bims-polyam 2020-08-09 papers

on Polyamines

Issue of 2020–08–09
seven papers selected by
Sebastian J. Hofer, University of Graz and Alexander Ivanov, Engelhardt Institute of Molecular Biology

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Anti-Tumor Efficacy of PD-1 Blockade.
Polyamine pathway activity promotes cysteine essentiality in cancer cells.
ESI-Q-TOF-MS determination of polyamines and related enzyme activity for elucidating cellular polyamine metabolism.
The rational discovery of multipurpose inhibitors of the ornithine decarboxylase.
Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed βH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.
The Neighboring Subunit Is Engaged to Stabilize the Substrate in the Active Site of Plant Arginases.
Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

Mol Cancer Ther. 2020 Aug 03. pii: molcanther.1116.2019. [Epub ahead of print]

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Anti-Tumor Efficacy of PD-1 Blockade.

Susan K Gilmour, Eric T Alexander, Kelsey Mariner, Julia Donnelly, Otto Phanstiel.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade, responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of co-treatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit anti-tumor immunity, and limit the efficacy of ICB. In this study we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the anti-tumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T-cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that co-treatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in cancer patients with resistant tumors.

DOI: https://doi.org/10.1158/1535-7163.MCT-19-1116
Nat Metab. 2020 Aug 03.

Polyamine pathway activity promotes cysteine essentiality in cancer cells.

Tong Zhang, Christin Bauer, Alice C Newman, Alejandro Huerta Uribe, Dimitris Athineos, Karen Blyth, Oliver D K Maddocks.

Cancer cells have high demands for non-essential amino acids (NEAAs), which are precursors for anabolic and antioxidant pathways that support cell survival and proliferation. It is well-established that cancer cells consume the NEAA cysteine, and that cysteine deprivation can induce cell death; however, the specific factors governing acute sensitivity to cysteine starvation are poorly characterized. Here, we show that that neither expression of enzymes for cysteine synthesis nor availability of the primary precursor methionine correlated with acute sensitivity to cysteine starvation. We observed a strong correlation between efflux of the methionine-derived metabolite methylthioadenosine (MTA) and sensitivity to cysteine starvation. MTA efflux results from genetic deletion of methylthioadenosine phosphorylase (MTAP), which is frequently deleted in cancers. We show that MTAP loss upregulates polyamine metabolism which, concurrently with cysteine withdrawal, promotes elevated reactive oxygen species and prevents cell survival. Our results reveal an unexplored metabolic weakness at the intersection of polyamine and cysteine metabolism.

DOI: https://doi.org/10.1038/s42255-020-0253-2
Anal Biochem. 2020 Jul 30. pii: S0003-2697(20)30363-8. [Epub ahead of print] 113831

ESI-Q-TOF-MS determination of polyamines and related enzyme activity for elucidating cellular polyamine metabolism.

Shun-Suke Moriya, Keijiro Samejima, Hideharu Taira, Kyoko Hiramatsu, Masao Kawakita.

  We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.

Keywords:  ESI-Q-TOF-MS; S-adenosylmethionine decarboxylase; ornithine decarboxylase; spermidine synthase; spermine synthase; stable isotope-labeling

DOI:  https://doi.org/10.1016/j.ab.2020.113831
FASEB J. 2020 Aug 07.

The rational discovery of multipurpose inhibitors of the ornithine decarboxylase.

Xiaoying Chai, Jingqiong Zhan, Jing Pan, Mengxi He, Bo Li, Jing Wang, Hongyan Ma, Yanlin Wang, Sen Liu.

  Metabolic reprograming is a hallmark of cancer, and the polyamine metabolic network is dysregulated in many cancers. Ornithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis in the polyamine metabolic network. In many cancer cells, ODC is over-expressed, so this enzyme has been an attracting anti-cancer drug target. In the catalysis axis (pathway), ODC converts ornithine to putrescine. Meanwhile, ODC's activity is regulated by protein-protein interactions (PPIs), including the ODC-OAZ1-AZIN1 PPI axis and its monomer-dimer equilibrium. Previous studies showed that when ODC's activity is inhibited, the PPIs might counteract the inhibition efficiency. Therefore, we proposed that multipurpose inhibitors that can simultaneously inhibit ODC's activity and perturb the PPIs would be very valuable as drug candidates and molecular tools. To discover multipurpose ODC inhibitors, we established a computational pipeline by combining positive screening and negative screening. We used this pipeline for the forward screening of multipurpose ligands that might inhibit ODC's activity, block ODC-OAZ1 interaction and enhance ODC non-functional dimerization. With a combination of different experimental assays, we identified three multipurpose ODC inhibitors. At last, we showed that one of these inhibitors is a promising drug candidate. This work demonstrated that our computational pipeline is useful for discovering multipurpose ODC inhibitors, and multipurpose inhibitors would be very valuable. Similar with ODC, there are a lot of proteins in human proteome that act as both enzymes and PPI components. Therefore, this work is not only presenting new molecular tools for polyamine study, but also providing potential insights and protocols for discovering multipurpose inhibitors to target more important protein targets.

Keywords:  inhibitor; negative screening; ornithine decarboxylase; protein-protein interaction; stabilizer

DOI:  https://doi.org/10.1096/fj.202001222R
Int J Mol Sci. 2020 Jul 30. pii: E5427. [Epub ahead of print]21(15):

Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed βH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

Carlo Mischiati, Giordana Feriotto, Claudio Tabolacci, Fabio Domenici, Sonia Melino, Ilaria Borromeo, Cinzia Forni, Angelo De Martino, Simone Beninati.

  In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward βH-crystallins and in particular to the βB2- and mostly in βB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of βB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Keywords:  FAD-PAO; TG2; cataract; protein post-translation modification; rabbit eye lens; spermidine

DOI:  https://doi.org/10.3390/ijms21155427
Front Plant Sci. 2020 ;11 987

The Neighboring Subunit Is Engaged to Stabilize the Substrate in the Active Site of Plant Arginases.

Bartosz Sekula.

  Arginine acts as a precursor of polyamines in plants in two known pathways, agmatine and ornithine routes. It is decarboxylated to agmatine by arginine decarboxylase, and then transformed to putrescine by the consecutive action of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Alternatively, it can be hydrolyzed to ornithine by arginase and then decarboxylated by ornithine decarboxylase to putrescine. Some plants lack a functional ornithine pathway, but all have one or two arginases that can have dual cellular localization, in mitochondria and plastids. It was recently shown that arginases from Arabidopsis thaliana and soybean act also as agmatinases, thus they can produce putrescine directly from agmatine. Therefore, arginase (together with arginine decarboxylase) can complement putrescine production in plastids, providing a third polyamine biosynthesis pathway in plants. Phylogenetic analysis suggests that arginases, highly conserved in the plant kingdom, create the only group of enzymes recognized in the family of ureohydrolases in plants. Arginases are metalloenzymes with binuclear manganese cluster in the active site. In this work, two arginases from A. thaliana and Medicago truncatula are structurally characterized and their binding properties are discussed. Crystal structures with bound ornithine show that plant hexameric arginases engage a long loop from the neighboring subunit to stabilize α-amino and carboxyl groups of the ligand. This unique ligand binding mode is unobserved in arginases from other domains of life. Structural analysis shows that substrate binding by residues from two neighboring subunits might also characterize some prokaryotic agmatinases. This feature of plant arginases is most likely the determinant of their ability to recognize not only arginine but also agmatine as their substrates, thus, to act as arginase and agmatinase.

Keywords:  agmatinase; arginine amidinohydrolase; polyamine biosynthesis; urea cycle; ureohydrolases

DOI:  https://doi.org/10.3389/fpls.2020.00987
Int J Mol Sci. 2020 Jul 31. pii: E5509. [Epub ahead of print]21(15):

Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

Meirong Chen, Zuoqi Gai, Chiaki Okada, Yuxin Ye, Jian Yu, Min Yao.

  The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288-P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.

Keywords:  IF5A; NAD+; deoxyhypusine synthase; hypusine modification; structure; translation factor

DOI:  https://doi.org/10.3390/ijms21155509