Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2

Chen, Xudong; Zhou, Mingze; Zhang, Sensen; Yin, Jian; Zhang, Ping; Xuan, Xujun; Wang, Peiyi; Liu, Zhiqiang; Zhou, Boda; Yang, Maojun

doi:10.1038/s41421-021-00334-6

Cited by 20 publications

(36 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The overlap in polyamine transport function among P5B-ATPases fits well with their close evolutionary relationship, conserved biochemical behavior of spontaneous autophosphorylation, their overlapping endo-/lysosomal localization, peculiar N-terminal topology and predicted three-dimensional structures (AlphaFold) [1,26]. In addition, all P5B-ATPases present a highly conserved substrate binding site that has been resolved in human ATP13A2 [27][28][29][30] and yeast Ypk9p [31] cryo-EM structures as a polyamine binding pocket.…”

Section: Discussionsupporting

confidence: 59%

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

Veen

Kourti

Ausloos

et al. 2023

Preprint

View full text Add to dashboard Cite

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). So 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 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 present elevated expression of a previously uncharacterized P5B-ATPase ATP13A4, which is responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells are more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, overexpression of ATP13A4 WT in MCF10A cells induces a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labelled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we establish ATP13A4 as a new polyamine transporter in the human PTS and show 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.

show abstract

Section: Discussionsupporting

confidence: 59%

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

Veen

Kourti

Ausloos

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…At the subcellular level, both proteins are localized in the endosomal pathway with a preferred late endo-/lysosomal distribution for ATP13A2 [12–14] and early/recycling endosomes for ATP13A3 [12,13,15]. We provided biochemical evidence that human ATP13A2 (hATP13A2) transports polyamines from late endo/lysosomes to the cytosol with a high affinity for SPM and SPD [13], which was confirmed at the structural level [16–20]. ATP13A2, and also the closely related ATP13A3, fulfill their polyamine transport function downstream of polyamine internalization via endocytosis [11,13].…”

Section: Introductionmentioning

confidence: 79%

“…hATP13A2 exhibits the highest affinity for SPM [13], which binds to a narrow channel-like substrate binding site at the luminal side of the protein [16–20]. Whether the polyamine pocket may also accommodate the bulkier BODIPY-SPM for subsequent transport remains unclear.…”

Section: Resultsmentioning

confidence: 99%

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

Houdou

Jacobs

Coene

et al. 2022

Preprint

View full text Add to dashboard Cite

Cells acquire the polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary action of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2 and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than ATP13A2. We also compared the uptake of new green-fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR) suggesting that isoform-specific polyamine probes can be designed.

show abstract

“…Previous studies obtained high-resolution structures of other P-type ATPases in the E1 state by vitrifying the wild-type ATPase directly 18 , 30 – 32 . However, we acquired a final EM map of hATP13A2 in the E1-like state at a resolution of approximately 5.6 Å with the same preparation protocol as that in previous studies 21 (Supplementary Fig. 2 ) by processing more than 10,000 movies with cryoSPARC 33 .…”

Section: Resultsmentioning

confidence: 99%

Conformational cycle of human polyamine transporter ATP13A2

Xue

et al. 2023

Nat Commun

View full text Add to dashboard Cite

Dysregulation of polyamine homeostasis strongly associates with human diseases. ATP13A2, which is mutated in juvenile-onset Parkinson’s disease and autosomal recessive spastic paraplegia 78, is a transporter with a critical role in balancing the polyamine concentration between the lysosome and the cytosol. Here, to better understand human ATP13A2-mediated polyamine transport, we use single-particle cryo-electron microscopy to solve high-resolution structures of human ATP13A2 in six intermediate states, including the putative E2 structure for the P5 subfamily of the P-type ATPases. These structures comprise a nearly complete conformational cycle spanning the polyamine transport process and capture multiple substrate binding sites distributed along the transmembrane regions, suggesting a potential polyamine transport pathway. Integration of high-resolution structures, biochemical assays, and molecular dynamics simulations allows us to obtain a better understanding of the structural basis of how hATP13A2 transports polyamines, providing a mechanistic framework for ATP13A2-related diseases.

show abstract

Cryo-EM structures and transport mechanism of human P5B type ATPase ATP13A2

Cited by 20 publications

References 62 publications

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

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

Conformational cycle of human polyamine transporter ATP13A2

Contact Info

Product

Resources

About