Douglas J. Lamont

University of Dundee

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Mechanism of Activation of AMPK by Cordycepin

Cell chemical biology (2020)

Simon A. Hawley Fiona A. Ross Fiona M. Russell Abdelmadjid Atrih Douglas J. Lamont

Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.

Cordycepin

10.1016/j.chembiol.2020.01.004

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Citations (51)

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

Eukaryotic Cell (2009)

Maria Lucia Sampaio Güther Kenneth L. Beattie Douglas J. Lamont John R. James Alan R. Prescott

ABSTRACT A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB 3 H 4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N -acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB 3 H 4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

Glycoconjugate

Galactose oxidase

10.1128/ec.00178-09

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Citations (25)

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

Journal of Biological Chemistry (2005)

Catherine Moss S. Matthews Douglas J. Lamont Colin Watts

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Deamidation

Endopeptidase

Antigen processing

10.1074/jbc.m501241200

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Citations (74)

Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix

PLoS neglected tropical diseases (2019)

Eliciane Cevolani Mattos Gisele André Baptista Canuto Nubia Carolina Manchola Rubens Daniel Miserani Magalhães Thomas W.M. Crozier

Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

Hexokinase

Phosphoglycerate kinase

10.1371/journal.pntd.0007103

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Citations (29)

Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

Molecular and Cellular Biology (2016)

Sharadha Dayalan Naidu Calum Sutherland Ying Zhang Ana Marı́a Risco Laureano de la Vega

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.

HSF1

Heat shock factor

Phosphorylation cascade

10.1128/mcb.00292-16

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Citations (70)

Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis

Cell Reports (2022)

Anees Rahman Cheratta Faisal Thayyullathil Simon A. Hawley Fiona A. Ross Abdelmajdid Atrih

AMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

AMP-Activated Protein Kinase

10.1016/j.celrep.2022.110761

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Citations (22)

DIA label-free proteomic analysis of murine bone-marrow-derived macrophages

STAR Protocols (2022)

Christa P. Baker Iain R. Phair Alejandro J. Brenes Abdelmadjid Atrih Dylan G. Ryan

Sample (material)

Mass cytometry

10.1016/j.xpro.2022.101725

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Citations (10)

Phenformin, But Not Metformin, Delays Development of T Cell Acute Lymphoblastic Leukemia/Lymphoma via Cell-Autonomous AMPK Activation

Cell Reports (2019)

Diana Vara‐Ciruelos Madhumita Dandapani Fiona M. Russell Katarzyna M. Grzes Abdelmadjid Atrih

AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers.

Phenformin

10.1016/j.celrep.2019.03.067

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Citations (60)

SL RNA overexpression leads to abnormal regulation of selected proteins and loss of virulence

Juliano S. Toledo Tânia Paula Aquino Defina Kenneth A. Beattie Douglas J. Lamont Ângela K. Cruz

Source

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Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals

Proceedings of the National Academy of Sciences (2010)

Michael McMahon Douglas J. Lamont Kenneth A. Beattie John D. Hayes

Recognition and repair of cellular damage is crucial if organisms are to survive harmful environmental conditions. In mammals, the Keap1 protein orchestrates this response, but how it perceives adverse circumstances is not fully understood. Herein, we implicate NO, Zn 2+ , and alkenals, endogenously occurring chemicals whose concentrations increase during stress, in this process. By combining molecular modeling with phylogenetic, chemical, and functional analyses, we show that Keap1 directly recognizes NO, Zn 2+ , and alkenals through three distinct sensors. The C288 alkenal sensor is of ancient origin, having evolved in a common ancestor of bilaterans. The Zn 2+ sensor minimally comprises H225, C226, and C613. The most recent sensor, the NO sensor, emerged coincident with an expansion of the NOS gene family in vertebrates. It comprises a cluster of basic amino acids (H129, K131, R135, K150, and H154) that facilitate S -nitrosation of C151. Taken together, our data suggest that Keap1 is a specialized sensor that quantifies stress by monitoring the intracellular concentrations of NO, Zn 2+ , and alkenals, which collectively serve as second messengers that may signify danger and/or damage.

KEAP1

Environmental stress

10.1073/pnas.1007387107

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Citations (406)