bims-mibica 2021-07-11 papers

bims-mibica

Biomed News

on Mitochondrial bioenergetics in cancer

Issue of 2021‒07‒11
twenty-nine papers selected by
Kelsey Fisher-Wellman
East Carolina University

PDE2 Inhibits PKA-Mediated Phosphorylation of TFAM to Promote Mitochondrial Ca2+-Induced Colorectal Cancer Growth.
Metabolic Enzyme DLST Promotes Tumor Aggression and Reveals a Vulnerability to OXPHOS Inhibition in High-Risk Neuroblastoma.
SIRT5 Is a Druggable Metabolic Vulnerability in Acute Myeloid Leukemia.
Independent of mitochondrial respiratory function, dietary nitrate attenuates HFD-induced lipid accumulation and mitochondrial ROS emission within the liver.
ONC212 is a novel mitocan acting synergistically with glycolysis inhibition in pancreatic cancer.
Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.
Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells.
Cloperastine inhibits esophageal squamous cell carcinoma proliferation in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation.
NQO1 Binds and Supports SIRT1 Function.
Skeletal muscle proteomes reveal downregulation of mitochondrial proteins in transition from prediabetes into type 2 diabetes.
Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity.
Pharmacologic targeting of Mcl-1 induces mitochondrial dysfunction and apoptosis in B-cell lymphoma cells in a TP53- and BAX-dependent manner.
Non-enzymatic lipid peroxidation initiated by photodynamic therapy drives a distinct ferroptosis-like cell death pathway.
Non-oncogene Addiction to SIRT5 in Acute Myeloid Leukemia.
Exosomal IDH1 increases the resistance of colorectal cancer cells to 5-Fluorouracil.
Extracellular Flux Analysis to Investigate the Impact of NF-κB on Mitochondrial Respiration in Colorectal Carcinoma (CRC).
TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia.
DNA repair protein APE1 degrades dysfunctional abasic mRNA in mitochondria affecting oxidative phosphorylation.
The UL16 protein of HSV-1 promotes the metabolism of cell mitochondria by binding to ANT2 protein.
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer.
Mitochondrial protein IF1 is a potential regulator of glucagon-like peptide (GLP-1) secretion function of the mouse intestine.
Thioredoxin reductase is a major regulator of metabolism in leukemia cells.
Metformin Antagonizes Ovarian Cancer Cells Malignancy Through MSLN Mediated IL-6/STAT3 Signaling.
Inhibition of SIRT1 Limits Self-Renewal and Oncogenesis by Inducing Senescence of Liver Cancer Stem Cells.
Sensitive detection of tumor mutations from blood and its application to immunotherapy prognosis.
A synthetic circuit for buffering gene dosage variation between individual mammalian cells.
Regulation of Mitochondrial Function by Natural Products for the Treatment of Metabolic Associated Fatty Liver Disease.
High-Fat Diet Impacts on Tumor Development in the Gut.
Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro.

Front Oncol. 2021 ;11 663778

PDE2 Inhibits PKA-Mediated Phosphorylation of TFAM to Promote Mitochondrial Ca2+-Induced Colorectal Cancer Growth.

Yilin Zhao, Yaya Wang, Jing Zhao, Zhaohui Zhang, Mingpeng Jin, Feng Zhou, Chao Jin, Jing Zhang, Jinliang Xing, Nan Wang, Xianli He, Tingting Ren.

  Growing evidence indicates that the dysregulation of mitochondrial calcium (Ca2+) plays a critical role in the growth of tumor cells, including colorectal cancer (CRC). However, the underling mechanism is not fully elucidated. In this study, the regulatory effects of mitochondrial Ca2+ on phosphodiesterase 2 (PDE2)/cAMP/PKA axis and the phosphorylation of mitochondrial transcription factor A (TFAM) as well as the growth of CRC cells were systematically investigated both in vitro and in vivo. Our findings demonstrated that MCU-induced mitochondrial Ca2+ uptake activated mitochondrial PDE2 in CRC cells. Moreover, overexpression MCU in CRC led to a 1.9-fold increase in Ca2+ uptake compared to control cells. However, knockdown of MCU resulted in 1.5-fould decrease in Ca2+ uptake in mitochondria compared to the controls. Activation of mitochondrial PDE2 significantly inhibited the activity of mitochondrial protein kinase A (PKA), which subsequently leads to decreased phosphorylation of TFAM. Our data further revealed that PKA regulates the phosphorylation of TFAM and promotes the degradation of phosphorylated TFAM. Thus, TFAM protein levels accumulated in mitochondria when the activity of PKA was inhibited. Overall, this study showed that the overexpression of MCU enhanced CRC growth through promoting the accumulation of TFAM proteins in mitochondria. Conversely, knockdown of MCU in CRC cells resulted in decreased CRC growth. Collectively, these data suggest that the mitochondrial Ca2+-activated PDE2/cAMP/PKA axis plays a key role in regulating TFAM stability and the growth of CRC cells.

Keywords:  colorectal cancer; mitochondrial Ca2+; mitochondrial calcium uniporter; mitochondrial transcription factor A; phosphodiesterase 2

DOI:  https://doi.org/10.3389/fonc.2021.663778
Cancer Res. 2021 Jul 07. pii: canres.2153.2020. [Epub ahead of print]

Metabolic Enzyme DLST Promotes Tumor Aggression and Reveals a Vulnerability to OXPHOS Inhibition in High-Risk Neuroblastoma.

Nicole M Anderson, Xiaodan Qin, Jennifer M Finan, Andrew Lam, Jacob Athoe, Rindert Missiaen, Nicolas Skuli, Annie Kennedy, Amandeep S Saini, Ting Tao, Shizhen Zhu, Itzhak Nissim, A Thomas Look, Guoliang Qing, M Celeste Simon, Hui Feng.

High-risk neuroblastoma remains therapeutically challenging to treat, and the mechanisms promoting disease aggression are poorly understood. Here we show that elevated expression of dihydrolipoamide S-succinyltransferase (DLST) predicts poor treatment outcome and aggressive disease in neuroblastoma patients. DLST is an E2 component of the a-ketoglutarate (a-KG) dehydrogenase complex, which governs the entry of glutamine into the tricarboxylic acid cycle (TCA) for oxidative decarboxylation. During this irreversible step, a-KG is converted into succinyl-CoA, producing NADH for oxidative phosphorylation (OXPHOS). Utilizing a zebrafish model of MYCN-driven neuroblastoma, we demonstrate that even modest increases in DLST expression promote tumor aggression, while monoallelic dlst loss impedes disease initiation and progression. DLST depletion in human MYCN-amplified neuroblastoma cells minimally affected glutamine anaplerosis and did not alter TCA cycle metabolites other than a-KG. However, DLST loss significantly suppressed NADH production and impaired OXPHOS, leading to growth arrest and apoptosis of neuroblastoma cells. Additionally, multiple inhibitors targeting the electron transport chain, including the potent IACS-010759 that is currently in clinical testing for other cancers, efficiently reduced neuroblastoma proliferation in vitro. IACS-010759 also suppressed tumor growth in zebrafish and mouse xenograft models of high-risk neuroblastoma. Together, these results demonstrate that DLST promotes neuroblastoma aggression and unveils OXPHOS as an essential contributor to high-risk neuroblastoma.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-2153
Cancer Discov. 2021 May;2(3): 266-287

SIRT5 Is a Druggable Metabolic Vulnerability in Acute Myeloid Leukemia.

Dongqing Yan, Anca Franzini, Anthony D Pomicter, Brayden J Halverson, Orlando Antelope, Clinton C Mason, Jonathan M Ahmann, Anna V Senina, Nadeem A Vellore, Courtney L Jones, Matthew S Zabriskie, Hein Than, Michael J Xiao, Alexandria van Scoyk, Ami B Patel, Phillip M Clair, William L Heaton, Shawn C Owen, Joshua L Andersen, Christina M Egbert, Julie A Reisz, Angelo D'Alessandro, James E Cox, Kevin C Gantz, Hannah M Redwine, Siddharth M Iyer, Jamshid S Khorashad, Nima Rajabi, Christian A Olsen, Thomas O'Hare, Michael W Deininger.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. SIGNIFICANCE: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.See related commentary by Li and Melnick, p. 198.

DOI: https://doi.org/10.1158/2643-3230.BCD-20-0168
Am J Physiol Endocrinol Metab. 2021 07 06.

Independent of mitochondrial respiratory function, dietary nitrate attenuates HFD-induced lipid accumulation and mitochondrial ROS emission within the liver.

Genevieve J DesOrmeaux, Heather L Petrick, Henver S Brunetta, Graham P Holloway.

  The liver is particularly susceptible to the detrimental effects of a high fat diet (HFD), rapidly developing lipid accumulation and impaired cellular homeostasis. Recently, dietary nitrate has been shown to attenuate HFD-induced whole body glucose intolerance and liver steatosis, however the underlying mechanism(s) remain poorly defined. In the current study we investigated the ability of dietary nitrate to minimize possible impairments in liver mitochondrial bioenergetics following 8 wk of HFD (60% fat) in male C57BL/6J mice. Consumption of a HFD caused whole-body glucose intolerance (p<0.0001), and within the liver, increased lipid accumulation (p<0.0001), mitochondrial-specific reactive oxygen species emission (p=0.007), and markers of oxidative stress. Remarkably, dietary nitrate attenuated almost all of these pathological responses. Despite the reduction in lipid accumulation and redox stress (reduced TBARS and nitrotyrosine), nitrate did not improve insulin signaling within the liver or whole-body pyruvate tolerance (p=0.313 HFD vs HFD+nitrate). Moreover, the beneficial effects of nitrate were independent of changes in weight gain, 5' AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) signaling, mitochondrial content, mitochondrial respiratory capacity and ADP sensitivity or antioxidant protein content. Combined, these data suggest nitrate supplementation represents a potential therapeutic strategy to attenuate hepatic lipid accumulation and decrease mitochondrial ROS emission following HFD, processes linked to improvements in whole-body glucose tolerance. However, the beneficial effects of nitrate within the liver do not appear to be a result of increased oxidative capacity or mitochondrial substrate sensitivity.

Keywords:  Mitochondria; ROS; high fat diet; liver; nitrate

DOI:  https://doi.org/10.1152/ajpendo.00610.2020
Mol Cancer Ther. 2021 Jun 17. pii: molcanther.MCT-20-0962-A.2020. [Epub ahead of print]

ONC212 is a novel mitocan acting synergistically with glycolysis inhibition in pancreatic cancer.

Isacco Ferrarini, Anna Louie, Lanlan Zhou, Wafik S El-Deiry.

ONC212 is a fluorinated-imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding-partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth-arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo. Thus, ONC212 is a novel mitocan targeting oxidative-metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0962
Nat Metab. 2021 Jul 05.

Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.

Elizabeth K Wiese, Sadae Hitosugi, Sharon T Loa, Annapoorna Sreedhar, Lindsey G Andres-Beck, Kiran Kurmi, Yuan-Ping Pang, Larry M Karnitz, Wilson I Gonsalves, Taro Hitosugi.

Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumours, compared to human LDHA-expressing tumours in mice, displayed resistance to the PKM2 activator. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates and inhibits LDHA following PKM2 activation.

DOI: https://doi.org/10.1038/s42255-021-00424-5
FEBS J. 2021 Jul 06.

Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells.

Peter F Doubleday, Luca Fornelli, Ioanna Ntai, Neil L Kelleher.

  Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC) and recent findings have demonstrated independent and non-redundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that expression of KRASG13D upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1, supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.

Keywords:  Quantitative proteomics; aspartate; colorectal cancer; metabolomics; mutant KRAS; urea cycle

DOI:  https://doi.org/10.1111/febs.16111
Cell Death Discov. 2021 Jun 21. 7(1): 166

Cloperastine inhibits esophageal squamous cell carcinoma proliferation in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation.

Bo Li, Yin Yu, Yanan Jiang, Lili Zhao, Ang Li, Mingzhu Li, Baoyin Yuan, Jing Lu, Ziming Dong, Jimin Zhao, Kangdong Liu.

Esophageal squamous cell carcinoma (ESCC) is a major type of esophageal cancer. The prognosis of patients with ESCC remains poor because of the high morbidity and mortality of the disease. One strategy for drug discovery for ESCC treatment or prevention is screening FDA-approved drugs. In the present study, we found that the antitussive agent cloperastine can inhibit the proliferation of ESCC cells. However, the underlying mechanism was unclear. To determine the mechanism of this inhibitory effect, we performed proteomic analysis using KYSE150 cells treated with cloperastine and DMSO. The results identified several down-regulated signaling pathways included those of three key proteins (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 1, NADH ubiquinone oxidoreductase subunit S5, and cytochrome C oxidase subunit 6B1) involved in oxidative phosphorylation. Meanwhile, we observed that oxidative phosphorylation in mitochondria was inhibited by the drug. Importantly, cloperastine suppressed ESCC growth in a xenograft mouse model in vivo. Our findings revealed that cloperastine inhibits the proliferation of ESCC in vivo and in vitro by suppressing mitochondrial oxidative phosphorylation.

DOI: https://doi.org/10.1038/s41420-021-00509-w
Front Pharmacol. 2021 ;12 671929

NQO1 Binds and Supports SIRT1 Function.

Peter Tsvetkov, Julia Adler, Romano Strobelt, Yaarit Adamovich, Gad Asher, Nina Reuven, Yosef Shaul.

  Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD+-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD+ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD+, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD+ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

Keywords:  NADH/NAD ratio; PGC1 alpha; SIRT1 activity; mitochondria stress; quinone oxidoreductase 1

DOI:  https://doi.org/10.3389/fphar.2021.671929
iScience. 2021 Jul 23. 24(7): 102712

Skeletal muscle proteomes reveal downregulation of mitochondrial proteins in transition from prediabetes into type 2 diabetes.

Tiina Öhman, Jaakko Teppo, Neeta Datta, Selina Mäkinen, Markku Varjosalo, Heikki A Koistinen.

  Skeletal muscle insulin resistance is a central defect in the pathogenesis of type 2 diabetes (T2D). Here, we analyzed skeletal muscle proteome in 148 vastus lateralis muscle biopsies obtained from men covering all glucose tolerance phenotypes: normal, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D. Skeletal muscle proteome was analyzed by a sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics technique. Our data indicate a downregulation in several proteins involved in mitochondrial electron transport or respiratory chain complex assembly already in IFG and IGT muscles, with most profound decreases observed in T2D. Additional phosphoproteomic analysis reveals altered phosphorylation in several signaling pathways in IFG, IGT, and T2D muscles, including those regulating glucose metabolic processes, and the structure of muscle cells. These data reveal several alterations present in skeletal muscle already in prediabetes and highlight impaired mitochondrial energy metabolism in the trajectory from prediabetes into T2D.

Keywords:  Diabetology; Molecular biology; Proteomics

DOI:  https://doi.org/10.1016/j.isci.2021.102712
EMBO Mol Med. 2021 Jul 07. e13086

Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity.

Vaishali Jayashankar, Elizabeth Selwan, Sarah E Hancock, Amandine Verlande, Maggie O Goodson, Kazumi H Eckenstein, Giedre Milinkeviciute, Brianna M Hoover, Bin Chen, Angela G Fleischman, Karina S Cramer, Stephen Hanessian, Selma Masri, Nigel Turner, Aimee L Edinger.

  Ceramide-induced mitochondrial fission drives high-fat diet (HFD)-induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide-induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide-induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6- and PIKfyve-dependent trafficking events, the synthetic sphingolipid SH-BC-893 blocked palmitate- and ceramide-induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in␣vitro. Similar benefits were observed in the tissues of HFD-fed mice. Within 4 h of oral administration, SH-BC-893 normalized mitochondrial morphology in the livers and brains of HFD-fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH-BC-893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH-BC-893 robustly and acutely blocks ceramide-induced mitochondrial dysfunction, correcting diet-induced obesity and its metabolic sequelae.

Keywords:  ceramide; high-fat diet; leptin resistance; mitochondrial fission; obesity

DOI:  https://doi.org/10.15252/emmm.202013086
Clin Cancer Res. 2021 Jul 07. pii: clincanres.0464.2021. [Epub ahead of print]

Pharmacologic targeting of Mcl-1 induces mitochondrial dysfunction and apoptosis in B-cell lymphoma cells in a TP53- and BAX-dependent manner.

Tingting Liu, Vi Lam, Elana Thieme, Duanchen Sun, Xin Wang, Fei Xu, Lili Wang, Olga V Danilova, Zheng Xia, Jeffrey W Tyner, Stephen E Kurtz, Alexey V Danilov.

PURPOSE: Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency.EXPERIMENTAL DESIGN: We explored the pre-clinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition.
RESULTS: Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of pro-survival proteins (e.g., Bcl-xL) in stromal conditions which mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice.
CONCLUSION: Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-0464
Redox Biol. 2021 Jun 23. pii: S2213-2317(21)00215-9. [Epub ahead of print]45 102056

Non-enzymatic lipid peroxidation initiated by photodynamic therapy drives a distinct ferroptosis-like cell death pathway.

Sufang Shui, Zenglu Zhao, Hao Wang, Marcus Conrad, Guoquan Liu.

  Ferroptosis is primarily triggered by a failure of the glutathione (GSH)-glutathione peroxidase 4 (GPX4) reductive system and associated overwhelming lipid peroxidation, in which enzymes regulating polyunsaturated fatty acid (PUFA) metabolism, and in particular acyl-CoA synthetase long chain family member 4 (ACSL4), are central. Here, we found that exogenous oxygen radicals generated by photodynamic therapy (PDT) can directly peroxidize PUFAs and initiate lipid autoxidation, coinciding with cellular GSH depletion. Different from canonical ferroptosis induced by RSL3 or erastin, PDT-initiated lipid peroxidation and ferroptotis-like cell death is independent of lipoxygenase (ALOXs) and ACSL4. Especially, this form of cell death modality can be triggered in malignant cells insensitive to or acquired resistance to canonical ferroptosis inducers. We also observed a distinct iron metabolism pathway in this PDT-triggered cell death modality, in which cytosolic labile iron is decreased probably due to its relocation to mitochondria. Inhibition of the mitochondrial Ca2+ and Fe2+ uniporter (MCU) effectively prevented PDT-triggered lipid peroxidation and subsequent cell death. Therefore, we tentatively term this distinct ferroptosis-like cell death as liperoptosis. Moreover, using the clinically approved photosensitizer Verteporfin, PDT inhibited tumor growth through inducing prevailing ferroptosis-like cell death in a mouse xenograft model. With its site-specific advantages, these findings highlight the value of using PDT to trigger lipid peroxidation and ferroptosis-like cell death in vivo, and will benefit exploring the exact molecular mechanism of immunological effects of PDT in cancer treatment.

Keywords:  Cell death; Ferroptosis; Liperoptosis; Lipid peroxidation; PDT

DOI:  https://doi.org/10.1016/j.redox.2021.102056
Cancer Discov. 2021 May;2(3): 198-200

Non-oncogene Addiction to SIRT5 in Acute Myeloid Leukemia.

Meng Li, Ari M Melnick.

In this issue of Blood Cancer Discovery, Yan and colleagues discovered that mitochondrial deacylase, SIRT5, is required in AML cells to support mitochondrial oxidative phosphorylation, maintain redox homeostasis, and drive glutaminolysis. The new SIRT5 inhibitor, NRD167, can efficiently target SIRT5 in AMLs at micromolar range and may constitute a novel therapeutic approach to improve clinical outcomes of patients with AML.See related article by Yan et al., p. 266.

DOI: https://doi.org/10.1158/2643-3230.BCD-21-0026
J Cancer. 2021 ;12(16): 4862-4872

Exosomal IDH1 increases the resistance of colorectal cancer cells to 5-Fluorouracil.

Hao Yang, Sha Xie, Beibei Liang, Qiqi Tang, Huanchen Liu, Dongliang Wang, Gang Huang.

  Chemoresistance challenges the clinical treatment of colorectal cancer and requires an urgent solution. Isocitrate dehydrogenase 1 (IDH1) is a key enzyme involved in glucose metabolism that mediates the malignant transformation of tumors. However, the mechanisms by which IDH1 is involved in colorectal cancer cell proliferation and drug resistance induction remain unclear. In this study, we found that IDH1 was highly expressed in human colorectal cancer tissues and could be used to indicate a high-grade tumor. In vitro gene overexpression and knockdown were used to determine whether IDH1 promoted the proliferation of the colorectal cancer cell line HCT8 and resistance to 5-Fluorouracil (5FU). Further studies have shown that the 5FU-resistant cell line, HCT8FU, secreted exosomes that contained a high level of IDH1 protein. The exosomal IDH1 derived from 5FU-resistant cells enhanced the resistance of 5FU-sensitive cells. Metabolic assays revealed that exosomes derived from 5FU-resistant cells promoted a decrease in the level of IDH1-mediated NADPH, which is associated with the development of 5FU resistance in colorectal cancer cells. Therefore, exosomal IDH1 may be the transmitter and driver of chemoresistance in colorectal cancer and a potential chemotherapy target.

Keywords:  5-Fluorouracil; IDH1; colorectal cancer; exosomes

DOI:  https://doi.org/10.7150/jca.58846
Methods Mol Biol. 2021 ;2366 293-303

Extracellular Flux Analysis to Investigate the Impact of NF-κB on Mitochondrial Respiration in Colorectal Carcinoma (CRC).

Daria Capece, Daniela Verzella, Federica Begalli, Jason Bennett, Daniel D'Andrea, Davide Vecchiotti, Francesca Zazzeroni, Guido Franzoso.

  The reprogramming of cell metabolism is a hallmark of cancer. NF-κB transcription factors coordinate the host defense responses to stress, injury, and infection. They also play a central role in oncogenesis, at least in part by regulating cell metabolism and the adaptation to energy stress conditions in various types of cancer, such as colorectal carcinoma (CRC). Here, we describe the XF Cell Mito Stress Test methodology aimed at characterizing the metabolic and bioenergetic profile of CRC cells following the silencing of the essential NF-κB subunit, RelA. This methodology may also be applied to other cancers to reveal novel core vulnerabilities of malignant cells.

Keywords:  Cancer; Colorectal carcinoma; Extracellular Flux Analysis; Metabolism; NF-κB

DOI:  https://doi.org/10.1007/978-1-0716-1669-7_18
Cancer Discov. 2021 Mar;2(2): 162-185

TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia.

Seongseok Yun, Nicole D Vincelette, Xiaoqing Yu, Gregory W Watson, Mario R Fernandez, Chunying Yang, Taro Hitosugi, Chia-Ho Cheng, Audrey R Freischel, Ling Zhang, Weimin Li, Hsinan Hou, Franz X Schaub, Alexis R Vedder, Ling Cen, Kathy L McGraw, Jungwon Moon, Daniel J Murphy, Andrea Ballabio, Scott H Kaufmann, Anders E Berglund, John L Cleveland.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism, and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML), and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by an MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML. SIGNIFICANCE: Alterations in epigenetic control are a hallmark of AML. This study establishes that a MYC-TFEB circuit controls AML differentiation and epigenetic programs by inducing IDH1/IDH2 and hydroxylation of 5-methylcytosine, that TFEB functions as a tumor suppressor in AML, and that this circuit is a targetable vulnerability in AML.See related commentary by Wu and Eisenman, p. 116.

DOI: https://doi.org/10.1158/2643-3230.BCD-20-0029
J Mol Biol. 2021 Jul 02. pii: S0022-2836(21)00349-1. [Epub ahead of print] 167125

DNA repair protein APE1 degrades dysfunctional abasic mRNA in mitochondria affecting oxidative phosphorylation.

Arianna Barchiesi, Veronica Bazzani, Agata Jabczynska, Lukasz S Borowski, Silke Oeljeklaus, Bettina Warscheid, Agnieszka Chacinska, Roman J Szczesny, Carlo Vascotto.

  APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.

Keywords:  Apurinic/Apyrimidinic Endonuclease 1; Mitochondria; Oxidative phosphorylation; RNA processing

DOI:  https://doi.org/10.1016/j.jmb.2021.167125
Sci Rep. 2021 Jul 07. 11(1): 14001

The UL16 protein of HSV-1 promotes the metabolism of cell mitochondria by binding to ANT2 protein.

Shiyu Li, Shuting Liu, Zhenning Dai, Qian Zhang, Yichao Xu, Youyu Chen, Zhenyou Jiang, Wenhua Huang, Hanxiao Sun.

Long-term studies have shown that virus infection affects the energy metabolism of host cells, which mainly affects the function of mitochondria and leads to the hydrolysis of ATP in host cells, but it is not clear how virus infection participates in mitochondrial energy metabolism in host cells. In our study, HUVEC cells were infected with HSV-1, and the differentially expressed genes were obtained by microarray analysis and data analysis. The viral gene encoding protein UL16 was identified to interact with host protein ANT2 by immunoprecipitation and mass spectrometry. We also reported that UL16 transfection promoted oxidative phosphorylation of glucose and significantly increased intracellular ATP content. Furthermore, UL16 was transfected into the HUVEC cell model with mitochondrial dysfunction induced by D-Gal, and it was found that UL16 could restore the mitochondrial function of cells. It was first discovered that viral protein UL16 could enhance mitochondrial function in mammalian cells by promoting mitochondrial metabolism. This study provides a theoretical basis for the prevention and treatment of mitochondrial dysfunction or the pathological process related to mitochondrial dysfunction.

DOI: https://doi.org/10.1038/s41598-021-93430-2
EMBO Mol Med. 2021 Jul 05. e13792

Targeting prominin2 transcription to overcome ferroptosis resistance in cancer.

Caitlin W Brown, Peter Chhoy, Dimpi Mukhopadhyay, Emmet R Karner, Arthur M Mercurio.

  Understanding how cancer cells resist ferroptosis is a significant problem that impacts ongoing efforts to stimulate ferroptosis as a therapeutic strategy. We reported that prominin2 is induced by ferroptotic stimuli and functions to resist ferroptotic death. Although this finding has significant implications for therapy, specific prominin2 inhibitors are not available. We rationalized that the mechanism by which prominin2 expression is induced by ferroptotic stress could be targeted, expanding the range of options to overcome ferroptosis resistance. Here, we show that that 4-hydroxynonenal (4HNE), a specific lipid metabolite formed from the products of lipid peroxidation stimulates PROM2 transcription by a mechanism that involves p38 MAP kinase-mediated activation of HSF1 and HSF1-dependent transcription of PROM2. HSF1 inhibitors sensitize a wide variety of resistant cancer cells to drugs that induce ferroptosis. Importantly, the combination of a ferroptosis-inducing drug and an HSF1 inhibitor causes the cytostasis of established tumors in mice, although neither treatment alone is effective. These data reveal a novel approach for the therapeutic induction of ferroptosis in cancer.

Keywords:  cancer; ferroptosis; heat shock factor 1; prominin2; therapy

DOI:  https://doi.org/10.15252/emmm.202013792
Acta Pharm Sin B. 2021 Jun;11(6): 1568-1577

Mitochondrial protein IF1 is a potential regulator of glucagon-like peptide (GLP-1) secretion function of the mouse intestine.

Ying Wang, Jiaojiao Zhang, Xinyu Cao, Yaya Guan, Shuang Shen, Genshen Zhong, Xiwen Xiong, Yanhong Xu, Xiaoying Zhang, Hui Wang, Jianping Ye.

  IF1 (ATPIF1) is a nuclear DNA-encoded mitochondrial protein whose activity is inhibition of the F1Fo-ATP synthase to control ATP production. IF1 activity remains unknown in the regulation of GLP-1 activity. In this study, IF1 was examined in the diet-induced obese mice using the gene knockout (If1-KO) mice. The mice gained more body weight on a high fat diet without a change in food intake. Insulin tolerance was impaired, but the oral glucose tolerance was improved through an increase in GLP-1 secretion. The KO mice exhibited an improved intestine structure, mitochondrial superstructure, enhanced mitophagy, reduced apoptosis and decreased adenine nucleotide translocase 2 (ANT2) protein in the intestinal epithelial cells together with preserved gut microbiota. The data suggest that GLP-1 secretion was enhanced in the obese If1-KO mice to preserve glucose tolerance through a signaling pathway of ANT2/mitochondria/L-cells/GLP-1/insulin. IF1 is a potential mitochondrial target for induction of GLP-1 secretion in L-cells.

Keywords:  ANT2; ATPIF1; GLP-1; Glucose tolerance; Insulin resistance; L-cells; Microbiota; Mitophagy

DOI:  https://doi.org/10.1016/j.apsb.2021.02.002
Oncogene. 2021 Jul 08.

Thioredoxin reductase is a major regulator of metabolism in leukemia cells.

Sheelarani Karunanithi, Ruifu Liu, Yongchun Hou, Giancarlo Gonzalez, Natasha Oldford, Anne Jessica Roe, Nethrie Idipilly, Kalpana Gupta, Chandra Sekhar Amara, Satwikreddy Putluri, Grace Kyueun Lee, Juan Valentin-Goyco, Lindsay Stetson, Stephen A Moreton, Vasanta Putluri, Shyam M Kavuri, Yogen Saunthararajah, Marcos de Lima, Gregory P Tochtrop, Nagireddy Putluri, David N Wald.

Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.

DOI: https://doi.org/10.1038/s41388-021-01924-0
Cell Transplant. 2021 Jan-Dec;30:30 9636897211027819

Metformin Antagonizes Ovarian Cancer Cells Malignancy Through MSLN Mediated IL-6/STAT3 Signaling.

Xu Yang, Mei Huang, Qin Zhang, Jiao Chen, Juan Li, Qian Han, Lu Zhang, JiaQi Li, Shuai Liu, YuLan Ma, Lan Li, Lei Yang, SiYing Zou, Bin Han.

  BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, and chemotherapy remains the cornerstone for ovarian cancer management. Due to the unsatisfactory prognosis, a better understanding of the underlying molecular carcinogenesis is urgently required.METHODS: Assays for determining cell growth, cell motility, and apoptosis were employed to evaluate the potential antitumor effects of metformin against ovarian cancer cells. Molecular biological methods were employed to explore the underlying mechanism. Human ovarian cancer samples and Gene Expression Profiling Interactive Analysis (GEPIA) dataset were used for uncovering the clinical significances of mesothelin (MSLN) on ovarian cancer.
RESULTS: In the present work, we found that metformin treatment led to cell growth and cell migration inhibition, and induced cell apoptosis. Metformin administration also impaired cancer cell stemness and the capillary-like structure formation capacity of SKOV3 cells. On mechanism, metformin treatment remarkably reduced mesothelin (MSLN) expression, downregulated IL-6/STAT3 signaling activity, subsequently resulted in VEGF and TGFβ1 expression. We also observed an oncogenic function of MSLN on ovarian cancer.
CONCLUSIONS: Collectively, our findings suggested that metformin exerts anticancer effects by suppressing ovarian cancer cell malignancy, which attributed to MSLN inhibition mediated IL6/STAT3 signaling and VEGF and TGFβ1 downregulation.

Keywords:  IL-6/STAT3; angiogenesis; mesothelin; metformin; ovarian cancer; stemness

DOI:  https://doi.org/10.1177/09636897211027819
J Hepatocell Carcinoma. 2021 ;8 685-699

Inhibition of SIRT1 Limits Self-Renewal and Oncogenesis by Inducing Senescence of Liver Cancer Stem Cells.

Min-Jun Wang, Jia-Jia Chen, Shao-Hua Song, Jing Su, Ling-Hao Zhao, Qing-Gui Liu, Tao Yang, Zhiwen Chen, Chang Liu, Zhi-Ren Fu, Yi-Ping Hu, Fei Chen.

  Purpose: Cancer stem cells (CSCs) have been considered involving in tumorigenesis, local recurrence, and therapeutic drug resistance of hepatocellular carcinoma (HCC). To investigate novel and effective methods for targeting hepatic CSCs is crucial for a permanent cure of liver cancer.Methods: The expression level of SIRT1 was detected in CSCs of HCC tissues and cancer cell lines. Expression of CSC markers, the self-renewal and tumorigenic ability of liver CSCs were analyzed with SIRT1 inhibition. Cellular senescence-related markers were used to detect CSCs senescence after inhibition of SIRT1.
Results: SIRT1 was highly expressed in CSCs of HCC cell lines and human HCC tissues. In vitro study revealed that decreasing of SIRT1 level significantly downregulated the stemness-associated genes of liver CSCs and reduced the CSC stemness properties. Also, downregulated SIRT1 suppressed liver CSCs proliferation by decreasing their self-renewal abilities. Furthermore, CSCs with decreased SIRT1 expression showed limited tumorigenicity and formed smaller HCC tumor in vivo. And SIRT1 decreased CSCs became more susceptible to chemotherapeutic drugs. Mechanistically, SIRT1 decreased CSCs became senescence through the activation of p53-p21 and p16 pathway. The data further indicated that the tumor formed from SIRT1-knockdown CSCs exhibited higher senescence-associated β-galactosidase (SA-β-Gal) activity but lower proliferative capacity.
Conclusion: Taken together, these findings pointed that induction of senescence in liver CSCs is an effective tumor suppression method for HCC, and SIRT1 may be served as a promising target for HCC treatment.

Keywords:  SIRT1; cellular senescence; hepatocellular carcinoma; liver cancer stem cells; self-renewal; stemness

DOI:  https://doi.org/10.2147/JHC.S296234
Nat Commun. 2021 Jul 07. 12(1): 4172

Sensitive detection of tumor mutations from blood and its application to immunotherapy prognosis.

Shuo Li, Zorawar S Noor, Weihua Zeng, Mary L Stackpole, Xiaohui Ni, Yonggang Zhou, Zuyang Yuan, Wing Hung Wong, Vatche G Agopian, Steven M Dubinett, Frank Alber, Wenyuan Li, Edward B Garon, Xianghong Jasmine Zhou.

Cell-free DNA (cfDNA) is attractive for many applications, including detecting cancer, identifying the tissue of origin, and monitoring. A fundamental task underlying these applications is SNV calling from cfDNA, which is hindered by the very low tumor content. Thus sensitive and accurate detection of low-frequency mutations (<5%) remains challenging for existing SNV callers. Here we present cfSNV, a method incorporating multi-layer error suppression and hierarchical mutation calling, to address this challenge. Furthermore, by leveraging cfDNA's comprehensive coverage of tumor clonal landscape, cfSNV can profile mutations in subclones. In both simulated and real patient data, cfSNV outperforms existing tools in sensitivity while maintaining high precision. cfSNV enhances the clinical utilities of cfDNA by improving mutation detection performance in medium-depth sequencing data, therefore making Whole-Exome Sequencing a viable option. As an example, we demonstrate that the tumor mutation profile from cfDNA WES data can provide an effective biomarker to predict immunotherapy outcomes.

DOI: https://doi.org/10.1038/s41467-021-24457-2
Nat Commun. 2021 07 05. 12(1): 4132

A synthetic circuit for buffering gene dosage variation between individual mammalian cells.

Jin Yang, Jihwan Lee, Michelle A Land, Shujuan Lai, Oleg A Igoshin, François St-Pierre.

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.

DOI: https://doi.org/10.1038/s41467-021-23889-0
Can J Gastroenterol Hepatol. 2021 ;2021 5527315

Regulation of Mitochondrial Function by Natural Products for the Treatment of Metabolic Associated Fatty Liver Disease.

Tingting Shi, Liping Yu, Rangxiao Zhuang, Jianjun Xi, Ruoyu He, Yidan Shao, Jinsong Huang, Shourong Liu, Xingxin Yang.

Metabolic associated fatty liver disease (MAFLD) is a multifactorial systemic disorder that occurs in the absence of excessive alcohol consumption. The disease is characterized by fatty degeneration and fat accumulation in liver parenchymal cells, the incidence of which is increasing annually, particularly in younger adults. MAFLD is caused by genetic and metabolism related disorders, of which mitochondrial dysfunction is the major contributor. Natural products can relieve MAFLD through restoring mitochondrial function. In this article, we describe the relationship between mitochondria and MAFLD and discuss the beneficial effects of natural products as a future anti-MAFLD strategy. Significance Statement. We herein propose that the development of mitochondrial regulators/nutrients from natural products can remedy mitochondrial dysfunction which represents an attractive strategy for the treatment of MAFLD. Furthermore, the mitochondrial regulation of natural products can provide new insight into the underlying mechanisms of action of natural products used for future MAFLD therapeutics.

DOI: https://doi.org/10.1155/2021/5527315
Trends Cancer. 2021 Jul 01. pii: S2405-8033(21)00135-7. [Epub ahead of print]

High-Fat Diet Impacts on Tumor Development in the Gut.

Milou S van Driel, Sanne M van Neerven, Louis Vermeulen.

  A high-fat diet (HFD) directly acts on intestinal stem cells by increasing their numbers and proliferation, resulting in an elevated risk of developing colorectal cancer (CRC). In a recent study, Mana et al. revealed that HFD-mediated intestinal tumor formation can be reduced by inhibiting fatty acid oxidation.

Keywords:  PPARs; colorectal cancer; high-fat diet; intestinal stem cells; tumor initiation

DOI:  https://doi.org/10.1016/j.trecan.2021.06.005
J Proteome Res. 2021 Jul 08.

Ultrafast and Reproducible Proteomics from Small Amounts of Heart Tissue Enabled by Azo and timsTOF Pro.

Timothy J Aballo, David S Roberts, Jake A Melby, Kevin M Buck, Kyle A Brown, Ying Ge.

  Global bottom-up mass spectrometry (MS)-based proteomics is widely used for protein identification and quantification to achieve a comprehensive understanding of the composition, structure, and function of the proteome. However, traditional sample preparation methods are time-consuming, typically including overnight tryptic digestion, extensive sample cleanup to remove MS-incompatible surfactants, and offline sample fractionation to reduce proteome complexity prior to online liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Thus, there is a need for a fast, robust, and reproducible method for protein identification and quantification from complex proteomes. Herein, we developed an ultrafast bottom-up proteomics method enabled by Azo, a photocleavable, MS-compatible surfactant that effectively solubilizes proteins and promotes rapid tryptic digestion, combined with the Bruker timsTOF Pro, which enables deeper proteome coverage through trapped ion mobility spectrometry (TIMS) and parallel accumulation-serial fragmentation (PASEF) of peptides. We applied this method to analyze the complex human cardiac proteome and identified nearly 4000 protein groups from as little as 1 mg of human heart tissue in a single one-dimensional LC-TIMS-MS/MS run with high reproducibility. Overall, we anticipate this ultrafast, robust, and reproducible bottom-up method empowered by both Azo and the timsTOF Pro will be generally applicable and greatly accelerate the throughput of large-scale quantitative proteomic studies. Raw data are available via the MassIVE repository with identifier MSV000087476.

Keywords:  bottom-up proteomics; human heart proteomics; photocleavable surfactant; quantitative proteomics; sample preparation

DOI:  https://doi.org/10.1021/acs.jproteome.1c00446