Monther Abu-Remaileh

All Publications

• Cell-Type Resolved Protein Atlas of Brain Lysosomes Identifies SLC45A1-Associated Disease as a Lysosomal Disorder. bioRxiv : the preprint server for biology Ghoochani, A., Heiby, J. C., Rawat, E. S., Medoh, U. N., Di Fraia, D., Dong, W., Gastou, M., Nyame, K., Laqtom, N. N., Gomez-Ospina, N., Ori, A., Abu-Remaileh, M. 2024

  Abstract

  Mutations in lysosomal genes cause neurodegeneration and neurological lysosomal storage disorders (LSDs). Despite their essential role in brain homeostasis, the cell-type-specific composition and function of lysosomes remain poorly understood. Here, we report a quantitative protein atlas of the lysosome from mouse neurons, astrocytes, oligodendrocytes, and microglia. We identify dozens of novel lysosomal proteins and reveal the diversity of the lysosomal composition across brain cell types. Notably, we discovered SLC45A1, mutations in which cause a monogenic neurological disease, as a neuron-specific lysosomal protein. Loss of SLC45A1 causes lysosomal dysfunction in vitro and in vivo. Mechanistically, SLC45A1 plays a dual role in lysosomal sugar transport and stabilization of V1 subunits of the V-ATPase. SLC45A1 deficiency depletes the V1 subunits, elevates lysosomal pH, and disrupts iron homeostasis causing mitochondrial dysfunction. Altogether, our work redefines SLC45A1-associated disease as a LSD and establishes a comprehensive map to study lysosome biology at cell-type resolution in the brain and its implications for neurodegeneration.

  View details for DOI 10.1101/2024.10.14.618295

  View details for PubMedID 39464040

  View details for PubMedCentralID PMC11507716

• The Bis(monoacylglycero)-phosphate Hypothesis: From Lysosomal Function to Therapeutic Avenues. Annual review of biochemistry Medoh, U. N., Abu-Remaileh, M. 2024

  Abstract

  Lysosomes catabolize and recycle lipids and other biological molecules to maintain cellular homeostasis in diverse nutrient environments. Lysosomal lipid catabolism relies on the stimulatory activity of bis(monoacylglycero)phosphate (BMP), an enigmatic lipid whose levels are altered across myriad lysosome-associated diseases. Here, we review the discovery of BMP over half a century ago and its structural properties that facilitate the activation of lipid hydrolases and recruitment of their coactivators. We further discuss the current, yet incomplete, understanding of BMP catabolism and anabolism. To conclude, we discuss its role in lysosome-associated diseases and the potential for modulating its levels by pharmacologically activating and inhibiting the BMP synthase to therapeutically target lysosomal storage disorders, drug-induced phospholipidosis, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, cancer, and viral infection.

  View details for DOI 10.1146/annurev-biochem-092823-113814

  View details for PubMedID 38603559

• APOE4/4 is linked to damaging lipid droplets in Alzheimer's diseasemicroglia. Nature Haney, M. S., Palovics, R., Munson, C. N., Long, C., Johansson, P. K., Yip, O., Dong, W., Rawat, E., West, E., Schlachetzki, J. C., Tsai, A., Guldner, I. H., Lamichhane, B. S., Smith, A., Schaum, N., Calcuttawala, K., Shin, A., Wang, Y., Wang, C., Koutsodendris, N., Serrano, G. E., Beach, T. G., Reiman, E. M., Glass, C. K., Abu-Remaileh, M., Enejder, A., Huang, Y., Wyss-Coray, T. 2024

  Abstract

  Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Abeta induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

  View details for DOI 10.1038/s41586-024-07185-7

  View details for PubMedID 38480892

• Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease. Molecular cell Nyame, K., Hims, A., Aburous, A., Laqtom, N. N., Dong, W., Medoh, U. N., Heiby, J. C., Xiong, J., Ori, A., Abu-Remaileh, M. 2024

  Abstract

  Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of thesephospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.

  View details for DOI 10.1016/j.molcel.2024.02.006

  View details for PubMedID 38447580

• The Batten disease gene product CLN5 is the lysosomal bis(monoacylglycero)phosphate synthase. Science (New York, N.Y.) Medoh, U. N., Hims, A., Chen, J. Y., Ghoochani, A., Nyame, K., Dong, W., Abu-Remaileh, M. 2023; 381 (6663): 1182-1189

  Abstract

  Lysosomes critically rely on bis(monoacylglycero)phosphate (BMP) to stimulate lipid catabolism, cholesterol homeostasis, and lysosomal function. Alterations in BMP levels in monogenic and complex neurodegeneration suggest an essential function in human health. However, the site and mechanism responsible for BMP synthesis have been subject to debate for decades. Here, we report that the Batten disease gene product CLN5 is the elusive BMP synthase (BMPS). BMPS-deficient cells exhibited a massive accumulation of the BMP synthesis precursor lysophosphatidylglycerol (LPG), depletion of BMP species, and dysfunctional lipid metabolism. Mechanistically, we found that BMPS mediated synthesis through an energy-independent base exchange reaction between two LPG molecules with increased activity on BMP-laden vesicles. Our study elucidates BMP biosynthesis and reveals an anabolic function of late endosomes/lysosomes.

  View details for DOI 10.1126/science.adg9288

  View details for PubMedID 37708259

• Golgi-IP, a tool for multimodal analysis of Golgi molecular content. Proceedings of the National Academy of Sciences of the United States of America Fasimoye, R., Dong, W., Nirujogi, R. S., Rawat, E. S., Iguchi, M., Nyame, K., Phung, T. K., Bagnoli, E., Prescott, A. R., Alessi, D. R., Abu-Remaileh, M. 2023; 120 (20): e2219953120

  Abstract

  The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signaling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid Golgi immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi-resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome, and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified proteins not previously associated with the Golgi. Metabolite profiling established the human Golgi metabolome and revealed the enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol, and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease.

  View details for DOI 10.1073/pnas.2219953120

  View details for PubMedID 37155866

• An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation. Science advances Scharenberg, S. G., Dong, W., Ghoochani, A., Nyame, K., Levin-Konigsberg, R., Krishnan, A. R., Rawat, E. S., Spees, K., Bassik, M. C., Abu-Remaileh, M. 2023; 9 (16): eadf8966

  Abstract

  Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.

  View details for DOI 10.1126/sciadv.adf8966

  View details for PubMedID 37075117

  View details for PubMedCentralID PMC10115416

• Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein. Cell chemical biology Armenta, D. A., Laqtom, N. N., Alchemy, G., Dong, W., Morrow, D., Poltorack, C. D., Nathanson, D. A., Abu-Remalieh, M., Dixon, S. J. 2022

  Abstract

  Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.

  View details for DOI 10.1016/j.chembiol.2022.10.006

  View details for PubMedID 36306785

• CLN3 is required for the clearance of glycerophosphodiesters from lysosomes. Nature Laqtom, N. N., Dong, W., Medoh, U. N., Cangelosi, A. L., Dharamdasani, V., Chan, S. H., Kunchok, T., Lewis, C. A., Heinze, I., Tang, R., Grimm, C., Dang Do, A. N., Porter, F. D., Ori, A., Sabatini, D. M., Abu-Remaileh, M. 2022

  Abstract

  Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.

  View details for DOI 10.1038/s41586-022-05221-y

  View details for PubMedID 36131016

• Isotope tracing in health and disease. Current opinion in biotechnology Dong, W., Rawat, E. S., Stephanopoulos, G., Abu-Remaileh, M. 2022; 76: 102739

  Abstract

  Biochemical characterization of metabolism provides molecular insights for understanding biology in health and disease. Over the past decades, metabolic perturbations have been implicated in cancer, neurodegeneration, and diabetes, among others. Isotope tracing is a technique that allows tracking of labeled atoms within metabolites through biochemical reactions. This technique has become an integral component of the contemporary metabolic research. Isotope tracing measures substrate contribution to downstream metabolites and indicates its utilization in cellular metabolic networks. In addition, isotopic labeling data are necessary for quantitative metabolicflux analysis. Here, we review recent work utilizing metabolic tracing to study health and disease, and highlight its application to interrogate subcellular, intercellular, and in vivo metabolism. We further discuss the current challenges and opportunities to expand the utility of isotope tracing to new research areas.

  View details for DOI 10.1016/j.copbio.2022.102739

  View details for PubMedID 35738210

• Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes. Immunity Wogram, E., Sümpelmann, F., Dong, W., Rawat, E., Fernández Maestre, I., Fu, D., Braswell, B., Khalil, A., Buescher, J. M., Mittler, G., Borner, G. H., Vlachos, A., Tholen, S., Schilling, O., Bell, G. W., Rambold, A. S., Akhtar, A., Schnell, O., Beck, J., Abu-Remaileh, M., Prinz, M., Jaenisch, R. 2024

  Abstract

  Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.

  View details for DOI 10.1016/j.immuni.2024.07.019

  View details for PubMedID 39151426

• CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice. Nature communications Colella, P., Sayana, R., Suarez-Nieto, M. V., Sarno, J., Nyame, K., Xiong, J., Pimentel Vera, L. N., Arozqueta Basurto, J., Corbo, M., Limaye, A., Davis, K. L., Abu-Remaileh, M., Gomez-Ospina, N. 2024; 15 (1): 5654

  Abstract

  Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

  View details for DOI 10.1038/s41467-024-49908-4

  View details for PubMedID 38969669

  View details for PubMedCentralID PMC11226701

• MFSD1 with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes. Nature cell biology Jungnickel, K. E., Guelle, O., Iguchi, M., Dong, W., Kotov, V., Gabriel, F., Debacker, C., Dairou, J., McCort-Tranchepain, I., Laqtom, N. N., Chan, S. H., Ejima, A., Sato, K., Massa López, D., Saftig, P., Mehdipour, A. R., Abu-Remaileh, M., Gasnier, B., Löw, C., Damme, M. 2024

  Abstract

  The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.

  View details for DOI 10.1038/s41556-024-01436-5

  View details for PubMedID 38839979

  View details for PubMedCentralID 5560103

• Sharing is caring: TMEM165 a Golgi calcium importer used by the lysosome. Trends in biochemical sciences Murchison, A. K., Abu-Remaileh, M. 2024

  Abstract

  Calcium is a crucial second messenger in the cell that is stored in organelles including lysosomes. Proteins that facilitate calcium entry to the lysosome were unknown. A recent report by Zajac et al. identified TMEM165 as a proton-activated calcium importer on the lysosome, thus discovering a key player in subcellular calcium homeostasis.

  View details for DOI 10.1016/j.tibs.2024.05.006

  View details for PubMedID 38816278

• Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity. eLife Yu, Y., Gao, S. M., Guan, Y., Hu, P. W., Zhang, Q., Liu, J., Jing, B., Zhao, Q., Sabatini, D. M., Abu-Remaileh, M., Jung, S. Y., Wang, M. C. 2024; 13

  Abstract

  Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMPK and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk and organism longevity.

  View details for DOI 10.7554/eLife.85214

  View details for PubMedID 38240316

• CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency. Research square Colella, P., Sayana, R., Suarez-Nieto, M. V., Sarno, J., Nyame, K., Xiong, J., Vera, L. N., Basurto, J. A., Corbo, M., Limaye, A., Davis, K. L., Abu-Remaileh, M., Gomez-Ospina, N. 2023

  Abstract

  Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.

  View details for DOI 10.21203/rs.3.rs-3263412/v1

  View details for PubMedID 37790525

  View details for PubMedCentralID PMC10543302

• APOE4/4 is linked to damaging lipid droplets in Alzheimer's microglia. bioRxiv : the preprint server for biology Haney, M. S., Pálovics, R., Munson, C. N., Long, C., Johansson, P., Yip, O., Dong, W., Rawat, E., West, E., Schlachetzki, J. C., Tsai, A., Guldner, I. H., Lamichhane, B. S., Smith, A., Schaum, N., Calcuttawala, K., Shin, A., Wang, Y. H., Wang, C., Koutsodendris, N., Serrano, G. E., Beach, T. G., Reiman, E. M., Glass, C. K., Abu-Remaileh, M., Enejder, A., Huang, Y., Wyss-Coray, T. 2023

  Abstract

  Several genetic risk factors for Alzheimer's Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aβ (fAβ) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.

  View details for DOI 10.1101/2023.07.21.549930

  View details for PubMedID 37546938

  View details for PubMedCentralID PMC10401952

• CYP4F2 is a human-specific determinant of circulating N-acyl amino acid levels. The Journal of biological chemistry Tanzo, J. T., Li, V. L., Wiggenhorn, A. L., Moya-Garzon, M. D., Wei, W., Lyu, X., Dong, W., Tahir, U. A., Chen, Z. Z., Cruz, D. E., Deng, S., Shi, X., Zheng, S., Guo, Y., Sims, M., Abu-Remaileh, M., Wilson, J. G., Gerszten, R. E., Long, J. Z., Benson, M. D. 2023: 104764

  Abstract

  N-acyl amino acids are a large family of circulating lipid metabolites that modulate energy expenditure and fat mass in rodents. However, little is known about the regulation and potential cardiometabolic functions of N-acyl amino acids in humans. Here, we analyze the cardiometabolic phenotype associations and genomic associations of four plasma N-acyl amino acids (N-oleoyl-leucine, N-oleoyl-phenylalanine, N-oleoyl-serine, and N-oleoyl-glycine) in 2,351 individuals from the Jackson Heart Study. We find that plasma levels of specific N-acyl amino acids are associated with cardiometabolic disease endpoints independent of free amino acid plasma levels and in patterns according to the amino acid head group. By integrating whole genome sequencing data with N-acyl amino acid levels, we identify that the genetic determinants of N-acyl amino acid levels also cluster according to amino acid head group. Furthermore, we identify the CYP4F2 locus as a genetic determinant of plasma N-oleoyl-leucine and N-oleoyl-phenylalanine levels in human plasma. In experimental studies, we demonstrate that CYP4F2-mediated hydroxylation of N-oleoyl-leucine and N-oleoyl-phenylalanine results in metabolic diversification and production of many previously unknown lipid metabolites with varying characteristics of the fatty acid tail group, including several that structurally resemble fatty acid hydroxy fatty acids (FAHFAs). These studies provide a structural framework for understanding the regulation and disease-associations of N-acyl amino acids in humans and identify that the diversity of this lipid signaling family can be significantly expanded through CYP4F-mediated ω-hydroxylation.

  View details for DOI 10.1016/j.jbc.2023.104764

  View details for PubMedID 37121548

• Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells. The Journal of biological chemistry Hsu, J., Fatuzzo, N., Weng, N., Michno, W., Dong, W., Kienle, M., Dai, Y., Pasca, A., Abu-Remaileh, M., Rasgon, N., Bigio, B., Nasca, C., Khosla, C. 2022: 102848

  Abstract

  In eukaryotes carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for post-translational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did β-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic post-translational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase (CRAT) is believed to catalyze acetyl group transfer from LAC to Coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase (CROT) as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

  View details for DOI 10.1016/j.jbc.2022.102848

  View details for PubMedID 36587768

• R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response. Nature Crossley, M. P., Song, C., Bocek, M. J., Choi, J., Kousorous, J., Sathirachinda, A., Lin, C., Brickner, J. R., Bai, G., Lans, H., Vermeulen, W., Abu-Remaileh, M., Cimprich, K. A. 2022

  Abstract

  R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1(refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the patternrecognition receptors cGAS and TLR3(ref.8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2(ref.9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.

  View details for DOI 10.1038/s41586-022-05545-9

  View details for PubMedID 36544021

• Lysosomal cathepsin D mediates endogenous mucin glycodomain catabolism in mammals. Proceedings of the National Academy of Sciences of the United States of America Pedram, K., Laqtom, N. N., Shon, D. J., Di Spiezio, A., Riley, N. M., Saftig, P., Abu-Remaileh, M., Bertozzi, C. R. 2022; 119 (39): e2117105119

  Abstract

  Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O-glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.

  View details for DOI 10.1073/pnas.2117105119

  View details for PubMedID 36122205

• Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration. Proceedings of the National Academy of Sciences of the United States of America Vest, R. T., Chou, C. C., Zhang, H., Haney, M. S., Li, L., Laqtom, N. N., Chang, B., Shuken, S., Nguyen, A., Yerra, L., Yang, A. C., Green, C., Tanga, M., Abu-Remaileh, M., Bassik, M. C., Frydman, J., Luo, J., Wyss-Coray, T. 2022; 119 (11): e2121609119

  Abstract

  SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.

  View details for DOI 10.1073/pnas.2121609119

  View details for PubMedID 35259016

• Ferroptosis regulation by the NGLY1/NFE2L1 pathway. Proceedings of the National Academy of Sciences of the United States of America Forcina, G. C., Pope, L., Murray, M., Dong, W., Abu-Remaileh, M., Bertozzi, C. R., Dixon, S. J. 2022; 119 (11): e2118646119

  Abstract

  SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

  View details for DOI 10.1073/pnas.2118646119

  View details for PubMedID 35271393

• Lessons from metabolic perturbations in lysosomal storage disorders for neurodegeneration CURRENT OPINION IN SYSTEMS BIOLOGY Medoh, U. N., Chen, J. Y., Abu-Remaileh, M. 2022; 29

  View details for DOI 10.1016/j.coisb.2021.100408

  View details for Web of Science ID 000853279900003

• Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition SCIENCE ADVANCES Fassl, A., Brain, C., Abu-Remaileh, M., Stukan, I., Butter, D., Stepien, P., Feit, A. S., Bergholz, J., Michowski, W., Otto, T., Sheng, Q., Loo, A., Michael, W., Tiedt, R., DeAngelis, C., Schiff, R., Jiang, B., Jovanovic, B., Nowak, K., Ericsson, M., Cameron, M., Gray, N., Dillon, D., Zhao, J. J., Sabatini, D. M., Jeselsohn, R., Brown, M., Polyak, K., Sicinski, P. 2020; 6 (25)

  View details for DOI 10.1126/sciadv.abb2210

  View details for Web of Science ID 000542299800037

• The microbiota programs DNA methylation to control intestinal homeostasis and inflammation. Nature microbiology Ansari, I., Raddatz, G., Gutekunst, J., Ridnik, M., Cohen, D., Abu-Remaileh, M., Tuganbaev, T., Shapiro, H., Pikarsky, E., Elinav, E., Lyko, F., Bergman, Y. 2020

  Abstract

  Although much research has been done on the diversity of the gut microbiome, little is known about how it influences intestinal homeostasis under normal and pathogenic conditions. Epigenetic mechanisms have recently been suggested to operate at the interface between the microbiota and the intestinal epithelium. We performed whole-genome bisulfite sequencing on conventionally raised and germ-free mice, and discovered that exposure to commensal microbiota induced localized DNA methylation changes at regulatory elements, which are TET2/3-dependent. This culminated in the activation of a set of 'early sentinel' response genes to maintain intestinal homeostasis. Furthermore, we demonstrated that exposure to the microbiota in dextran sodium sulfate-induced acute inflammation results in profound DNA methylation and chromatin accessibility changes at regulatory elements, leading to alterations in gene expression programs enriched in colitis- and colon-cancer-associated functions. Finally, by employing genetic interventions, we show that microbiota-induced epigenetic programming is necessary for proper intestinal homeostasis in vivo.

  View details for DOI 10.1038/s41564-019-0659-3

  View details for PubMedID 32015497

• Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation. Molecular cell Weber, R. A., Yen, F. S., Nicholson, S. P., Alwaseem, H. n., Bayraktar, E. C., Alam, M. n., Timson, R. C., La, K. n., Abu-Remaileh, M. n., Molina, H. n., Birsoy, K. n. 2020

  Abstract

  The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.

  View details for DOI 10.1016/j.molcel.2020.01.003

  View details for PubMedID 31983508

• Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science advances Fassl, A. n., Brain, C. n., Abu-Remaileh, M. n., Stukan, I. n., Butter, D. n., Stepien, P. n., Feit, A. S., Bergholz, J. n., Michowski, W. n., Otto, T. n., Sheng, Q. n., Loo, A. n., Michael, W. n., Tiedt, R. n., DeAngelis, C. n., Schiff, R. n., Jiang, B. n., Jovanovic, B. n., Nowak, K. n., Ericsson, M. n., Cameron, M. n., Gray, N. n., Dillon, D. n., Zhao, J. J., Sabatini, D. M., Jeselsohn, R. n., Brown, M. n., Polyak, K. n., Sicinski, P. n. 2020; 6 (25): eabb2210

  Abstract

  Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.

  View details for DOI 10.1126/sciadv.abb2210

  View details for PubMedID 32704543

  View details for PubMedCentralID PMC7360435

• Structural basis for the docking of mTORC1 on the lysosomal surface. Science (New York, N.Y.) Rogala, K. B., Gu, X., Kedir, J. F., Abu-Remaileh, M., Bianchi, L. F., Bottino, A. M., Dueholm, R., Niehaus, A., Overwijn, D., Fils, A. P., Zhou, S. X., Leary, D., Laqtom, N. N., Brignole, E. J., Sabatini, D. M. 2019; 366 (6464): 468-475

  Abstract

  The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP-RagC/D•GDP) permits mTORC1 association. We used cryo-electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.

  View details for DOI 10.1126/science.aay0166

  View details for PubMedID 31601708

• WWOX somatic ablation in skeletal muscles alters glucose metabolism. Molecular metabolism Abu-Remaileh, M. n., Abu-Remaileh, M. n., Akkawi, R. n., Knani, I. n., Udi, S. n., Pacold, M. E., Tam, J. n., Aqeilan, R. I. 2019; 22: 132–40

  Abstract

  WWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism.We re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis.Several WWOX variants were found to be strongly associated with MetS disorders. In mouse models, somatic ablation of Wwox in skeletal muscle (WwoxΔSKM) results in weight gain, glucose intolerance, and insulin resistance. Furthermore, WwoxΔSKM mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1α) in SKM.Our studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes.Genetics, Metabolic Syndrome, Diabetes.

  View details for DOI 10.1016/j.molmet.2019.01.010

  View details for PubMedID 30755385

  View details for PubMedCentralID PMC6437662

• MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo. Proceedings of the National Academy of Sciences of the United States of America Bayraktar, E. C., Baudrier, L. n., Özerdem, C. n., Lewis, C. A., Chan, S. H., Kunchok, T. n., Abu-Remaileh, M. n., Cangelosi, A. L., Sabatini, D. M., Birsoy, K. n., Chen, W. W. 2019; 116 (1): 303–12

  Abstract

  Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

  View details for DOI 10.1073/pnas.1816656115

  View details for PubMedID 30541894

  View details for PubMedCentralID PMC6320505

• High-fat diet enhances stemness and tumorigenicity of intestinal progenitors (vol 531, pg 53, 2016) NATURE Beyaz, S., Mana, M. D., Roper, J., Kedrin, D., Saadatpour, A., Hong, S., Bauer-Rowe, K. E., Xifaras, M. E., Akkad, A., Arias, E., Pinello, L., Katz, Y., Shinagare, S., Abu-Remaileh, M., Mihaylova, M. M., Lamming, D. W., Dogum, R., Guo, G., Bell, G. W., Selig, M., Nielsen, G., Gupta, N., Ferrone, C. R., Deshpande, V., Yuan, G., Orkin, S. H., Sabatini, D. M., Yilmaz, O. H. 2018; 560 (7717): E26

  Abstract

  In Fig. 4e of this Article, the labels for 'Control' and 'HFD' were reversed ('Control' should have been labelled blue rather than purple, and 'HFD' should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for 'V' and 'GW' were reversed ('V' should have been labelled blue rather than purple, and 'GW' should have been labelled purple instead of blue). The original figure has been corrected online.

  View details for DOI 10.1038/s41586-018-0187-y

  View details for Web of Science ID 000441115200013

  View details for PubMedID 29849139

• Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging. Cell stem cell Mihaylova, M. M., Cheng, C. W., Cao, A. Q., Tripathi, S. n., Mana, M. D., Bauer-Rowe, K. E., Abu-Remaileh, M. n., Clavain, L. n., Erdemir, A. n., Lewis, C. A., Freinkman, E. n., Dickey, A. S., La Spada, A. R., Huang, Y. n., Bell, G. W., Deshpande, V. n., Carmeliet, P. n., Katajisto, P. n., Sabatini, D. M., Yilmaz, Ö. H. 2018; 22 (5): 769–78.e4

  Abstract

  Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here we show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration.

  View details for DOI 10.1016/j.stem.2018.04.001

  View details for PubMedID 29727683

  View details for PubMedCentralID PMC5940005

• Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates. eLife Yu, Z. n., Surface, L. E., Park, C. Y., Horlbeck, M. A., Wyant, G. A., Abu-Remaileh, M. n., Peterson, T. R., Sabatini, D. M., Weissman, J. S., O'Shea, E. K. 2018; 7

  Abstract

  Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

  View details for DOI 10.7554/eLife.36620

  View details for PubMedID 29745899

  View details for PubMedCentralID PMC6021172

• NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science (New York, N.Y.) Wyant, G. A., Abu-Remaileh, M. n., Frenkel, E. M., Laqtom, N. N., Dharamdasani, V. n., Lewis, C. A., Chan, S. H., Heinze, I. n., Ori, A. n., Sabatini, D. M. 2018; 360 (6390): 751–58

  Abstract

  The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.

  View details for DOI 10.1126/science.aar2663

  View details for PubMedID 29700228

  View details for PubMedCentralID PMC6020066

• Histidine catabolism is a major determinant of methotrexate sensitivity. Nature Kanarek, N. n., Keys, H. R., Cantor, J. R., Lewis, C. A., Chan, S. H., Kunchok, T. n., Abu-Remaileh, M. n., Freinkman, E. n., Schweitzer, L. D., Sabatini, D. M. 2018; 559 (7715): 632–36

  Abstract

  The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.

  View details for DOI 10.1038/s41586-018-0316-7

  View details for PubMedID 29995852

  View details for PubMedCentralID PMC6082631

• Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science (New York, N.Y.) Abu-Remaileh, M. n., Wyant, G. A., Kim, C. n., Laqtom, N. N., Abbasi, M. n., Chan, S. H., Freinkman, E. n., Sabatini, D. M. 2017; 358 (6364): 807–13

  Abstract

  The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+-adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux.

  View details for DOI 10.1126/science.aan6298

  View details for PubMedID 29074583

  View details for PubMedCentralID PMC5704967

• Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase. Cell Cantor, J. R., Abu-Remaileh, M. n., Kanarek, N. n., Freinkman, E. n., Gao, X. n., Louissaint, A. n., Lewis, C. A., Sabatini, D. M. 2017; 169 (2): 258–72.e17

  Abstract

  A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.

  View details for DOI 10.1016/j.cell.2017.03.023

  View details for PubMedID 28388410

  View details for PubMedCentralID PMC5421364

• mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient. Cell Wyant, G. A., Abu-Remaileh, M. n., Wolfson, R. L., Chen, W. W., Freinkman, E. n., Danai, L. V., Vander Heiden, M. G., Sabatini, D. M. 2017; 171 (3): 642–54.e12

  Abstract

  The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.

  View details for DOI 10.1016/j.cell.2017.09.046

  View details for PubMedID 29053970

  View details for PubMedCentralID PMC5704964

• KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature Wolfson, R. L., Chantranupong, L. n., Wyant, G. A., Gu, X. n., Orozco, J. M., Shen, K. n., Condon, K. J., Petri, S. n., Kedir, J. n., Scaria, S. M., Abu-Remaileh, M. n., Frankel, W. N., Sabatini, D. M. 2017; 543 (7645): 438–42

  Abstract

  The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.

  View details for DOI 10.1038/nature21423

  View details for PubMedID 28199306

  View details for PubMedCentralID PMC5360989

• Embryonic Stem Cell (ES)-Specific Enhancers Specify the Expression Potential of ES Genes in Cancer. PLoS genetics Aran, D., Abu-Remaileh, M., Levy, R., Meron, N., Toperoff, G., Edrei, Y., Bergman, Y., Hellman, A. 2016; 12 (2): e1005840

  Abstract

  Cancers often display gene expression profiles resembling those of undifferentiated cells. The mechanisms controlling these expression programs have yet to be identified. Exploring transcriptional enhancers throughout hematopoietic cell development and derived cancers, we uncovered a novel class of regulatory epigenetic mutations. These epimutations are particularly enriched in a group of enhancers, designated ES-specific enhancers (ESSEs) of the hematopoietic cell lineage. We found that hematopoietic ESSEs are prone to DNA methylation changes, indicative of their chromatin activity states. Strikingly, ESSE methylation is associated with gene transcriptional activity in cancer. Methylated ESSEs are hypermethylated in cancer relative to normal somatic cells and co-localized with silenced genes, whereas unmethylated ESSEs tend to be hypomethylated in cancer and associated with reactivated genes. Constitutive or hematopoietic stem cell-specific enhancers do not show these trends, suggesting selective reactivation of ESSEs in cancer. Further analyses of a hypomethylated ESSE downstream to the VEGFA gene revealed a novel regulatory circuit affecting VEGFA transcript levels across cancers and patients. We suggest that the discovered enhancer sites provide a framework for reactivation of ES genes in cancer.

  View details for DOI 10.1371/journal.pgen.1005840

  View details for PubMedID 26886256

  View details for PubMedCentralID PMC4757527

• A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Nature chemical biology Pacold, M. E., Brimacombe, K. R., Chan, S. H., Rohde, J. M., Lewis, C. A., Swier, L. J., Possemato, R. n., Chen, W. W., Sullivan, L. B., Fiske, B. P., Cho, S. n., Freinkman, E. n., Birsoy, K. n., Abu-Remaileh, M. n., Shaul, Y. D., Liu, C. M., Zhou, M. n., Koh, M. J., Chung, H. n., Davidson, S. M., Luengo, A. n., Wang, A. Q., Xu, X. n., Yasgar, A. n., Liu, L. n., Rai, G. n., Westover, K. D., Vander Heiden, M. G., Shen, M. n., Gray, N. S., Boxer, M. B., Sabatini, D. M. 2016; 12 (6): 452–58

  Abstract

  Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

  View details for DOI 10.1038/nchembio.2070

  View details for PubMedID 27110680

  View details for PubMedCentralID PMC4871733

• High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature Beyaz, S. n., Mana, M. D., Roper, J. n., Kedrin, D. n., Saadatpour, A. n., Hong, S. J., Bauer-Rowe, K. E., Xifaras, M. E., Akkad, A. n., Arias, E. n., Pinello, L. n., Katz, Y. n., Shinagare, S. n., Abu-Remaileh, M. n., Mihaylova, M. M., Lamming, D. W., Dogum, R. n., Guo, G. n., Bell, G. W., Selig, M. n., Nielsen, G. P., Gupta, N. n., Ferrone, C. R., Deshpande, V. n., Yuan, G. C., Orkin, S. H., Sabatini, D. M., Yilmaz, Ö. H. 2016; 531 (7592): 53–58

  Abstract

  Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

  View details for DOI 10.1038/nature17173

  View details for PubMedID 26935695

  View details for PubMedCentralID PMC4846772

• Chronic inflammation induces a novel epigenetic program that is conserved in intestinal adenomas and in colorectal cancer. Cancer research Abu-Remaileh, M., Bender, S., Raddatz, G., Ansari, I., Cohen, D., Gutekunst, J., Musch, T., Linhart, H., Breiling, A., Pikarsky, E., Bergman, Y., Lyko, F. 2015; 75 (10): 2120-30

  Abstract

  Chronic inflammation represents a major risk factor for tumor formation, but the underlying mechanisms have remained largely unknown. Epigenetic mechanisms can record the effects of environmental challenges on the genome level and could therefore play an important role in the pathogenesis of inflammation-associated tumors. Using single-base methylation maps and transcriptome analyses of a colitis-induced mouse colon cancer model, we identified a novel epigenetic program that is characterized by hypermethylation of DNA methylation valleys that are characterized by low CpG density and active chromatin marks. This program is conserved and functional in mouse intestinal adenomas and results in silencing of active intestinal genes that are involved in gastrointestinal homeostasis and injury response. Further analyses reveal that the program represents a prominent feature of human colorectal cancer and can be used to correctly classify colorectal cancer samples with high accuracy. Together, our results show that inflammatory signals establish a novel epigenetic program that silences a specific set of genes that contribute to inflammation-induced cellular transformation.

  View details for DOI 10.1158/0008-5472.CAN-14-3295

  View details for PubMedID 25808873

• An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis. Cell Birsoy, K. n., Wang, T. n., Chen, W. W., Freinkman, E. n., Abu-Remaileh, M. n., Sabatini, D. M. 2015; 162 (3): 540–51

  Abstract

  The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation.

  View details for DOI 10.1016/j.cell.2015.07.016

  View details for PubMedID 26232224

  View details for PubMedCentralID PMC4522279

• Aberrant DNA methylation in ES cells. PloS one Ludwig, G., Nejman, D., Hecht, M., Orlanski, S., Abu-Remaileh, M., Yanuka, O., Sandler, O., Marx, A., Roberts, D., Benvenisty, N., Bergman, Y., Mendelsohn, M., Cedar, H. 2014; 9 (5): e96090

  Abstract

  Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.

  View details for DOI 10.1371/journal.pone.0096090

  View details for PubMedID 24852222

  View details for PubMedCentralID PMC4031077

• Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/β-catenin signalling. The EMBO journal Abu-Remaileh, M., Gerson, A., Farago, M., Nathan, G., Alkalay, I., Zins Rousso, S., Gur, M., Fainsod, A., Bergman, Y. 2010; 29 (19): 3236-48

  Abstract

  Although the transcriptional regulatory events triggered by Oct-3/4 are well documented, understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and biochemical studies that suggest an unexpected novel strategy for Oct-3/4-dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct-3/4 specifically interacts with nuclear β-catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct-3/4-mediated control of β-catenin stability has an important function in regulating ES cell motility. Down-regulation of Oct-3/4 increases β-catenin protein levels, enhancing Wnt signalling and initiating invasive cellular activity characteristic of epithelial-mesenchymal transition. Our data suggest a novel mode of regulation by which a delicate balance between β-catenin, Tcf3 and Oct-3/4 regulates maintenance of stem cell identity. Altering the balance between these proteins can direct cell fate decisions and differentiation.

  View details for DOI 10.1038/emboj.2010.200

  View details for PubMedID 20736927

  View details for PubMedCentralID PMC2957205

• De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nature structural & molecular biology Epsztejn-Litman, S., Feldman, N., Abu-Remaileh, M., Shufaro, Y., Gerson, A., Ueda, J., Deplus, R., Fuks, F., Shinkai, Y., Cedar, H., Bergman, Y. 2008; 15 (11): 1176-1183

  Abstract

  The pluripotency-determining gene Oct3/4 (also called Pou5f1) undergoes postimplantation silencing in a process mediated by the histone methyltransferase G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates numerous early-embryonic genes by bringing about heterochromatinization of methylated histone H3K9 and de novo DNA methylation. Genetic studies in differentiating embryonic stem cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization but still allows de novo methylation, whereas biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin domain, by recruiting Dnmt3a and Dnmt3b independently of its histone methyltransferase activity. These modifications seem to be programmed for carrying out two separate biological functions: histone methylation blocks target-gene reactivation in the absence of transcriptional repressors, whereas DNA methylation prevents reprogramming to the undifferentiated state.

  View details for DOI 10.1038/nsmb.1476

  View details for PubMedID 18953337

  View details for PubMedCentralID PMC2581722