Cellular Pool Research Articles

Imatinib and Dasatinib Provoke Mitochondrial Dysfunction Leading to Oxidative Stress in C2C12 Myotubes and Human RD Cells

Tyrosine kinase inhibitors (TKIs) can cause skeletal muscle toxicity in patients, but the underlying mechanisms are mostly unclear. The goal of the current study was to better characterize the role of mitochondria in TKI-associated myotoxicity. We exposed C2C12 murine myoblasts and myotubes as well as human rhabdomyosarcoma cells (RD cells) for 24 h to imatinib (1–100 µM), erlotinib (1–20 µM), and dasatinib (0.001–100 µM). In C2C12 myoblasts, imatinib was membrane toxic at 50 µM and depleted the cellular ATP pool at 20 µM. In C2C12 myotubes exposed to imatinib, ATP depletion started at 50 µM whereas membrane toxicity was not detectable. In myoblasts and myotubes exposed to dasatinib, membrane toxicity started at 0.5 µM and 2 µM, respectively, and the ATP drop was visible at 0.1 µM and 0.2 µM, respectively. When RD cells were exposed to imatinib, ATP depletion started at 20 µM whereas membrane toxicity was not detectable. Dasatinib was membrane toxic at 20 µM and depleted the cellular ATP pool already at 0.5 µM. Erlotinib was not toxic in both cell models. Imatinib (20 µM) and dasatinib (1 µM) reduced complex I activity in both cell models. Moreover, the mitochondrial membrane potential (Δψm) was dissipated for both TKIs in myotubes. In RD cells, the Δψm was reduced only by dasatinib. Both TKIs increased mitochondrial superoxide accumulation and decreased the mitochondrial copy number in both cell lines. In consequence, they increased protein expression of superoxide dismutase (SOD) 2 and thioredoxin 2 and cleavage of caspase 3, indicating apoptosis in C2C12 myotubes. Moreover, in both cell models, the mRNA expression of Sod1 and Sod2 increased when RD cells were exposed to dasatinib. Furthermore, dasatinib increased the mRNA expression of atrogin-1 and murf-1, which are important transcription factors involved in muscle atrophy. The mRNA expression of atrogin-1 increased also in RD cells exposed to imatinib. In conclusion, imatinib and dasatinib are mitochondrial toxicants in mouse C2C12 myotubes and human RD cells. Mitochondrial superoxide accumulation induced by these two TKIs is due to the inhibition of complex I and is probably related to impaired mitochondrial and myocyte proliferation.

Antagonism between splicing and microprocessor complex dictates the serum-induced processing of lnc-MIRHG for efficient cell cycle reentry

Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG. Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG. Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.

The Pentose Phosphate Pathway Dynamics in Cancer and Its Dependency on Intracellular pH.

The Pentose Phosphate Pathway (PPP) is one of the key metabolic pathways occurring in living cells to produce energy and maintain cellular homeostasis. Cancer cells have higher cytoplasmic utilization of glucose (glycolysis), even in the presence of oxygen; this is known as the “Warburg Effect”. However, cytoplasmic glucose utilization can also occur in cancer through the PPP. This pathway contributes to cancer cells by operating in many different ways: (i) as a defense mechanism via the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to prevent apoptosis, (ii) as a provision for the maintenance of energy by intermediate glycolysis, (iii) by increasing genomic material to the cellular pool of nucleic acid bases, (iv) by promoting survival through increasing glycolysis, and so increasing acid production, and (v) by inducing cellular proliferation by the synthesis of nucleic acid, fatty acid, and amino acid. Each step of the PPP can be upregulated in some types of cancer but not in others. An interesting aspect of this metabolic pathway is the shared regulation of the glycolytic and PPP pathways by intracellular pH (pHi). Indeed, as with glycolysis, the optimum activity of the enzymes driving the PPP occurs at an alkaline pHi, which is compatible with the cytoplasmic pH of cancer cells. Here, we outline each step of the PPP and discuss its possible correlation with cancer.

Pollen-Specific Protein PSP231 Activates Callose Synthesis to Govern Male Gametogenesis and Pollen Germination.

Spatiotemporally regulated callose deposition is an essential, genetically programmed phenomenon that promotes pollen development and functionality. Severe male infertility is associated with deficient callose biosynthesis, highlighting the significance of intact callose deposition in male gametogenesis. The molecular mechanism that regulates the crucial role of callose in production of functional male gametophytes remains completely unexplored. Here, we provide evidence that the gradual upregulation of a previously uncharacterized cotton (Gossypium hirsutum) pollen-specific SKS-like protein (PSP231), specifically at the post pollen-mitosis stage, activates callose biosynthesis to promote pollen maturation. Aberrant PSP231 expression levels caused by either silencing or overexpression resulted in late pollen developmental abnormalities and male infertility phenotypes in a dose-dependent manner, highlighting the importance of fine-tuned PSP231 expression. Mechanistic analyses revealed that PSP231 plays a central role in triggering and fine-tuning the callose synthesis and deposition required for pollen development. Specifically, PSP231 protein sequesters the cellular pool of RNA-binding protein GhRBPL1 to destabilize GhWRKY15 mRNAs, turning off GhWRKY15-mediated transcriptional repression of GhCalS4/GhCalS8 and thus activating callose biosynthesis in pollen. This study showed that PSP231 is a key molecular switch that activates the molecular circuit controlling callose deposition toward pollen maturation and functionality and thereby safeguards agricultural crops against male infertility.

Cluster exchange reactivity of [2Fe-2S]-bridged heterodimeric BOLA1-GLRX5.

Mitochondrial BOLA1 is known to form a [2Fe-2S] cluster-bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe-4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1-GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3-GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe-2S]-bridged BOLA1-GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe-2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo-formation following delivery from [2Fe-2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe-2S] BOLA1-GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe-2S]-bridged BOLA1 homodimer (lacking a partner GLRX). While the holo-heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity.

Chemical Priming by Isothiocyanates Protects Against Intoxication by Products of the Mustard Oil Bomb.

In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.

SAMHD1-mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

Extremely-low frequency magnetic field exposure for simulating geomagnetic pulsations in Alexandrium pacificum and Gymnodinium catenatum cultures

Growth and chain formation in cultures of the chain-forming dinoflagellates Alexandrium pacificum and Gymnodinium catenatum were previously found to be susceptible to space weather variables. A clock drive was used to deliver a frequency of 0.5 Hz and central amplitude of 7 µT in order to perform in vitro simulation of geomagnetic pulsations (composed of extremely low-frequency magnetic fields, ELFMF) which occur during high geomagnetic activity (GMA) periods. Short-term exposure (hours) to this ELFMF increased relative cell growth around 10 nT of naturally occurring GMA. Relative growth outside these intervals gradually approached 0% or was negative for G. catenatum. Differential survival to a subsequent shock was inversely related to growth, and minimal survival coincided with the same 10 nT interval. Relative growth and survival displayed opposite hormetic curves towards GMA: inverted U-shaped for growth, and J-shaped for survival. After exposure to this ELFMF, positive phototaxis response was not lost, but the percentage of cells swimming was slightly reduced. Long-term exposure (days) increased relative growth in A. pacificum but reduced in G. catenatum when low GMA was taking place. These alterations in growth were both associated with a reduction in the cellular pool of mycosporine-like amino acids (MAAs). MAAs that are more susceptible to oxidation were more reduced than those resistant, highlighting that an ELFMF can act by increasing cellular oxidative stress status. The higher susceptibility of G. catenatum found is in compliance with the previous association of its natural populations at the western Iberia coast with periods of solar activity minima and GMA minima.

590-P: Modulation of Adenine Phosphoribosyltransferase-Mediated Purine Salvage to Accelerate Diabetic Wound Healing

Aim: Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. Our aim was to evaluate the role of APRT in diabetic wound healing. Methods: The molecular mechanism of APRT-mediated cell survival was validated by the haploid HAP1 cell. The adenylate nucleotides in NIH-3T3 fibroblast response to adenine was monitoring by HPLC-MS/MS method. The effects of cell behaviors were evaluated by mice NIH-3T3 fibroblast and human epithelial keratinocytes. Genetically diabetic mice received surgery for full-thickness of the cutaneous wound and the adenine addition as the substrate for APRT on the wound. Results: In the wild type and APRT knockout HAP1 cell, our data revealed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. The time-course of monitoring the total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. In mice NIH-3T3 fibroblasts and human NHEK keratinocytes, adenine elevated cellular ATP levels in a dose-dependent manner and facilitated cell migration. The up-regulated APRT found in wound skin was correlated with the demands of wound repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. Conclusions: Our data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. This study provided an explanation for the up-regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation. Disclosure G. Young: Employee; Self; Energenesis-Biomedical. J. Lin: Employee; Self; Energenesis-Biomedical. Y. Cheng: Employee; Self; Energenesis-Biomedical. H. Chen: Board Member; Self; Energenesis-Biomedical.

Decreased, Deformed, Defective-How HIV-1 Vpu Targets Peroxisomes.

Peroxisomes are found in essentially all eukaryotic cells and have been described as important hubs in innate sensing and the induction of type III interferons upon viral infection. Nevertheless, it remains poorly investigated how viral pathogens modulate biogenesis or function of peroxisomes to evade innate sensing and restriction. In a recent study, Hobman and colleagues found that the accessory viral protein u (Vpu) of HIV-1 inhibits peroxisome activity by depleting cellular peroxisome pools. This depletion could be ascribed to a Vpu-dependent induction of four microRNAs (miRNAs) that suppress the expression of peroxisomal biogenesis factors PEX2, PEX7, PEX11B, and PEX13. Although the downstream effects on antiretroviral gene expression and HIV-1 replication remain to be determined, these findings provide important insights into peroxisome biogenesis and the modulation of cell organelles by HIV-1 Vpu.

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

SummaryGenome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

Medicinal Thiols: Current Status and New Perspectives.

The thiol (-SH) functional group is found in a number of drug compounds and confers a unique combination of useful properties. Thiol-containing drugs can reduce radicals and other toxic electrophiles, restore cellular thiol pools, and form stable complexes with heavy metals such as lead, arsenic, and copper. Thus, thiols can treat a variety of conditions by serving as radical scavengers, GSH prodrugs, or metal chelators. Many of the compounds discussed here have been in use for decades, yet continued exploration of their properties has yielded new understanding in recent years, which can be used to optimize their clinical application and provide insights into the development of new treatments. The purpose of this narrative review is to highlight the biochemistry of currently used thiol drugs within the context of developments reported in the last five years. More specifically, this review focuses on thiol drugs that represent the standard of care for their associated conditions, including N-acetylcysteine, 2,3-meso-dimercaptosuccinic acid, British anti-Lewisite, D-penicillamine, amifostine, and others. Reports of novel dosing regimens, delivery strategies, and clinical applications for these compounds were examined with an eye toward emerging approaches to address a wide range of medical conditions in the future.

Abstract B37: Development of inhibitors of the activated form of KRAS G12C

Abstract Activating mutations in RAS proteins occur in ~1/3 of human cancers. These mutations impair the ability of the protein to hydrolyze GTP to GDP. As a result, mutant RAS proteins exist predominantly in the GTP-bound state, which directly activates aberrant downstream signaling via interaction with effectors such as RAF. Most RAS mutations occur at glycine 12 of the KRAS isoform. One such mutation, KRAS G12C, is particularly common in non-small cell lung cancer where it is found in ~15% of lung adenocarcinomas. Recent efforts have targeted KRAS G12C in the GDP-bound state; however, direct pharmacologic inhibition of active, GTP-bound KRAS G12C has proved challenging. Here, we deployed a novel SMARTTM (Small Molecule Assisted Receptor Targeting) platform to advance covalent compounds that selectively inhibit GTP-bound KRAS G12C. Using a mechanism reminiscent of the natural products rapamycin and cyclosporine, these compounds promote formation of a novel inhibitory ternary complex consisting of cyclophilin A (CypA, an abundant immunophilin present in all human cells), the SMART inhibitor, and GTP-KRAS G12C. Structure-based design of the SMART inhibitor yielded potent covalent inhibitors of GTP-KRAS G12C that exhibit >100-fold selectivity for mutant KRAS G12C over WT KRAS. Structural analysis of the ternary complex revealed that the covalent linkage between the SMART inhibitor and the mutant cysteine of KRAS occurred in the context of extensive interactions between CypA, the SMART inhibitor, and GTP-KRAS G12C that provide significant binding affinity (KI = 2.5 μM). The GTP-KRAS G12C|Inhibitor|CypA complex directly occluded effector binding, and as such, the compounds disrupted the KRAS-RAF interaction in biochemical assays. This activity was dependent on CypA, underlining the importance of the KRAS G12C| CypA protein-protein interaction in driving target engagement. In cell-based studies, SMART inhibitors crosslinked KRAS G12C and potently inhibited ERK phosphorylation and cell growth in G12C mutant tumor cell lines but had no effect on non-G12C bearing tumor cells. CRISPR knockout of cellular CypA confirmed that these activities were dependent on the presence of endogenous CypA. Importantly, SMART inhibitors bind directly to active, GTP-KRAS G12C and thus, their activity does not rely on trapping KRAS G12C in the inactive GDP-bound state. As a result, the cellular potency of SMART inhibitors with respect to crosslinking, pERK inhibition, and growth inhibition was maintained in the presence of growth factor treatments that reduce the cellular GDP-KRAS G12C pool. In contrast, we found that the activity of a previously described GDP-KRAS G12C targeting inhibitor was attenuated by growth factor treatment. To our knowledge, these are the first examples of mutant-selective KRAS inhibitors that target the active, GTP-bound state of KRAS G12C. We are currently optimizing the drug-like properties of these SMART inhibitors and evaluating their activity in in vivo models. Citation Format: Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Siminia Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Cindy C. Benod, Alan S. Mann, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Roy M. Pollock, Meizhong Jin. Development of inhibitors of the activated form of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B37.

Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions

The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHOD), and the F1F0-ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.

A widespread toxin−antitoxin system exploiting growth control via alarmone signaling

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

Signs of a metabolon in action

Biosynthetic Enzymes Eukaryotic cells have a heterogeneous cytoplasm, with compartments large and small, membrane bound or not. Enzymes that catalyze the de novo synthesis of purine nucleotides, which are needed in rapidly dividing cells, are known to assemble into loosely associated, multienzyme structures called purinosomes, but the extent to which these structures are metabolically active has been less certain. Pareek et al. performed metabolomics to trace how purines are synthesized within purinosomes and used sophisticated mass spectrometry imaging to directly observe hotspots of metabolic activity within frozen HeLa cells (see the Perspective by Alexandrov). They found evidence for metabolic channeling between enzymes, which limits equilibration of intermediates formed in purinosomes with the bulk cellular metabolite pool. This process occurs specifically within purinosomes associated with mitochondria, because the input metabolites, glycine, aspartate, and formate, come from mitochondrial metabolism. Such channeling may help cells control the ratio and abundance of purine nucleotides. Science , this issue p. [283][1]; see also p. [241][2] [1]: /lookup/doi/10.1126/science.aaz6465 [2]: /lookup/doi/10.1126/science.abb3094

Short Communication: Elevated Labile Iron Levels in CD4 and CD8 T Cells from HIV-Positive Individuals with Undetectable Viral Load.

Iron is a key factor at various stages of HIV life cycle and determines the progression of HIV infection. Data about cellular labile iron pool (LIP) in the settings of contemporary antiretroviral therapy (cART) are lacking. Yet LIP is directly related to the generation of reactive oxygen species, and may contribute to immune activation, dysfunction, and exhaustion. Using multiparameter flow cytometry, we evaluated LIP in CD4 and CD8 T cells from HIV+ patients with sustained viral suppression (SVS) as a result of continuous long-term cART. Based on the recovery of CD4/CD8 ratio, two patients' subgroups were defined: A (n = 26), CD4/CD8 > 0.9, and B (n = 37), CD4/CD8 < 0.9, with significantly differing CD4 absolute count (AC) (mean 752 vs. 571 cells/μL, p < .05). Although hemoglobin and serum iron had recovered in all patients, CD4 T cell LIP and CD8 T cell LIP were significantly higher than that of controls, both in the subgroup with complete (A) and with incomplete (B) immune recovery [mean CD4 mean fluorescence intensity (ΔMFI) 318.7 and 777.8 vs. 157.6; mean CD8 ΔMFI 359.5 and 628.7 vs. 179.2, analysis of variance p < .05 for both]. CD4 LIP correlated inversely with CD4 AC (R = -0.4, p < .01), and both CD4 LIP and CD8 LIP-with CD4/CD8 ratio (R = -0.4, p < .01). Thus, increased CD4 T cell LIP and CD8 T cell LIP in the settings of SVS and immune recovery are a sensitive marker of residual immune activation and may predict immune exhaustion in long-term cART-treated patients.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.