IU Indianapolis ScholarWorks :: Browsing by Subject "Stress response"

Browsing by Subject "Stress response"

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Asparagine: A Metabolite to Be Targeted in Cancers
(MDPI, 2021-06-19) Jiang, Jie; Batra, Sandeep; Zhang, Ji; Pediatrics, School of Medicine
Amino acids play central roles in cancer progression beyond their function as building blocks for protein synthesis. Thus, targeting amino acid acquisition and utilization has been proved to be therapeutically beneficial in various pre-clinical models. In this regard, depletion of circulating asparagine, a nonessential amino acid, by L-asparaginase has been used in treating pediatric acute lymphoblastic leukemia (ALL) for decades. Of interest, unlike most solid tumor cells, ALL cells lack the ability to synthesize their own asparagine de novo effectively. However, only until recently, growing evidence suggests that solid tumor cells strive to acquire adequate amounts of asparagine to support tumor progression. This process is subjected to the regulation at various levels, including oncogenic signal, tumor-niche interaction, intratumor heterogeneity and dietary accessibility. We will review the literature on L-asparaginase-based therapy as well as recent understanding of asparagine metabolism in solid tumor progression, with the hope of shedding light into a broader cancer therapeutic strategy by perturbing its acquisition and utilization.
Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii
(Elsevier, 2024) Dey, Vishakha; Holmes, Michael J.; Bastos, Matheus S.; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
Translational control mechanisms modulate the microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we determined that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for the cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.
Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response
(American Society for Biochemistry and Molecular Biology, 2021-10) Jin, Danni; Wek, Sheree A.; Kudlapur, Nathan T.; Cantara, William A.; Bakhtina, Marina; Wek, Ronald C.; Musier-Forsyth, Karin; Biochemistry and Molecular Biology, School of Medicine
Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNAGlu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.
DLK signaling in axotomized neurons triggers complement activation and loss of upstream synapses
(Elsevier, 2024) Asghari Adib, Elham; Shadrach, Jennifer L.; Reilly-Jankowiak, Lauren; Dwivedi, Manish K.; Rogers, Abigail E.; Shahzad, Shameena; Passino, Ryan; Giger, Roman J.; Pierchala, Brian A.; Collins, Catherine A.; Anatomy, Cell Biology and Physiology, School of Medicine
Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss are currently unknown. Here, we delineate a critical role for neuronal kinase dual leucine zipper kinase (DLK)/MAP3K12, which becomes activated in axotomized neurons. Studies with conditional knockout mice indicate that DLK signaling activation in injured MNs triggers the induction of phagocytic microglia and synapse loss. Aspects of the DLK-regulated response include expression of C1q first from the axotomized MN and then later in surrounding microglia, which subsequently phagocytose presynaptic components of upstream synapses. Pharmacological ablation of microglia inhibits the loss of cholinergic C boutons from axotomized MNs. Together, the observations implicate a neuronal mechanism, governed by the DLK, in the induction of inflammation and the removal of synapses.
Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii
(American Society for Microbiology, 2018-10-24) Augusto, Leonardo; Martynowicz, Jennifer; Staschke, Kirk A.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.
Genes Differentially Expressed by Haemophilus ducreyi during Anaerobic Growth Significantly Overlap Those Differentially Expressed during Experimental Infection of Human Volunteers
(American Society for Microbiology, 2022) Brothwell, Julie A.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. In humans, H. ducreyi is found in the anaerobic environment of an abscess; previous studies comparing bacterial gene expression levels in pustules with the inocula (∼4-h aerobic mid-log-phase cultures) identified several upregulated differentially expressed genes (DEGs) that are associated with anaerobic metabolism. To determine how H. ducreyi alters its gene expression in response to anaerobiosis, we performed RNA sequencing (RNA-seq) on both aerobic and anaerobic broth cultures harvested after 4, 8, and 18 h of growth. Principal-coordinate analysis (PCoA) plots showed that anaerobic growth resulted in distinct transcriptional profiles compared to aerobic growth. During anaerobic growth, early-time-point comparisons (4 versus 8 h) identified few DEGs at a 2-fold change in expression and a false discovery rate (FDR) of <0.01. By 18 h, we observed 18 upregulated and 16 downregulated DEGs. DEGs involved in purine metabolism, the uptake and use of alternative carbon sources, toxin production, nitrate reduction, glycine metabolism, and tetrahydrofolate synthesis were upregulated; DEGs involved in electron transport, thiamine biosynthesis, DNA recombination, peptidoglycan synthesis, and riboflavin synthesis or modification were downregulated. To examine whether transcriptional changes that occur during anaerobiosis overlap those that occur during infection of human volunteers, we compared the overlap of DEGs obtained from 4 h of aerobic growth to 18 h of anaerobic growth to those found between the inocula and pustules in previous studies; the DEGs significantly overlapped. Thus, a major component of H. ducreyi gene regulation in vivo involves adaptation to anaerobiosis. IMPORTANCE: In humans, H. ducreyi resides in the anaerobic environment of an abscess and appears to upregulate genes involved in anaerobic metabolism. How anaerobiosis alone affects gene transcription in H. ducreyi is unknown. Using RNA-seq, we investigated how anaerobiosis affects gene transcription over time compared to aerobic growth. Our results suggest that a substantial component of H. ducreyi gene regulation in vivo overlaps the organism's response to anaerobiosis in vitro. Our data identify potential therapeutic targets that could be inhibited during in vivo growth.
Mechanisms of Toxoplasma gondii persistence and latency
(Oxford University Press, 2012) Sullivan, William J., Jr.; Jeffers, Victoria; Pharmacology and Toxicology, School of Medicine
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes opportunistic disease, particularly in immunocompromised individuals. Central to its transmission and pathogenesis is the ability of the proliferative stage (tachyzoite) to convert into latent tissue cysts (bradyzoites). Encystment allows Toxoplasma to persist in the host, and affords the parasite a unique opportunity to spread to new hosts without proceeding through its sexual stage, which is restricted to felids. Bradyzoite tissue cysts can cause reactivated toxoplasmosis if host immunity becomes impaired. A greater understanding of the molecular mechanisms orchestrating bradyzoite development is needed to better manage the disease. Here we will review key studies that have contributed to our knowledge about this persistent form of the parasite and how to study it, with a focus on how cellular stress can signal for the reprogramming of gene expression needed during bradyzoite development.
Promoter demethylation of the asparagine synthetase gene is required for ATF4-dependent adaptation to asparagine depletion
(Elsevier, 2019-12-06) Jiang, Jie; Srivastava, Sankalp; Seim, Gretchen; Pavlova, Natalya N.; King, Bryan; Zou, Lihua; Zhang, Chi; Zhong, Minghua; Feng, Hui; Kapur, Reuben; Wek, Ronald C.; Fan, Jing; Zhang, Ji; Pediatrics, School of Medicine
Tumor cells adapt to nutrient-limited environments by inducing gene expression that ensures adequate nutrients to sustain metabolic demands. For example, during amino acid limitations, ATF4 in the amino acid response induces expression of asparagine synthetase (ASNS), which provides for asparagine biosynthesis. Acute lymphoblastic leukemia (ALL) cells are sensitive to asparagine depletion, and administration of the asparagine depletion enzyme l-asparaginase is an important therapy option. ASNS expression can counterbalance l-asparaginase treatment by mitigating nutrient stress. Therefore, understanding the mechanisms regulating ASNS expression is important to define the adaptive processes underlying tumor progression and treatment. Here we show that DNA hypermethylation at the ASNS promoter prevents its transcriptional expression following asparagine depletion. Insufficient expression of ASNS leads to asparagine deficiency, which facilitates ATF4-independent induction of CCAAT-enhancer-binding protein homologous protein (CHOP), which triggers apoptosis. We conclude that chromatin accessibility is critical for ATF4 activity at the ASNS promoter, which can switch ALL cells from an ATF4-dependent adaptive response to ATF4-independent apoptosis during asparagine depletion. This work may also help explain why ALL cells are most sensitive to l-asparaginase treatment compared with other cancers.
Ribosome Elongation Stall Directs Gene-specific Translation in the Integrated Stress Response
(American Society for Biochemistry and Molecular Biology, 2016-03-18) Young, Sara K.; Palam, Lakshmi Reddy; Wu, Cheng; Sachs, Matthew S.; Wek, Ronald C.; Department of Biochemistry & Molecular Biology, IU School of Medicine
Upon exposure to environmental stress, phosphorylation of the α subunit of eIF2 (eIF2α-P) represses global protein synthesis, coincident with preferential translation of gene transcripts that mitigate stress damage or alternatively trigger apoptosis. Because there are multiple mammalian eIF2 kinases, each responding to different stress arrangements, this translational control scheme is referred to as the integrated stress response (ISR). Included among the preferentially translated mRNAs induced by eIF2α-P is that encoding the transcription factor CHOP (DDIT3/GADD153). Enhanced levels of CHOP promote cell death when ISR signaling is insufficient to restore cell homeostasis. Preferential translation of CHOP mRNA occurs by a mechanism involving ribosome bypass of an inhibitory upstream ORF (uORF) situated in the 5'-leader of the CHOP mRNA. In this study, we used biochemical and genetic approaches to define the inhibitory features of the CHOP uORF and the biological consequences of loss of the CHOP uORF on CHOP expression during stress. We discovered that specific sequences within the CHOP uORF serve to stall elongating ribosomes and prevent ribosome reinitiation at the downstream CHOP coding sequence. As a consequence, deletion of the CHOP uORF substantially increases the levels and modifies the pattern of induction of CHOP expression in the ISR. Enhanced CHOP expression leads to increased expression of key CHOP target genes, culminating in increased cell death in response to stress.
TgIF2K-B Is an eIF2α Kinase in Toxoplasma gondii That Responds to Oxidative Stress and Optimizes Pathogenicity
(American Society for Microbiology, 2021-01-26) Augusto, Leonardo; Martynowicz, Jennifer; Amin, Parth H.; Carlson, Kenneth R.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not yet clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eukaryotic initiation factor 2 (eIF2). Here, we show that the Toxoplasma eIF2 kinase TgIF2K-B is activated in response to oxidative stress and affords protection. Knockout of the TgIF2K-B gene, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite's ability to establish persistent infection in its host.IMPORTANCE Toxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite's oxidative stress response and its ability to persist in the host as a latent infection.

Browsing by Subject "Stress response"

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