bims-polyam 2022-10-09 papers

Issue of 2022–10–09
five papers selected by
Sebastian J. Hofer, University of Graz

ACS Omega. 2022 Sep 27. 7(38): 34665-34675

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

X Edward Zhou, Chad R Schultz, Kelly Suino Powell, Amy Henrickson, Jared Lamp, Joseph S Brunzelle, Borries Demeler, Irving E Vega, André S Bachmann, Karsten Melcher.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

DOI: https://doi.org/10.1021/acsomega.2c04702

JCI Insight. 2022 Oct 04. pii: e158879. [Epub ahead of print]

Pseudomonas aeruginosa utilises the host-derived polyamine spermidine to facilitate antimicrobial tolerance.

Chowdhury M Hasan, Sian Pottenger, Angharad E Green, Adrienne A Cox, Jack S White, Trevor Jones, Craig Winstanley, Aras Kadioglu, Megan H Wright, Daniel R Neill, Joanne L Fothergill.

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.

Keywords: Bacterial infections; Drug therapy; Infectious disease; Microbiology; Polyamines

DOI: https://doi.org/10.1172/jci.insight.158879

J Am Chem Soc. 2022 Oct 06.

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout.

Raphael R Steimbach, Corey J Herbst-Gervasoni, Severin Lechner, Tracy Murray Stewart, Glynis Klinke, Johannes Ridinger, Magalie N E Géraldy, Gergely Tihanyi, Jackson R Foley, Ulrike Uhrig, Bernhard Kuster, Gernot Poschet, Robert A Casero, Guillaume Médard, Ina Oehme, David W Christianson, Nikolas Gunkel, Aubry K Miller.

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

DOI: https://doi.org/10.1021/jacs.2c05030

Clin Transl Immunology. 2022 ;11(9): e1419

Autophagic flux restoration of senescent T cells improves antitumor activity of TCR-engineered T cells.

Chaoting Zhang, Yizhe Sun, Shance Li, Luyan Shen, Xia Teng, Yefei Xiao, Ping Zhou, Zheming Lu.

Objectives: Although adoptive cell therapy with T-cell receptor-engineered T cells (TCR-Ts) has mediated effective antitumor responses in several cancers, senescence of T cells could impair the therapeutic effect of TCR-Ts. Thus, it is essential to elucidate the characteristics of senescent TCR-Ts and how to subsequently improve their antitumor effect. Here, we focused on the influence of autophagy on TCR-Ts, since autophagy is tightly associated with the regulation of T-cell activation, proliferation and differentiation.
Methods: We first evaluated autophagy level of senescent TCR-Ts, and then the senescent TCR-Ts were expanded in vitro for 7 days with and without spermidine treatment, respectively. Furthermore, the proliferative potential, phenotypical characteristics and functionality of the propagated senescent TCR-Ts were analysed in vitro and in vivo after 7-day ex vivo expansion.
Results: We found that autophagic flux of senescent TCR-T cells was significantly impaired. The restoration of autophagic flux via spermidine treatment reduced the expression of inhibitory immunoreceptors (PD-1, TIM-3 or LAG-3), enhanced proliferation and effector functions and subsequently demonstrated the superior in vitro and in vivo antitumor activity of TCR-Ts.
Conclusion: These data suggest that spermidine treatment presents an opportunity to improve the antitumor effect of TCR-Ts for the treatment of solid tumors.

Keywords: TCR‐T; T‐cell receptor; autophagy; senescence; spermidine

DOI: https://doi.org/10.1002/cti2.1419

NPJ Precis Oncol. 2022 Oct 07. 6(1): 70

Pharmacometabolomics reveals urinary diacetylspermine as a biomarker of doxorubicin effectiveness in triple negative breast cancer.

Thomas J Velenosi, Kristopher W Krausz, Keisuke Hamada, Tiffany H Dorsey, Stefan Ambs, Shogo Takahashi, Frank J Gonzalez.

Triple-negative breast cancer (TNBC) patients receive chemotherapy treatment, including doxorubicin, due to the lack of targeted therapies. Drug resistance is a major cause of treatment failure in TNBC and therefore, there is a need to identify biomarkers that determine effective drug response. A pharmacometabolomics study was performed using doxorubicin sensitive and resistant TNBC patient-derived xenograft (PDX) models to detect urinary metabolic biomarkers of treatment effectiveness. Evaluation of metabolite production was assessed by directly studying tumor levels in TNBC-PDX mice and human subjects. Metabolic flux leading to biomarker production was determined using stable isotope-labeled tracers in TNBC-PDX ex vivo tissue slices. Findings were validated in 12-h urine samples from control (n = 200), ER+/PR+ (n = 200), ER+/PR+/HER2+ (n = 36), HER2+ (n = 81) and TNBC (n = 200) subjects. Diacetylspermine was identified as a urine metabolite that robustly changed in response to effective doxorubicin treatment, which persisted after the final dose. Urine diacetylspermine was produced by the tumor and correlated with tumor volume. Ex vivo tumor slices revealed that doxorubicin directly increases diacetylspermine production by increasing tumor spermidine/spermine N1-acetyltransferase 1 expression and activity, which was corroborated by elevated polyamine flux. In breast cancer patients, tumor diacetylspermine was elevated compared to matched non-cancerous tissue and increased in HER2+ and TNBC compared to ER+ subtypes. Urine diacetylspermine was associated with breast cancer tumor volume and poor tumor grade. This study describes a pharmacometabolomics strategy for identifying cancer metabolic biomarkers that indicate drug response. Our findings characterize urine diacetylspermine as a non-invasive biomarker of doxorubicin effectiveness in TNBC.

DOI: https://doi.org/10.1038/s41698-022-00313-4