The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.

Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency, which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiolcleavable biotin-assisted TurboID proteomics achieved remarkable intraorganelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

Enumeration and phenotypic profiling of circulating tumor cells (CTCs) can give important information about tumor progression, presence of therapeutic targets, and metastatic potential. New and informative cancer-specific biomarkers are being discovered at a rapid pace, so there is a strong need for tools that enable investigator driven assays to best study and utilize these biomarkers. Through the RareCyte platform, we provide sensitive and specific assays that are optimized and validated for CTC enumeration and biomarker expression. RarePlex Developer Kits enable the addition of user-selected antibodies against biomarkers of interest to a CTC detection assay. Here we demonstrate the application of RarePlex Developer Kits to study the presence of a variety of cancer related biomarkers. Using the Developer strategy, we present results for several biomarkers, including HER2, ER, PR, EGFR, Ki67, AR, ARv7, PDL1, and PSMA. We also characterized clinical samples from prostate (AR and ARv7) and breast (HER2 and ER) cancer patients. The biomarkers demonstrated proper localization on or within model CTC control cells when using default antigen retrieval and fixation conditions. For each biomarker, fluorescence intensity cut-offs that segregated negative and positive cell lines were statistically defined to maximize classification accuracy. For clinical samples, breast and prostate cancer sample staining showed expected localization based on available clinical information. In conclusion, RarePlex Developer Kits provide a flexible tool for custom CTC assay development that enables researchers to develop assays in their own lab for characterization of phenotypic heterogeneity. Citation Format: Edward Lo, Daniel Campton, Arturo Ramirez, Lillian Costandy, Brady Gardner, Ryan Houston, Heather Itamoto, Jeffery L. Werbin, VK Gadi, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel E. Sabath, Eric P. Kaldjian, Tad George. Investigation of custom biomarkers on circulating tumor cells from clinical samples using RarePlex® Developer Kits [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6442.

The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs. Various stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programmes. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.

Abstract Circulating tumor cells (CTCs) can provide information on drug target expression, response to therapy, and disease prognosis from a non-invasive blood draw. Currently, investigating biomarkers on CTCs is difficult due to challenges of developing multiplexed assays that also identify rare cells. Presence of the androgen receptor splice variant ARv7 in prostate cancer cells is associated with resistance to second generation anti-androgen therapies. We report here the analytical validation of an immunofluorescence assay for characterization of ARv7 protein expression on CTCs using the RareCyte platform - an end-to-end platform that combines CTC sample preparation, multiparameter fluorescence staining, digital imaging, and single cell retrieval. Blood samples spiked with positive and negative cell lines for ARv7 expression were processed using the AccuCyte Sample Preparation System. Slides were auto-stained by immunofluorescence with the RarePlex ARv7 CTC Panel Kit comprised of a three-channel CTC detection base plus an ARv7 biomarker channel. The detection base consists of a nuclear dye, anti-CD45 antibody to exclude white blood cells, and cocktailed antibodies to cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM). Stained slides were imaged with the CyteFinder Instrument. CTCs were identified using machine learning-based algorithms and confirmed by user review. Mean fluorescence intensity (MFI) measurements were used as a metric for ARv7 expression on confirmed CTCs. Analytic validation studies of the AR-V7 CTC assay were performed using 22RV1 (high), LNCaP (low), and BT-474 (negative) cell lines. Performance characteristics tested for ARv7 included accuracy, sensitivity, specificity, repeatability, and inter-stainer run coefficient of variation. Performance metrics for CTC recovery were calculated on spike-in and clinical samples. For recovery calculations, the number of CTCs found with the ARv7 assay was compared to the number of CTCs found with the CTC detection base assay. An ARv7 MFI threshold that segregated negative and positive cell lines was statistically defined. This threshold identified 80% of 22RV1 cells as positive for ARv7, 97% of BT-474 cells as negative, with an overall accuracy of 90%. When the assay was applied to clinical prostate cancer samples, staining with proper nuclear localization was observed. CTC recovery was at least as high with the ARv7 assay as with the base CTC detection assay. Citation Format: Daniel Campton, Edward Lo, Lillian Costandy, Brady Gardner, Ryan Houston, Jeffery L. Werbin, Kyla Teplitz, Daniel E. Sabath, Alisa C. Clein, Celestia S. Higano, Tanisha M. Mojica, Kristin Province, Eric P. Kaldjian, Arturo B. Ramirez, Tad George. Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5384.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.