN2, PERK, HRI, and PKR. GCN2 is activated by stress due to amino acid deprivation. Amino acids are crucial for cell survival, and cancer cells, require higher amounts than normal cells, particularly depending on conditionally essential amino acids like glutamine, asparagine, and arginine. GCN2 activation acts as a key mechanism for cancer cells to overcome amino acid deficiencies, making GCN2 a potential target, especially in conjunction with anticancer drugs like asparaginase. In this study, we aimed to identify a potential GCN2 inhibitor to explore a promising anti-cancer approach in sarcoma, a rare cancer with no new therapeutic alternatives. Method The activity verification of ISR kinases was characterized using in-vitro enzyme assays. In-vitro panel assays were employed to determine the selectivity profiling for oncology kinases. The modulation of the ISR pathway was validated using a sarcoma cell line (HT-1080) and a cell line derived from sarcoma patients. Western blot or ELISA assays were employed to measure phospho-GCN2, ATF4, phospho-eIF2a, and CHOP under both basal conditions and amino acid starvation conditions. Gene expression profiling (GEP) of sarcoma patient tissue samples(n=17) was conducted through RNA sequencing (RNAseq) analysis. Results AST-05X is a selective and potent GCN2 inhibitor. AST-05X inhibits the expression of GCN2 and ISR downstream factors (p-eIF2a, ATF, CHOP) in vitro cell-based experiments under amino acid deprivation. In addition, based on the results of ISR-related gene expression profiling GEP analysis, we identified potential CDx biomarkers for evaluating the diagnosis and treatment response of sarcoma. Conclusion This study is currently investigating the efficacy of the drug in sarcoma patient-derived xenograft models. AST-05X is a GCN2 inhibitor that has the potential to validate the inhibition of GCN2 as a promising anti-cancer approach for sarcoma, a rare cancer with limited therapeutic alternatives. Citation Format: Youngki Choi, Jinback Lim, Hyo-Hyun Park, Jinho Kang, Hyosong Kim, Kumhee Yun, Hun Jung. AST-05X: A novel GCN2 inhibitor as a promising anti-cancer approach for sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1873.

METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation

METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions. Mechanistically, we determined that the METTL16 knockdown represses the expression of extrinsic death receptors at both transcription and translation levels. Depletion of METTL16 prevented protein synthesis of GCN2, resulting in diminished ATF4 expression in a GCN2-eIF2α-dependent manner. Reduction of ATF4 further declined the expression of apoptotic receptor protein DR5. Meanwhile, METTL16 deficiency directly hampered protein synthesis of FADD and DR5, thereby impairing apoptosis and promoting cancer cell survival. Taken together, our study provides novel evidence for the involvement of METTL16 in regulating cancer progression, suggesting that METTL16 as a potential therapeutic target for cancer treatment.

OTUD3 suppresses the mTORC1 signaling by deubiquitinating KPTN.

Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitin-proteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3K-AKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors.

Hepatitis E Virus Protease Inhibits the Activity of Eukaryotic Initiation Factor 2-Alpha Kinase 4 and Promotes Virus Survival.

Multiple mechanisms exist in a cell to cope with stress. Four independent stress-sensing kinases constitute the integrated stress response machinery of the mammalian cell, and they sense the stress signals and act by phosphorylating the eukaryotic initiation factor 2α (eIF2α) to arrest cellular translation. Eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4) is one of the four kinases and is activated under conditions of amino acid starvation, UV radiation, or RNA virus infection, resulting in shutdown of global translation. An earlier study in our laboratory constructed the protein interaction network of the hepatitis E virus (HEV) and identified eIF2AK4 as a host interaction partner of the genotype 1 (g1) HEV protease (PCP). Here, we report that PCP's association with the eIF2AK4 results in inhibition of self-association and concomitant loss of kinase activity of eIF2AK4. Site-directed mutagenesis of the 53rd phenylalanine residue of PCP abolishes its interaction with the eIF2AK4. Further, a genetically engineered HEV-expressing F53A mutant PCP shows poor replication efficiency. Collectively, these data identify an additional property of the g1-HEV PCP protein, through which it helps the virus in antagonizing eIF2AK4-mediated phosphorylation of the eIF2α, thus contributing to uninterrupted synthesis of viral proteins in the infected cells. IMPORTANCE Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. It causes chronic infection in organ transplant patients. Although the disease is self-limiting in normal individuals, it is associated with high mortality (~30%) in pregnant women. In an earlier study, we identified the interaction between the genotype 1 HEV protease (PCP) and cellular eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4). Since eIF2AK4 is a sensor of the cellular integrated stress response machinery, we evaluated the significance of the interaction between PCP and eIF2AK4. Here, we show that PCP competitively associates with and interferes with self-association of the eIF2AK4, thereby inhibiting its kinase activity. Lack of eIF2AK4 activity prevents phosphorylation-mediated inactivation of the cellular eIF2α, which is essential for initiation of cap-dependent translation. Thus, PCP behaves as a proviral factor, promoting uninterrupted synthesis of viral proteins in infected cells, which is crucial for survival and proliferation of the virus.

Nutritional compensation of the circadian clock is a conserved process influenced by gene expression regulation and mRNA stability.

Compensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Known compensation effectors directly regulate core clock factors to buffer the oscillator's period length from variables in the environment. Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Using the filamentous fungus Neurospora crassa, we performed a genetic screen under glucose and amino acid starvation conditions to identify new regulators of Nutritional Compensation. Our screen uncovered 16 novel mutants, and together with 4 mutants characterized in prior work, a model emerges where Nutritional Compensation of the fungal clock is achieved at the levels of transcription, chromatin regulation, and mRNA stability. However, eukaryotic circadian Nutritional Compensation is completely unstudied outside of Neurospora. To test for conservation in cultured human cells, we selected top hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian orthologs, and characterized the cell lines with respect to compensation. We find that the wild-type mammalian clock is also compensated across a large range of external glucose concentrations, as observed in Neurospora, and that knocking down the mammalian orthologs of the Neurospora compensation-associated genes CPSF6 or SETD2 in human cells also results in nutrient-dependent period length changes. We conclude that, like Temperature Compensation, Nutritional Compensation is a conserved circadian process in fungal and mammalian clocks and that it may share common molecular determinants.

Effects of Essential Amino Acid Deficiency on General Control Nonderepressible 2/Eukaryotic Initiation Factor 2 Signaling and Proteomic Changes in Primary Bovine Mammary Epithelial Cells.

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal–Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA–Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2–99.8% viability indicated low cell death rates. Proliferation (range, 1.02–1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.

An AMPKα2-specific phospho-switch controls lysosomal targeting for activation.

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy.

Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1.

Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding.IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD.

Mutation of C9ORF72 is the most prevalent defect in amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD)1. Together with hexanucleotide repeat expansion2,3, haploinsufficiency of C9ORF72 contributes to neuronal dysfunction4–6. We determined the structure of the C9orf72-SMCR8-WDR41 complex by cryo-EM. C9orf72 and SMCR8 are both longin-DENN domain proteins7, while WDR41 is a beta-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and SMCR8DENN drive lysosomal localization in amino acid starvation. The structure suggested that C9orf72-SMCR8 was a small GTPase activating protein (GAP). We found that C9orf72-SMCR8-WDR41 is a GAP for Arf family small GTPases. These data rationalize the function of C9orf72 both in normal physiology and in ALS/FTD.

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp0 strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp0 strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp0 suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp0 backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.

Stringent response governs the oxidative stress resistance and virulence of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

Ssb2 is a novel factor in regulating synthesis and degradation of Gcn4 in Saccharomyces cerevisiae.

Yeast cells respond to environmental stress by inducing the master regulator Gcn4 to control genes involved in biosynthesis of amino acids and purine pathways. Gcn4 is a member of the basic leucine Zipper family and binds directly as a homodimer to a conserved regulatory region of target genes. Ssb2 was discovered to rescue the mutant Gcn4 which has a point mutation that decreases DNA-binding affinity. Ssb2 is part of the Hsp70 protein family responsible for protein quality control and it is thought that Ssb2 assists the passage of nascent polypeptide chains from the ribosomes. To characterize the mechanism behind the rescue of the mutant gcn4 phenotype, transcriptional activity and protein levels of Gcn4 were analyzed. We found that Ssb2 improved the expression of Gcn4 target genes by increasing the DNA-binding affinity of gcn4 mutants to target gene promoters under conditions of amino acid starvation. Gcn4 levels increased at both translational and post-translational levels without regulating GCN4 steady-state mRNA levels. We also found that the nuclear export signal of Ssb2 is required for interaction with Gcn4 and rescue of the gcn4 mutant phenotype. These findings suggest that Ssb2 is a critical factor that modulates Gcn4 functions in the nucleus and cytosol.

Deletion of Nrip1 Extends Female Mice Longevity, Increases Autophagy, and Delays Cell Senescence.

Using age of female sexual maturation as a biomarker, we previously identified nuclear receptor interacting protein 1 (Nrip1) as a candidate gene that may regulate aging and longevity. In the current report, we found that the deletion of Nrip1 can significantly extend longevity of female mice (log-rank test, p = .0004). We also found that Nrip1 expression is altered differently in various tissues during aging and under diet restriction. Remarkably, Nrip1 expression is elevated with aging in visceral white adipose tissue (WAT), but significantly reduced after 4 months of diet restriction. However, in gastrocnemius muscle, Nrip1 expression is significantly upregulated after the diet restriction. In mouse embryonic fibroblasts, we found that the deletion of Nrip1 can suppress fibroblast proliferation, enhance autophagy under normal culture or amino acid starvation conditions, as well as delay oxidative and replicative senescence. Importantly, in WAT of old animals, the deletion of the Nrip could significantly upregulate autophagy and reduce the number of senescent cells. These results suggest that deleting Nrip1 can extend female longevity, but tissue-specific deletion may have varying effects on health span. The deletion of Nrip1 in WAT may delay senescence in WAT and extend health span.

Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4.

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion.

Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs.

Manipulation of components that control feeding behavior in Drosophila melanogaster increases sensitivity to amino acid starvation.

Feeding is a complex behavior that is regulated by several internal mechanisms. Neuropeptides are able to survey quantities of stored energy and inform the organism if nutrient intake is required. In addition to this homeostatic regulation, a post-feeding reward system positively reinforces feeding. Slight adjustments to either system can tilt the balance to affect the energy reserves and survivorship in times of nutrient adversity. Neuropeptide F (NPF), a homolog of the mammalian neuropeptide Y, acts to induce feeding within the homeostatic regulation of this behavior. Drosophila and other insects bear a shorter form of NPF known as short NPF (sNPF) that can influence feeding. A neural hormone regulator, the dopamine transporter (DAT), works to clear dopamine from the synapses. This action may manipulate the post-feeding reward circuit in that lowered dopamine levels depress feeding, and excess dopamine levels encourage feeding. Here, we have overexpressed and impaired the activities of NPF, sNPF, and DAT in Drosophila, and we examined their ability to survive during conditions of amino acid starvation. Too much or too little NPF or sNPF, which are key players in homeostatic feeding regulation, leads to increased sensitivity to amino acid starvation and diminished survivorship when compared to controls. When DAT, a member of the post-feeding reward system, is either overexpressed or reduced via mutation, Drosophila has increased sensitivity to amino acid starvation. Taken together, these results indicate that subtle variation in the expression of key components of these systems impacts survivorship during adverse nutrient conditions.

Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants.

Some bioflocculants composed of extracellular polymeric substances are produced under peculiar conditions. Bacillus licheniformis CGMCC2876 is a microorganism that secretes both extracellular polysaccharides (EPS) and poly-gamma-glutamic acid (γ-PGA) under stress conditions. In this work, SWATH acquisition LC-MS/MS method was adopted for differential proteomic analysis of B. licheniformis, aiming at determining the bacterial stress mechanism. Compared with LB culture, 190 differentially expressed proteins were identified in B. licheniformis CGMCC2876 cultivated in EPS culture, including 117 up-regulated and 73 down-regulated proteins. In γ-PGA culture, 151 differentially expressed proteins, 89 up-regulated and 62 down-regulated, were found in the cells. Up-regulated proteins involved in amino acid biosynthesis were found to account for 43% and 41% of the proteomes in EPS and γ-PGA cultivated cells, respectively. Additionally, a series of proteins associated with amino acid degradation were found to be repressed under EPS and γ-PGA culture conditions. Transcriptional profiling via the qPCR detection of selected genes verified the proteomic analysis. Analysis of free amino acids in the bacterial cells further suggested the presence of amino acid starvation conditions. EPS or γ-PGA was synthesized to alleviate the effect of amino acid limitation in B. licheniformis. This study identified a stress response mechanism in the synthesis of macromolecules in B. licheniformis, providing potential culture strategies to improve the production of two promising bioflocculants.

PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.

State transitions in the TORC1 signaling pathway and information processing in Saccharomyces cerevisiae.

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases-Gtr1, Gtr2, and Rho1-bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9-dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell.