Polyamine Transport System Research Articles

Polyamine Derived Photosensitizer: A Novel Approach for Photodynamic Therapy of Cancer.

Polyamines play a pivotal role in cancer cell proliferation. The excessive polyamine requirement of these malignancies is satisfied through heightened biosynthesis and augmented extracellular uptake via the polyamine transport system (PTS) present on the cell membrane. Meanwhile, photodynamic therapy (PDT) emerges as an effective anti-cancer treatment devoid of drug resistance. Recognizing these intricacies, our study devised a novel polyamine-derived photosensitizer (PS) for targeted photodynamic treatment, focusing predominantly on pancreatic cancer cells. We synthesized and evaluated novel spermine-derived fluorescent probes (N2) and PS (N3), exhibiting selectivity towards pancreatic cancer cells via PTS. N3 showed minimal dark toxicity but significant phototoxicity upon irradiation, effectively causing cell death in vitro. A significant reduction in tumor volume was observed post-treatment with no pronounced dark toxicity using the pancreatic cancer CDX mouse model, affirming the therapeutic potential of N3. Overall, our findings introduce a promising new strategy for cancer treatment, highlighting the potential of polyamine-derived PSs in PDT.

A PET probe targeting polyamine transport system for precise tumor diagnosis and therapy

Polyamine metabolism dysregulation is a hallmark of many cancers, offering a promising avenue for early tumor theranostics. This study presents the development of a nuclear probe derived from spermidine (SPM) for dual-purpose tumor PET imaging and internal radiation therapy. The probe, radiolabeled with either [68Ga]Ga for diagnostic applications or [177Lu]Lu for therapeutic use, was synthesized with exceptional purity, stability, and specific activity. Extensive testing involving 12 different tumor cell lines revealed remarkable specificity towards B16 melanoma cells, showcasing outstanding tumor localization and target-to-non-target ratio. Mechanistic investigations employing polyamines, non-labeled precursor, and polyamine transport system (PTS) inhibitor, consistently affirmed the probeʼs targetability through recognition of the PTS. Notably, while previous reports indicated PTS upregulation in various tumor types for targeted therapy, this study observed no positive signals, highlighting a concentration-dependent discrepancy between targeting for therapy and diagnosis. Furthermore, when labeled with [177Lu], the probe demonstrated its therapeutic potential by effectively controlling tumor growth and extending mouse survival. Investigations into biodistribution, excretion, and biosafety in healthy humans laid a robust foundation for clinical translation. This study introduces a versatile SPM-based nuclear probe with applications in precise tumor theranostics, offering promising prospects for clinical implementation.

AMXT-1501 targets membrane phospholipids against Gram-positive and -negative multidrug-resistant bacteria

ABSTRACT The rapid proliferation of multidrug-resistant (MDR) bacterial pathogens poses a serious threat to healthcare worldwide. Carbapenem-resistant (CR) Enterobacteriaceae, which have near-universal resistance to available antimicrobials, represent a particularly concerning issue. Herein, we report the identification of AMXT-1501, a polyamine transport system inhibitor with antibacterial activity against Gram-positive and -negative MDR bacteria. We observed minimum inhibitory concentration (MIC)50/MIC90 values for AMXT-1501 in the range of 3.13–12.5 μM (2.24–8.93 μg /mL), including for methicillin-resistant Staphylococcus aureus (MRSA), CR Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. AMXT-1501 was more effective against MRSA and CR E. coli than vancomycin and tigecycline, respectively. Subinhibitory concentrations of AMXT-1501 reduced the biofilm formation of S. aureus and Enterococcus faecalis. Mechanistically, AMXT-1501 exposure damaged microbial membranes and increased membrane permeability and membrane potential by binding to cardiolipin (CL) and phosphatidylglycerol (PG). Importantly, AMXT-1501 pressure did not induce resistance readily in the tested pathogens.

ATP13A4 Upregulation Drives the Elevated Polyamine Transport System in the Breast Cancer Cell Line MCF7.

Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). Thus far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether the upregulation of candidate polyamine transporters of the P5B transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells presented elevated expression of a previously uncharacterized P5B-ATPase, ATP13A4, which was responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells were more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, the overexpression of ATP13A4 WT in MCF10A cells induced a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labeled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we established ATP13A4 as a new polyamine transporter in the human PTS and showed that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS.

Abstract 1589: Characterization of polyaminohydroxamic acids and polyaminobenzamides as inhibitors of HDAC10

Abstract Elevated polyamine levels are essential for continual proliferation of cancer cells. Cytosolic histone deacetylase-10 (HDAC10) specifically deacetylates the modified polyamine N8-acetylspermidine (N8-AcSpd), of which extracellular sources can be abundant, particularly in the colonic lumen. We previously demonstrated that exogenously supplied N8-AcSpd can support tumor cell growth through HDAC10-mediated deacetylation into spermidine, which is then redistributed within the polyamine interconversion pathway to maintain homeostasis.In the current study, we investigate the utility of polyamine-based hydroxamic acids (PAHAs) and benzamides (PABAs) as pharmacological inhibitors of HDAC10-mediated polyamine deacetylation. Incorporation of the polyamine moiety enables these compounds to enter the cell selectively through the polyamine transport system, which is upregulated in cancer cells. A total of 42 compounds, including 21 PAHAs and 21 PABAs, were screened for HDAC10 inhibition using a cell-based growth assay in which polyamine-depleted, growth-arrested cells are exposed to N8-AcSpd: in the presence of HDAC10 activity, this polyamine is deacetylated and proliferation is restored. Several compounds in our screen inhibited HDAC10 activity in this assay, as indicated by a lack of growth rescue, and were selected for further analysis in which treatment was expanded over a range of doses, thereby allowing a dose-dependent evaluation and comparison of sensitivity. HPLC-based analysis of cell lysates functionally confirmed that deacetylation of N8-AcSpd into spermidine was inhibited by the test compounds. Finally, specificity of the selected PAHAs and PABAs for HDAC10 was evaluated by immunoblotting for increased acetylation of the substrates of related HDACs, including histones H3 and H4, and alpha-tubulin. These studies provide a basis for future drug design strategies targeting the polyamine deacetylase activity of HDAC10 as an antitumor strategy, which may be particularly important in the context of polyamine-blocking therapies. Citation Format: Tracy Murray Stewart, Jackson R. Foley, Patrick M. Woster, Robert A. Casero. Characterization of polyaminohydroxamic acids and polyaminobenzamides as inhibitors of HDAC10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1589.

Novel green fluorescent polyamines to analyze ATP13A2 and ATP13A3 activity in the mammalian polyamine transport system.

Novel green fluorescent polyamines to analyze ATP13A2 and ATP13A3 activity in the mammalian polyamine transport system.

Elucidating the Role of Chmp1 Overexpression in the Transport of Polyamines in Drosophila melanogaster.

Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS.

Polyamine-Based Nanostructures Share Polyamine Transport Mechanisms with Native Polyamines and Their Analogues: Significance for Polyamine-Targeted Therapy.

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including cell proliferation, survival, and protection from oxidative stress. Polyamine homeostasis is tightly regulated through coordinated biosynthesis, catabolism, and transport. Due to their continual proliferation, cancer cells maintain elevated intracellular polyamine pools. Both polyamine metabolism and transport are commonly dysregulated in cancer, and as such, polyamine analogues are a promising strategy for exploiting the increased polyamine requirement of cancer cells. One potential polyamine analogue resistance mechanism is the downregulation of the poorly defined polyamine transport system. Recent advances in nanomedicine have produced nanostructures with polyamine analogue-based backbones (nanopolyamines). Similar nanostructures with non-polyamine backbones have been shown to be transported by endocytosis. As these polyamine-based nanoparticles could be a method for polyamine analogue delivery that bypasses polyamine transport, we designed the current studies to determine the efficacy of polyamine-based nanoparticles in cells lacking intact polyamine transport. Utilizing polyamine transport-deficient derivatives of lung adenocarcinoma lines, we demonstrated that cells unable to transport natural polyamines were also resistant to nanopolyamine-induced cytotoxicity. This resistance was a result of transport-deficient cells being incapable of importing and accumulating nanopolyamines. Pharmacological modulation of polyamine transport confirmed these results in polyamine transport competent cells. These studies provide additional insight into the polyamine transport pathway and suggest that receptor-mediated endocytosis is a likely mechanism of transport for higher-order polyamines, polyamine analogues and the nanopolyamines.

Nanospermidine in Combination with Nanofenretinide Induces Cell Death in Neuroblastoma Cell Lines.

A new strategy to cause cell death in tumors might be the increase of intracellular polyamines at concentrations above their physiological values to trigger the production of oxidation metabolites at levels exceeding cell tolerance. To test this hypothesis, we prepared nanospermidine as a carrier for spermidine penetration into the cells, able to escape the polyamine transport system that strictly regulates intracellular polyamine levels. Nanospermidine was prepared by spermidine encapsulation in nanomicelles and was characterized by size, zeta potential, loading, dimensional stability to dilution, and stability to spermidine leakage. Antitumor activity, ROS production, and cell penetration ability were evaluated in vitro in two neuroblastoma cell lines (NLF and BR6). Nanospermidine was tested as a single agent and in combination with nanofenretinide. Free spermidine was also tested as a comparison. The results indicated that the nanomicelles successfully transported spermidine into the cells inducing cell death in a concentration range (150–200 μM) tenfold lower than that required to provide similar cytotoxicity with free spermidine (1500–2000 μM). Nanofenretinide provided a cytostatic effect in combination with the lowest nanospermidine concentrations evaluated and slightly improved nanospermidine cytotoxicity at the highest concentrations. These data suggest that nanospermidine has the potential to become a new approach in cancer treatment. At the cellular level, in fact, it exploits polyamine catabolism by means of biocompatible doses of spermidine and, in vivo settings, it can exploit the selective accumulation of nanomedicines at the tumor site. Nanofenretinide combination further improves its efficacy. Furthermore, the proven ability of spermidine to activate macrophages and lymphocytes suggests that nanospermidine could inhibit immunosuppression in the tumor environment.

The Loss of ALDH9A1 Is a Significant Source of Endogenous DNA Damage Which May be Reversed By the Inhibition of Polyamine Transport System

Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome and is caused by impaired DNA interstrand crosslink repair. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage likely play an important role in the pathogenesis of FA.We previously identified loss of ALDH9A1 as a significant source of endogenous DNA damage using a metabolism-focused CRISPR knockout (KO) screen. This finding was validated using Jurkat cells as well as human hematopoietic stem and progenitor cells. Here, we present updates of our project.To determine whether endogenous DNA damage was induced by the combined loss of FANCD2 and ALDH9A1, we investigated markers of DNA damage in bulk-edited cells. We found that the numbers of chromosomal breaks, 53BP1 foci, and gamma-H2AX foci were increased in FANCD2-/-ALDH9A1-/- cells, compared with single KO or wildtype (WT) controls, in the absence of an exogenous DNA damaging agent. These findings are consistent with spontaneously increased basal levels of DNA damage in FANCD2-/-ALDH9A1-/- cells.To study in vivo BMF and tumorigenesis phenotypes of ALDH9A1 deficiency in the setting of FA, we generated a mouse model. Fanca-/-Aldh9a1-/- mice showed the lowest frequency of long-term hematopoietic stem cells and lineage-negative Sca-1-positive cKit-positive cells, however the differences were not significant compared with control groups. While we did not observe aplastic anemia or leukemia, we found a higher incidence of solid tumors, most notably ovarian tumors and hepatocellular carcinoma in aged Fanca-/-Aldh9a1-/- mice. This suggests that the level of endogenous reactive aldehydes created by ALDH9A1 deficiency in mouse is not high enough to cause full-blown hematopoietic phenotypes, most likely due to redundant detoxification pathways. However, in tissues where ALDH9A1 is active and non-redundant, the low level of endogenous DNA damage accumulating over time causes solid tumors.To identify the specific reactive aldehydes responsible for DNA damage in ALDH9A1 deficiency, we performed a growth selection screen using FANCD2-/-ALDH9A1-/- cells. We found that the loss of ATP13A3 conferred survival advantage to FANCD2-/-ALDH9A1-/- cells. ATP13A3 transports endocytosed polyamines into the cytosol where polyamines can be metabolized by serum amine oxidases into 3-aminopropanal, a reactive aminoaldehyde. 3-aminopropanal also undergoes spontaneous decomposition to acrolein, a well-known reactive aldehyde carcinogen. Finding that the loss of ATP13A3 rescues that FANCD2-/-ALDH9A1-/- cells indicates that 3-aminopropanal and/or acrolein induces endogenous DNA damage requiring the Fanconi anemia pathway function for repair.Finally, to determine the contribution of ALDH9A1 variants to clinical manifestations of FA patients, we performed targeted sequencing of DNA from FA patients. We identified five missense variants, four of which had high CADD scores (>20). ALDH9A1 cDNA containing missense variants with high CADD scores expressed in ALDH9A1-/- Jurkat cells resulted in lower protein expression than the WT cDNA. Cell culture supernatant from cells expressing the variant cDNAs also had increased aldehyde levels as assessed by fluorometric assays, suggesting decreased enzymatic activity of the variant proteins. The patients with ALDH9A1 missense variants with high CADD scores had either early hematologic onset of FA (n=3; two patients before age 1 and one patient before age 4) or AML (n=1).In conclusion, we showed that the loss of ALDH9A1 generates endogenous DNA damage necessitating the FA pathway for its repair. Synthetic lethality caused by the combined loss of FANCD2 and ALDH9A1 was rescued by the loss of ATP13A3, which suggests that 3-aminopropanal is the culprit aminoaldehyde that accumulates in ALDH9A1-deficient cells and results in DNA damage. Functionally deleterious ALDH9A1 variants were observed in some FA patients with early onset of disease suggesting that ALDH9A1 could be a modifier of FA in humans. DisclosuresSridhar: Deciphera Pharmaceuticals: Current Employment; CRISPR Therapeutics: Ended employment in the past 24 months. White: Regeneron Pharmaceuticals: Current Employment. Smogorzewska: Rocket Pharmaceuticals: Research Funding.

Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome.

Snyder Robinson Syndrome (SRS) is a rare disease associated with a defective spermine synthase gene and low intracellular spermine levels. In this study, a spermine replacement therapy was developed using a spermine prodrug that enters cells via the polyamine transport system. The prodrug was comprised of three components: a redox-sensitive quinone "trigger", a "trimethyl lock (TML)" aryl "release mechanism", and spermine. The presence of spermine in the design facilitated uptake by the polyamine transport system. The quinone-TML motifs provided a redox-sensitive agent, which upon intracellular reduction generated a hydroquinone, which underwent intramolecular cyclization to release free spermine and a lactone byproduct. Rewardingly, most SRS fibroblasts treated with the prodrug revealed a significant increase in intracellular spermine. Administering the spermine prodrug through feeding in a Drosophila model of SRS showed significant beneficial effects. In summary, a spermine prodrug is developed and provides a lead compound for future spermine replacement therapy experiments.

Synthesis and biological evaluation of chalcone-polyamine conjugates as novel vectorized agents in colorectal and prostate cancer chemotherapy.

The aim of this study was to synthesize chalcone-polyamine conjugates in order to enhance bioavailability and selectivity of chalcone core towards cancer cells, using polyamine-based vectors. Indeed, it is well-known that polyamine transport system is upregulated in tumor cells. 3',4,4',5'-tetramethoxychalcone was selected as parent chalcone since it was found to be an efficient anti-proliferative agent on various cancer cells. A series of five chalcone-polyamine conjugates was obtained using the 4-bromopropyloxy-3',4',5'-trimethoxychalcone as a key intermediate. Chalcone core and polyamine tails were fused through an amine bond. These conjugates were found to possess a marked invitro antiproliferative effect against colorectal (HT-29 and HCT-116) and prostate cancer (PC-3 and DU-145) cell lines. The most active conjugate (compound 8b) was then chosen for further biological evaluations to elucidate mechanisms responsible for its antiproliferative activity. Investigations on cell cycle distribution revealed that this conjugate can prevent the proliferation of human colorectal and prostate cancer cells by blocking the cell cycle at the G1 and G2 phase, respectively. Flow cytometry analysis revealed a sub-G1 peak, characteristic of apoptotic cell population and our inquiries highlighted apoptosis induction at early and later stages through several pro-apoptotic markers. Therefore, this chalcone-N1-spermidine conjugate could be considered as a promising agent for colon and prostatic cancer adjuvant therapy.

Investigating how amine structure influences drug-amine ion-pair formation and uptake via the polyamine transporter in A549 lung cells

Transiently associating amines with therapeutic agents through the formation of ion-pairs has been established both in vitro and in vivo as an effective means to systemically direct drug delivery to the lung via the polyamine transport system (PTS). However, there remains a need to better understand the structural traits required for effective PTS uptake of drug ion-pairs. This study aimed to use a structurally related series of amine counterions to investigate how they influenced the stability of theophylline ion-pairs and their active uptake in A549 cells. Using ethylamine (mono-amine), ethylenediamine (di-amine), spermidine (tri-amine) and spermine (tetra-amine) as counterions the ion-pair affinity was shown to increase as the number of protonated amine groups in the counterion structure increased. The mono and diamines generated a single hydrogen bond and the weakest ion-pair affinities (pKFTIR: 1.32 ± 0.04 and 1.43 ± 0.02) whereas the polyamines produced two hydrogen bonds and thus the strongest ion-pair affinities (pKFTIR: 1.93 ± 0.05 and 1.96 ± 0.04). In A549 cells depleted of endogenous polyamines using α-difluoromethylornithine (DFMO), the spermine-theophylline uptake was significantly increased (p < 0.05) compared to non-amine depleted cells and this evidenced the active PTS sequestering of the ion-pair. The mono-amine and di-amine failed to enhance theophylline uptake in these A549 cells, but the tri-amine and tetra-amine both almost doubled the theophylline uptake into the cells when compared to the uptake of free drug. As the data indicated that polyamines with at least 3 amines were required to form ion-pairs that could enhance A549 cell uptake, it suggested that at least two amines were required to physically stabilise the ion-pair and one to interact with the PTS.

Harnessing the polyamine transport system to treat BRAF inhibitor-resistant melanoma

ABSTRACT BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine ( AP ) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy ( AP ) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.

Deletion of Polyamine Transport Protein PotD Exacerbates Virulence in Glaesserella (Haemophilus) parasuis in the Form of Non-biofilm-generated Bacteria in a Murine Acute Infection Model

ABSTRACT Polyamines are small, polycationic molecules with a hydrocarbon backbone and multiple amino groups required for optimal cell growth. The potD gene, belonging to the ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system, playing a pivotal role in bacterial metabolism and growth. The swine pathogen Glaesserella parasuis possesses an intact pot operon, and the studies presented here mainly examined the involvement of PotD in Glaesserella pathogenesis. A potD-deficient mutant was constructed using a virulent G. parasuis strain SC1401 by natural transformation; immuno-electron microscopy was used to identify the subcellular location of native PotD protein; an electron microscope was adopted to inspect biofilm and bacterial morphology; immunofluorescence technique was employed to study cellular adhesion, the levels of inflammation and apoptosis. The TSA++-pre-cultured mutant strain showed a significantly reduced adhesion capacity to PK-15 and MLE-12 cells. Likewise, we also found attenuation in virulence using murine models focusing on the clinical sign, H&E, and IFA for inflammation and apoptosis. However, when the mutant was grown in TSB++, virulence recovered to normal levels, along with a high level of radical oxygen species formation in the host. The expression of PotD could actively stimulate the production of ROS in Raw 264.7. Our data suggested that PotD from G. parasuis has a high binding potential to polyamine, and is essential for the full bacterial virulence within mouse models. However, the virulence of the potD mutant is highly dependent on its TSA++ culture conditions rather than on biofilm-formation.

ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.

Polyamine Transport Assay Using Reconstituted Yeast Membranes.

ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020 ) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.

Tailoring of silica-based nanoporous pod by spermidine multi-activity

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy.

ATP13A2 deficiency disrupts lysosomal polyamine export.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia1-and early-onset Parkinson's disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromiseslysosomes3. However, the transport function of ATP13A2in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK-MAPK and NF-κB Signal Pathways through TLR4.

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL–1β, IL–6, and TNF–α using qPCR and ELISA. Blocking signal-regulated JNK–MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK–MAPK and RelA(p65) pathways.