Publications by authors named "David Lombard"

67 Publications

ER stress protein PERK promotes inappropriate innate immune responses and pathogenesis during RSV infection.

J Leukoc Biol 2021 Apr 18. Epub 2021 Apr 18.

Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

The activation of dendritic cells (DC) during respiratory viral infections is central to directing the immune response and the pathologic outcome. In these studies, the effect of RSV infection on development of ER stress responses and the impact on innate immunity was examined. The upregulation of ER stress was closely associated with the PERK pathway through the upregulation of CHOP in RSV infected DC. The inhibition of PERK corresponded with decreased EIF2a phosphorylation but had no significant effect on Nrf2 in DC, two primary pathways regulated by PERK. Subsequent studies identified that by blocking PERK activity in infected DC an altered ER stress response and innate cytokine profile was observed with the upregulation of IFNβ and IL-12, coincident to the down regulation of IL-1β. When mitochondria respiration was assessed in PERK deficient DC there were increased dysfunctional mitochondria after RSV infection that resulted in reduced oxygen consumption rates (OCR) and ATP production indicating altered cellular metabolism. Use of a CD11c targeted genetic deleted murine model, RSV infection was characterized by reduced inflammation and diminished mucus staining as well as reduced mucus-associated gene gob5 expression. The assessment of the cytokine responses showed decreased IL-13 and IL-17 along with diminished IL-1β in the lungs of PERK deficient infected mice. When PERK-deficient animals were assessed in parallel for lung leukocyte numbers, animals displayed significantly reduced myeloid and activated CD4 and CD8 T cell numbers. Thus, the PERK activation pathway may provide a rational target for altering the severe outcome of an RSV infection through modifying immune responses.
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http://dx.doi.org/10.1002/JLB.3A0520-322RRDOI Listing
April 2021

Melanoma models for the next generation of therapies.

Cancer Cell 2021 Feb 4. Epub 2021 Feb 4.

Center for Cancer Research, NCI, NIH, 37 Convent Drive, Bethesda, MD 20892, USA. Electronic address:

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
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http://dx.doi.org/10.1016/j.ccell.2021.01.011DOI Listing
February 2021

An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells.

Sci Rep 2020 10 12;10(1):17030. Epub 2020 Oct 12.

Human and Animal Physiology, Wageningen University & Research, Wageningen, 6708 WD, The Netherlands.

Succinylation is a novel post-translational modification identified on many proteins and is involved in multiple biological processes. Succinylation levels are dynamically regulated, balanced by succinylation and desuccinylation processes, and are closely connected to metabolic state in vivo. Sirtuins have been shown to possess NAD-dependent desuccinylation activity in vitro and in vivo, among which the desuccinylation activity of SIRT5 is most extensively studied. Our understanding of the response of succinylation levels to different metabolic conditions, is hampered by the lack of a fast NAD-dependent desuccinylation assay in a physiological context. In the present study, we therefore optimized and validated a fluorescence-based assay for measuring NAD-dependent desuccinylation activity in cell lysates. Our results demonstrated that shorter and stricter reaction time was critical to approach the initial rate of NAD-dependent desuccinylation activity in crude cell lysate systems, as compared to the desuccinylation reaction of purified His-SIRT5. Analysis of desuccinylation activity in SIRT5 knockout HEK293T cells confirmed the relevance of SIRT5 in cellular desuccinylation activity, as well as the presence of other NAD-dependent desuccinylase activities. In addition, we were able to analyse desuccinylation and deacetylation activity in multiple cell lines using this assay. We showed a remarkably higher desuccinylase activity, but not deacetylase activity, in proliferative cultured muscle and adipose cells in comparison with their differentiated counterparts. Our results reveal an alteration in NAD-dependent desuccinylation activity under different metabolic states.
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http://dx.doi.org/10.1038/s41598-020-72833-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7552388PMC
October 2020

High-throughput small molecule screening reveals Nrf2-dependent and -independent pathways of cellular stress resistance.

Sci Adv 2020 Oct 2;6(40). Epub 2020 Oct 2.

Department of Pathology, University of Michigan, Ann Arbor, MI, USA.

Aging is the dominant risk factor for most chronic diseases. Development of antiaging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in , although not in testing performed in worms and flies with a more restricted set of compounds. Transcriptomic analysis and genetic studies implicated Nrf2/SKN-1 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Small molecules identified in this work may represent attractive tools to elucidate mechanisms of stress resistance in mammalian cells.
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http://dx.doi.org/10.1126/sciadv.aaz7628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852388PMC
October 2020

Canagliflozin extends life span in genetically heterogeneous male but not female mice.

JCI Insight 2020 11 5;5(21). Epub 2020 Nov 5.

Sam and Ann Barshop Institute for Longevity and Aging Studies and Departments of Physiology and Molecular Medicine, UT Health San Antonio, San Antonio, Texas, USA; South Texas Veterans Healthcare System, San Antonio, Texas, USA.

Canagliflozin (Cana) is an FDA-approved diabetes drug that protects against cardiovascular and kidney diseases. It also inhibits the sodium glucose transporter 2 by blocking renal reuptake and intestinal absorption of glucose. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing Cana at 180 ppm at 7 months of age until their death. Cana extended median survival of male mice by 14%. Cana also increased by 9% the age for 90th percentile survival, with parallel effects seen at each of 3 test sites. Neither the distribution of inferred cause of death nor incidental pathology findings at end-of-life necropsies were altered by Cana. Moreover, although no life span benefits were seen in female mice, Cana led to lower fasting glucose and improved glucose tolerance in both sexes, diminishing fat mass in females only. Therefore, the life span benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in male mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e., slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.
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http://dx.doi.org/10.1172/jci.insight.140019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710304PMC
November 2020

High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions.

Commun Biol 2020 May 29;3(1):271. Epub 2020 May 29.

Rogel Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA.

Metabolic flux technology with the Seahorse bioanalyzer has emerged as a standard technique in cellular metabolism studies, allowing for simultaneous kinetic measurements of respiration and glycolysis. Methods to extend the utility and versatility of the metabolic flux assay would undoubtedly have immediate and wide-reaching impacts. Herein, we describe a platform that couples the metabolic flux assay with high-content fluorescence imaging to simultaneously provide means for normalization of respiration data with cell number; analyze cell cycle distribution; and quantify mitochondrial content, fragmentation state, membrane potential, and mitochondrial reactive oxygen species. Integration of fluorescent dyes directly into the metabolic flux assay generates a more complete data set of mitochondrial features in a single assay. Moreover, application of this integrated strategy revealed insights into mitochondrial function following PGC1a and PRC1 inhibition in pancreatic cancer and demonstrated how the Rho-GTPases impact mitochondrial dynamics in breast cancer.
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http://dx.doi.org/10.1038/s42003-020-0988-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260371PMC
May 2020

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mol Biol Cell 2020 05 26;31(11):1103-1111. Epub 2020 Mar 26.

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for or exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.
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http://dx.doi.org/10.1091/mbc.E19-10-0565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353162PMC
May 2020

Sirtuin 1 regulates mitochondrial function and immune homeostasis in respiratory syncytial virus infected dendritic cells.

PLoS Pathog 2020 02 27;16(2):e1008319. Epub 2020 Feb 27.

Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, Michigan, United States of America.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1), a NAD+ dependent deacetylase, has been associated with induction of autophagy, reprogramming cellular metabolism, and regulating immune mediators. In this study, we investigated the role of SIRT1 in bone marrow dendritic cell (BMDC) function during RSV infection. SIRT1 deficient (SIRT1 -/-) BMDC showed a defect in mitochondrial membrane potential (Δ⍦m) that worsens during RSV infection. This defect in Δ⍦m caused the generation of elevated levels of reactive oxygen species (ROS). Furthermore, the oxygen consumption rate (OCR) was reduced as assessed in Seahorse assays, coupled with lower levels of ATP in SIRT1-/- DC. These altered responses corresponded to altered innate cytokine responses in the SIRT1-/- DC in response to RSV infection. Reverse Phase Protein Array (RPPA) functional proteomics analyses of SIRT1-/- and WT BMDC during RSV infection identified a range of differentially regulated proteins involved in pathways that play a critical role in mitochondrial metabolism, autophagy, oxidative and ER stress, and DNA damage. We identified an essential enzyme, acetyl CoA carboxylase (ACC1), which plays a central role in fatty acid synthesis and had significantly increased expression in SIRT1-/- DC. Blockade of ACC1 resulted in metabolic reprogramming of BMDC that ameliorated mitochondrial dysfunction and reduced pathologic innate immune cytokines in DC. The altered DC responses attenuated Th2 and Th17 immunity allowing the appropriate generation of anti-viral Th1 responses both in vitro and in vivo during RSV infection thus reducing the enhanced pathogenic responses. Together, these studies identify pathways critical for appropriate DC function and innate immunity that depend on SIRT1-mediated regulation of metabolic processes.
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http://dx.doi.org/10.1371/journal.ppat.1008319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046194PMC
February 2020

"MPNST Epigenetics"-Response.

Mol Cancer Res 2019 10;17(10):2140

Department of Pathology, University of Michigan, Ann Arbor, Michigan.

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http://dx.doi.org/10.1158/1541-7786.MCR-19-0788DOI Listing
October 2019

SIRT3 Regulates Macrophage-Mediated Inflammation in Diabetic Wound Repair.

J Invest Dermatol 2019 12 15;139(12):2528-2537.e2. Epub 2019 Jun 15.

Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA; Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA. Electronic address:

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.
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http://dx.doi.org/10.1016/j.jid.2019.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185380PMC
December 2019

Malignant Peripheral Nerve Sheath Tumors: From Epigenome to Bedside.

Mol Cancer Res 2019 07 25;17(7):1417-1428. Epub 2019 Apr 25.

Department of Pathology and Institute of Gerontology, University of Michigan, Ann Arbor, Michigan.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas typically developing in the context of neurofibromatosis type 1 (NF-1). With the exception of surgical resection, these tumors are resistant to all current therapies, and unresectable, recurrent, or metastatic tumors are considered incurable. Preclinical studies have identified several novel candidate molecular targets for therapeutic intervention, but, to date, targeted therapies have proven ineffective. Recent studies have identified recurrent mutations in polycomb repressive complex 2 (PRC2) core components, embryonic ectoderm development protein (EED) and suppressor of zeste 12 homolog (SUZ12), in MPNST. These mutations result in global loss of the histone H3 lysine 27 trimethylation epigenetic mark, normally deposited by PRC2, and subsequent gain in acetylation at this residue. This altered chromatin state has been shown to promote MPNST malignancy; however, acetylation at this residue sensitizes MPNSTs to BRD4 and bromodomain and extra-terminal domain inhibition. Interestingly, the catalytic component of PRC2, enhancer of zeste homolog 2 (EZH2), is not mutated in MPNST, hinting that a noncanonical, PRC2-independent function of EZH2 may play a role in this cancer. This review examines the pathobiology of MPNST, the contribution of PRC2 subunits to this process, and the prospects for PRC2-related therapies for this cancer. IMPLICATIONS: Identification of mutations in the PRC2 components EED and SUZ12 in the majority of MPNSTs may imply noncanonical oncogenic activities of the intact component, EZH2, and provide new opportunities for therapeutic intervention.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610818PMC
July 2019

Combined MAPK Pathway and HDAC Inhibition Breaks Melanoma.

Cancer Discov 2019 Apr;9(4):469-471

Department of Pathology, University of Michigan, Ann Arbor, Michigan.

In this issue, Maertens and colleagues demonstrate that HDAC3 inhibition potentiates the effects of MAPK pathway inhibitors in melanoma, including difficult-to-treat - and -driven tumors, with expression serving as a biomarker for responsiveness to the BRAF/MEK/HDAC inhibitor combination. Mechanistically, this triple cocktail suppresses expression of genes involved in DNA repair, leading to enhanced killing of melanoma cells..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446927PMC
April 2019

A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells.

Cell Rep 2019 03;26(11):3061-3075.e6

Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Division of Gynecology-Oncology, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; Division of Hematology-Oncology, Department of Internal Medicine, Division of Gynecology-Oncology, Department of Obstetrics and Gynecology, and UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA; Magee-Womens Research Institute, Pittsburgh, PA, USA. Electronic address:

Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target. Although it remains controversial which of the 19 ALDH family members drive chemotherapy resistance, ALDH1A family members have been primarily linked with chemotherapy resistant and stemness. We identified two ALDH1A family selective inhibitors (ALDH1Ai). ALDH1Ai preferentially kills CD133 ovarian cancer stem-like cells (CSCs). ALDH1Ai induce necroptotic CSC death, mediated, in part, by the induction of mitochondrial uncoupling proteins and reduction in oxidative phosphorylation. ALDH1Ai is highly synergistic with chemotherapy, reducing tumor initiation capacity and increasing tumor eradication in vivo. These studies link ALDH1A with necroptosis and confirm the family as a critical therapeutic target to overcome chemotherapy resistance and improve patient outcomes.
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http://dx.doi.org/10.1016/j.celrep.2019.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061440PMC
March 2019

Association of the POT1 Germline Missense Variant p.I78T With Familial Melanoma.

JAMA Dermatol 2019 05;155(5):604-609

Experimental Cancer Genetics, The Wellcome Trust Sanger Institute, Hinxton, England.

Importance: The protection of telomeres 1 protein (POT1) is a critical component of the shelterin complex, a multiple-protein machine that regulates telomere length and protects telomere ends. Germline variants in POT1 have been linked to familial melanoma, and somatic mutations are associated with a range of cancers including cutaneous T-cell lymphoma (CTCL).

Objective: To characterize pathogenic variation in POT1 in families with melanoma to inform clinical management.

Design, Setting, And Participants: In this case study and pedigree evaluation, analysis of the pedigree of 1 patient with melanoma revealed a novel germline POT1 variant (p.I78T, c.233T>C, chromosome 7, g.124870933A>G, GRCh38) that was subsequently found in 2 other pedigrees obtained from the GenoMEL Consortium.

Main Outcomes And Measures: (1) Identification of the POT1 p.I78T variant; (2) evaluation of the clinical features and characteristics of patients with this variant; (3) analysis of 3 pedigrees; (4) genomewide single-nucleotide polymorphism genotyping of germline DNA; and (5) a somatic genetic analysis of available nevi and 1 melanoma lesion.

Results: The POT1 p.I78T variant was found in 3 melanoma pedigrees, all of persons who self-reported as being of Jewish descent, and was shown to disrupt POT1-telomere binding. A UV mutation signature was associated with nevus and melanoma formation in POT1 variant carriers, and somatic mutations in driver genes such as BRAF, NRAS, and KIT were associated with lesion development in these patients.

Conclusions And Relevance: POT1 p.I78T is a newly identified, likely pathogenic, variant meriting screening for in families with melanoma after more common predisposition genes such as CDKN2A have been excluded. It could also be included as part of gene panel testing.
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http://dx.doi.org/10.1001/jamadermatol.2018.3662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506889PMC
May 2019

Mitochondrial Deacetylase SIRT3 Plays an Important Role in Donor T Cell Responses after Experimental Allogeneic Hematopoietic Transplantation.

J Immunol 2018 12 2;201(11):3443-3455. Epub 2018 Nov 2.

Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109;

Allogeneic hematopoietic cell transplantation (allo-HCT) through its graft-versus-tumor (GVT) effects is a curative therapy against many hematological malignancies. However, GVT is linked to harmful graft-versus-host disease (GVHD) after allo-HCT. Both GVT and GVHD require allogeneic T cell responses, which is an energetically costly process that causes oxidative stress. Sirtuin 3 (SIRT3), a mitochondrial histone deacetylase (HDAC), plays an important role in cellular processes through inhibition of reactive oxygen species (ROS). Nonmitochondrial class of HDACs regulate T cell responses, but the role of mitochondrial HDACs, specifically SIRT3, on donor T cell responses after allo-HCT remains unknown. In this study, we report that SIRT3-deficient (SIRT3) donor T cells cause reduced GVHD severity in multiple clinically relevant murine models. The GVHD protective effect of allogeneic SIRT3 T cells was associated with a reduction in their activation, reduced CXCR3 expression, and no significant impact on cytokine secretion or cytotoxic functions. Intriguingly, the GVHD protective effect of SIRT3 T cells was associated with a reduction in ROS production, which is contrary to the effect of SIRT3 deficiency on ROS production in other cells/tissues and likely a consequence of their deficient activation. Notably, the reduction in GVHD in the gastrointestinal tract was not associated with a substantial reduction in the GVT effect. Collectively, these data reveal that SIRT3 activity promotes allogeneic donor T cell responses and ROS production without altering T cell cytokine or cytolytic functions and identify SIRT3 as a novel target on donor T cells to improve outcomes after allo-HCT.
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http://dx.doi.org/10.4049/jimmunol.1800148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240608PMC
December 2018

Functions of the sirtuin deacylase SIRT5 in normal physiology and pathobiology.

Crit Rev Biochem Mol Biol 2018 06 11;53(3):311-334. Epub 2018 Apr 11.

a Department of Pathology , University of Michigan , Ann Arbor , MI , USA.

Sirtuins are NAD-dependent protein deacylases/ADP-ribosyltransferases that have emerged as candidate targets for new therapeutics to treat metabolic disorders and other diseases, including cancer. The sirtuin SIRT5 resides primarily in the mitochondrial matrix and catalyzes the removal of negatively charged lysine acyl modifications; succinyl, malonyl, and glutaryl groups. Evidence has now accumulated to document the roles of SIRT5 as a significant regulator of cellular homeostasis, in a context- and cell-type specific manner, as has been observed previously for other sirtuin family members. SIRT5 regulates protein substrates involved in glycolysis, the TCA cycle, fatty acid oxidation, electron transport chain, ketone body formation, nitrogenous waste management, and ROS detoxification, among other processes. SIRT5 plays pivotal roles in cardiac physiology and stress responses and is involved in the regulation of numerous aspects of myocardial energy metabolism. SIRT5 is implicated in neoplasia, as both a tumor promoter and suppressor in a context-specific manner, and may serve a protective function in the setting of neurodegenerative disorders. Here, we review the current understanding of functional impacts of SIRT5 on its metabolic targets, and its molecular functions in both normal and pathological conditions. Finally, we will discuss the potential utility of SIRT5 as a drug target and also summarize the current status, progress, and challenges in developing small molecule compounds to modulate SIRT5 activity with high potency and specificity.
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http://dx.doi.org/10.1080/10409238.2018.1458071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233320PMC
June 2018

Sirtuin 6 Builds a Wall Against Inflammation, Trumping Diabetes.

Diabetes 2017 10;66(10):2535-2537

Department of Pathology, University of Michigan, Ann Arbor, MI

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http://dx.doi.org/10.2337/dbi17-0025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5606319PMC
October 2017

Emerging Roles for SIRT5 in Metabolism and Cancer.

Antioxid Redox Signal 2018 03 26;28(8):677-690. Epub 2017 Oct 26.

1 Department of Pathology, University of Michigan , Ann Arbor, Michigan.

Significance: Developing evidence in the literature suggests that sirtuin 5 (SIRT5) may be involved in metabolic reprogramming, an emerging hallmark of cancer by which neoplastic cells reconfigure their metabolism to support the anabolic demands of rapid cell division. SIRT5 is one of the seven members of the nicotinamide adenine dinucleotide-dependent sirtuin family of lysine deacetylases. It removes succinyl, malonyl, and glutaryl groups from protein targets within the mitochondrial matrix and other subcellular compartments. SIRT5 substrates include a number of proteins integral to metabolism. Recent Advances: New work has begun to elucidate the roles of SIRT5 in glycolysis, tricarboxylic acid cycle, fatty acid oxidation, nitrogen metabolism, pentose phosphate pathway, antioxidant defense, and apoptosis.

Critical Issues: In this study, we summarize biological functions of SIRT5 reported in normal tissues and in cancer and discuss potential mechanisms whereby SIRT5 may impact tumorigenesis, particularly focusing on its reported roles in metabolic reprogramming. Finally, we review current efforts to target SIRT5 pharmacologically.

Future Directions: The biological significance of SIRT5 has been elucidated in the context of only an extremely small fraction of its targets and interactors. There is no doubt that further studies in this area will provide a wealth of insights into functions of SIRT5 and its targets in normal and neoplastic cells. Antioxid. Redox Signal. 28, 677-690.
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http://dx.doi.org/10.1089/ars.2017.7264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824490PMC
March 2018

ACSF3 and Mal(onate)-Adapted Mitochondria.

Cell Chem Biol 2017 Jun;24(6):649-650

Ben May Department for Cancer Research, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

In this issue of Cell Chemical Biology, Bowman and colleagues show that the mitochondrial enzyme ACSF3 generates malonyl-CoA from malonate, in turn regulating metabolic flux and mitochondrial protein malonylation (Bowman et al., 2017). The study reveals a mechanism to generate mitochondrial malonyl-CoA and how this molecule impacts mitochondrial biology.
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http://dx.doi.org/10.1016/j.chembiol.2017.06.004DOI Listing
June 2017

Cycling around Lysine Modifications.

Trends Biochem Sci 2017 07 6;42(7):501-503. Epub 2017 Jun 6.

Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA; Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Recent studies have revealed the existence of a plethora of previously unknown protein acyl-lysine modifications, affecting the functions of targets involved in diverse cellular processes. A recent study from the Hirschey laboratory has provided new chemical insights into the mechanisms of protein acylation.
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http://dx.doi.org/10.1016/j.tibs.2017.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518615PMC
July 2017

For Certain, SIRT4 Activities!

Trends Biochem Sci 2017 07 3;42(7):499-501. Epub 2017 Jun 3.

Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA; Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Despite the fact that SIRT4 regulates important biological processes, its primary enzymatic activity has remained ambiguous. A recent study by Anderson, Huynh et al. has uncovered deacylase activities of SIRT4 towards newly described lysine modifications derived from reactive acyl-CoAs generated in leucine catabolism.
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http://dx.doi.org/10.1016/j.tibs.2017.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518788PMC
July 2017

SIRT3 as a regulator of hepatic autophagy.

Hepatology 2017 09 20;66(3):700-702. Epub 2017 Jul 20.

Department of Molecular and Integrative Physiology, Michigan Medicine, University of Michigan, Ann Arbor, MI.

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http://dx.doi.org/10.1002/hep.29271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570665PMC
September 2017

Generation and Purification of Catalytically Active Recombinant Sirtuin5 (SIRT5) Protein.

Methods Mol Biol 2016 ;1436:241-57

Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA.

Sirtuin-family deacylases promote health and longevity in mammals. The sirtuin SIRT5 localizes predominantly to the mitochondrial matrix. SIRT5 preferentially removes negatively charged modifications from its target lysines: succinylation, malonylation, and glutarylation. It regulates protein substrates involved in glucose oxidation, ketone body formation, ammonia detoxification, fatty acid oxidation, and ROS management. Like other sirtuins, SIRT5 has recently been linked with neoplasia. Therefore, targeting SIRT5 pharmacologically could conceivably provide new avenues for treatment of metabolic disease and cancer, necessitating development of SIRT5-selective modulators. Here we describe the generation of SIRT5 bacterial expression plasmids, and their use to express and purify catalytically active and inactive forms of SIRT5 protein from E. coli. Additionally, we describe an approach to assay the catalytic activity of purified SIRT5, potentially useful for identification and validation of SIRT5-specific modulators.
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http://dx.doi.org/10.1007/978-1-4939-3667-0_16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890613PMC
December 2017

Metabolic Regulation of Gene Expression by Histone Lysine β-Hydroxybutyrylation.

Mol Cell 2016 04;62(2):194-206

Ben May Department for Cancer Research, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

Here we report the identification and verification of a β-hydroxybutyrate-derived protein modification, lysine β-hydroxybutyrylation (Kbhb), as a new type of histone mark. Histone Kbhb marks are dramatically induced in response to elevated β-hydroxybutyrate levels in cultured cells and in livers from mice subjected to prolonged fasting or streptozotocin-induced diabetic ketoacidosis. In total, we identified 44 histone Kbhb sites, a figure comparable to the known number of histone acetylation sites. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbhb is a mark enriched in active gene promoters and that the increased H3K9bhb levels that occur during starvation are associated with genes upregulated in starvation-responsive metabolic pathways. Histone β-hydroxybutyrylation thus represents a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and diverse functions of β-hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.
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http://dx.doi.org/10.1016/j.molcel.2016.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540445PMC
April 2016

Finding Ponce de Leon's Pill: Challenges in Screening for Anti-Aging Molecules.

F1000Res 2016 29;5. Epub 2016 Mar 29.

Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA; Institute of Gerontology, University of Michigan, Ann Arbor, MI, 48109, USA.

Aging is characterized by the progressive accumulation of degenerative changes, culminating in impaired function and increased probability of death. It is the major risk factor for many human pathologies - including cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases - and consequently exerts an enormous social and economic toll. The major goal of aging research is to develop interventions that can delay the onset of multiple age-related diseases and prolong healthy lifespan (healthspan). The observation that enhanced longevity and health can be achieved in model organisms by dietary restriction or simple genetic manipulations has prompted the hunt for chemical compounds that can increase lifespan. Most of the pathways that modulate the rate of aging in mammals have homologs in yeast, flies, and worms, suggesting that initial screening to identify such pharmacological interventions may be possible using invertebrate models. In recent years, several compounds have been identified that can extend lifespan in invertebrates, and even in rodents. Here, we summarize the strategies employed, and the progress made, in identifying compounds capable of extending lifespan in organisms ranging from invertebrates to mice and discuss the formidable challenges in translating this work to human therapies.
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http://dx.doi.org/10.12688/f1000research.7821.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813637PMC
April 2016

Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples.

Anal Bioanal Chem 2016 Jan 3;408(3):721-31. Epub 2015 Dec 3.

Department of Chemistry, University of Michigan, 930 N University Ave, Ann Arbor, MI, 48109, USA.

Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that catalyzes removal of post-translational modifications, such as succinyl and malonyl moieties, on lysine residues. In light of SIRT5's roles in regulating metabolism, and its reported oncogenic functions, SIRT5 modulators would be valuable tools for basic biological research and perhaps clinically. Several fluorescence assays for sirtuin modulators have been developed; however, the use of fluorogenic substrates has the potential to cause false positive results due to interactions of engineered substrates with enzyme or test compounds. Therefore, development of high-throughput screening (HTS) assays based on other methods is valuable. In this study, we report the development of a SIRT5 assay using microchip electrophoresis (MCE) for identification of SIRT5 modulators. A novel SIRT5 substrate based on succinate dehydrogenase (SDH) was developed to allow rapid and efficient separation of substrate and product peptide. To achieve high throughput, samples were injected onto the microchip using a droplet-based scheme. By coupling this approach to existing HTS sample preparation workflows, 1408 samples were analyzed at 0.5 Hz in 46 min. Using a 250 ms separation time, eight MCE injections could be made from each sample generating >11,000 electropherograms during analysis. Of the 1280 chemicals tested, eight were identified as inhibiting SIRT5 activity by at least 70% and verified by dose-response analysis.
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http://dx.doi.org/10.1007/s00216-015-9206-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712073PMC
January 2016

SIRT3 Deacetylates Ceramide Synthases: IMPLICATIONS FOR MITOCHONDRIAL DYSFUNCTION AND BRAIN INJURY.

J Biol Chem 2016 Jan 30;291(4):1957-1973. Epub 2015 Nov 30.

the Department of Neuroscience, Medical University of South Carolina, Charleston, South Carolina 29425,; From the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401,. Electronic address:

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2, and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2, and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, -2, and -6 are hyperacetylated in the mitochondria of SIRT3-null mice, and SIRT3 directly deacetylates the ceramide synthases in a NAD(+)-dependent manner that increases enzyme activity. Investigation of the SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3-mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR-induced blockade of the electron transport chain at the level of complex III, attenuated mitochondrial outer membrane permeabilization, and decreased reactive oxygen species generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation reduced the brain injury after IR. These data support the hypothesis that IR triggers SIRT3-dependent deacetylation of ceramide synthases and the elevation of ceramide, which could inhibit complex III, leading to increased reactive oxygen species generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest an important role of SIRT3 in mitochondrial dysfunction and brain injury after experimental stroke.
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http://dx.doi.org/10.1074/jbc.M115.668228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722471PMC
January 2016

Acetyl-ed question in mitochondrial biology?

EMBO J 2015 Nov 14;34(21):2597-600. Epub 2015 Sep 14.

Department of Pathology, University of Michigan, Ann Arbor, MI, USA.

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http://dx.doi.org/10.15252/embj.201592927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641526PMC
November 2015

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation.

Mol Cell Proteomics 2015 Nov 28;14(11):3056-71. Epub 2015 Aug 28.

From the Ben May Department of Cancer Research, University of Chicago, Chicago, Illinois 60637, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China,

The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD-/- fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases.
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http://dx.doi.org/10.1074/mcp.M115.048850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4638046PMC
November 2015

Sirtuin 1 Regulates Dendritic Cell Activation and Autophagy during Respiratory Syncytial Virus-Induced Immune Responses.

J Immunol 2015 Aug 8;195(4):1637-46. Epub 2015 Jul 8.

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1), an NAD(+)-dependent deacetylase, has been associated with the induction of autophagy and the regulation of inflammatory mediators. We found that Sirt1 was upregulated in mouse lung after RSV infection. Infected animals that received EX-527, a selective SIRT1 inhibitor, displayed exacerbated lung pathology, with increased mucus production, elevated viral load, and enhanced Th2 cytokine production. Gene expression analysis of isolated cell populations revealed that Sirt1 was most highly upregulated in RSV-treated dendritic cells (DCs). Upon RSV infection, EX-527-treated DCs, Sirt1 small interfering RNA-treated DCs, or DCs from conditional knockout (Sirt1(f/f)-CD11c-Cre(+)) mice showed downregulated inflammatory cytokine gene expression and attenuated autophagy. Finally, RSV infection of Sirt1(f/f)-CD11c-Cre(+) mice resulted in altered lung and lymph node cytokine responses, leading to exacerbated pathology. These data indicate that SIRT1 promotes DC activation associated with autophagy-mediated processes during RSV infection, thereby directing efficient antiviral immune responses.
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http://dx.doi.org/10.4049/jimmunol.1500326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530059PMC
August 2015