Zinc transporter ZIP10 forms a heteromer with ZIP6 which regulates embryonic development and cell migration

There is growing evidence that zinc and its transporters are involved in cell migration during development and in cancer. In the present study, we show that zinc transporter ZIP10 (SLC39A10) stimulates cell motility and proliferation, both in mammalian cells and in the zebrafish embryo. This is associated with inactivation of GSK-3α and -3ß and downregulation of E-cadherin (CDH1). Morpholino-mediated knock-down of zip10 causes delayed epiboly and deformities of the head, eye, heart and tail. Furthermore, zip10 deficiency results in overexpression of cdh1, zip6 and stat3, the latter gene product driving transcription of both zip6 and zip10. The non-reduntant requirement of Zip6 and Zip10 for epithelial to mesenchymal transition (EMT) is consistent with our finding that they exist as a heteromer. We postulate that a subset of ZIPs carrying PrP-like ectodomains, including ZIP6 and ZIP10, are integral to cellular pathways and plasticity programs, such as EMT

PrP and its Ancestral Relatives ZIP6 and ZIP10 Interact with NCAM1, Altering its Molecular Environment and Post-translational Modifications during Epithelial-to-mesenchymal Transition

The prion protein (PrP) was recently found to be evolutionarily linked to a subfamily of ZIP transporters which possess a PrP-like domain. A member of this subfamily, ZIP6, is of particular interest as separate studies have shown that morpholino knockdowns of ZIP6 or PrP in zebrafish leads to an impairment in gastrulation, a process dependent on epithelial-to-mesenchymal transition (EMT). Furthermore, the neural cell adhesion molecule (NCAM1), a known interactor of PrP, has itself been described as a mediator of EMT. Based on these findings, we hypothesized that both PrP and ZIP6 play crucial roles in the process of EMT by controlling the environment surrounding NCAM. We determined that ZIP6 forms a heteromeric complex with ZIP10 that affects NCAM1â s integration into adhesion complexes while also mediating its phosphorylation during EMT. Meanwhile, PrP was found to have a unique role in controlling the polysialylation of NCAM1 during EMT.M.Sc.2017-11-21 00:00:0

The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis.

Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP

PrP-deficiency affects expression of a subset of proteins undergoing pronounced expression levels changes during EMT.

<p>List of proteins exhibiting >20% level differences in comparison of global proteomes of TGFB1-treated stable PrP kd versus wt NMuMG cells (dataset II). Coverage: percentages of primary structure of covered by peptide-to-spectrum matches; # Peptides: number of peptides matched to a given protein entry (note that instances of the same peptide being identified with different modifications counted separately in this tally); Count: number of TMT signature ion distributions, which passed stringent filtering criteria and were used for relative quantitation. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133741#pone.0133741.s003" target="_blank">S1 Table</a> for complete list of proteins identified, including control samples, confidence scores and statistical measures.</p

Quantitative mass spectrometry identifies perturbed ‘response to metal ions’ and EMT markers, including NCAM1, affected in PrP-deficient cells.

<p>(a) Design of quantitative global proteome comparisons giving rise to datasets I to III. (b) Workflow of global proteome analyses conducted by comparative mass spectrometry. Note that this workflow was executed 3 times to generated datasets I to III, with the ‘x’ being replaced by the respective condition specified at the top of this panel. To facilitate comparison of datasets, the three experiments differed in the biological samples which were labeled with even-numbered TMT reagents. All three datasets shared the use of wt NMuMG cell extracts following 48 h TGFB1 exposure as reference samples labeled with odd-numbered TMT reagents. (c) Example graph depicting post-acquisition filtering of datasets and benchmarks of mass spectrometry analysis (shown for dataset I). (d) Profound overlap amongst top 200 proteins whose levels are most changed during EMT or following stable PrP kd. (e) Exposure of NMuMG cells to TGFB1 causes changes to proteins whose KEGG annotations identify them as players in pathways that contribute to ‘focal adhesion’ formation and ‘actin cytoskeleton regulation’. (f) Direct comparison of global proteomes of wt and stable PrP kd NMuMG cells following TGFB1 exposure identifies highly significant perturbations in biological processes with ‘response to inorganic substance’ and ‘response to metal ions’ GO annotations.</p

Inhibition of CTNNB1-dependent transcription phenocopies loss of PSA in NMuMG cells.

<p>(a) Comparison of global proteomes of stable PrP kd clones versus wt NMuMG cells and stable versus transient PrP-deficient cell clones. Of the total of 1421 proteins quantified in all global proteome analyses, relative levels of 41 proteins were changed by more than 20% in the direct comparisons. Four and three proteins had prior GO annotations, which identified them as ‘DNA binding’ and/or ‘Transcriptional regulators’. Based on these annotations, only β-catenin emerged as a DNA-binding transcriptional regulator whose levels are also changed during EMT. Note also that the level changes between stable kd cells and wt or transient kd NMuMG cells turned out to be equidirectional for all proteins whose levels changed more than 20%. (b) Transient kd of CTNNB1 or inhibitor-based disruption of protein-protein interactions between CTNNB1 and TCF or CBP reduces polysialylation of NCAM1. (c) Stable PrP ko or kd in NMuMG cells altered nuclear levels of SNAI1 and p133-CREB, developmental transcription factors known to interact with CTNNB1. Lamin A served as a nuclear reporter protein in these experiments, indicating both enrichment levels of nuclear fractions and equal protein loading. (d) Quantitation of nuclear levels of SNAI1 and p133-CREB in stable PrP ko or kd NMuMG clones versus wild-type or transient PrP kd NMuMG cells. The asterisks indicate significant differences in levels of SNAI1 (p = 0.029) and p133-CREB (p = 0.029) in cells that support or are impaired in NCAM1 polysialylation during EMT. (e) Cartoon depicting signaling pathways which may underlie differences in NCAM1 polysialylation in stable PrP-deficient cells.</p

Stable PrP-deficiency prevents EMT-dependent polysialylation of NCAM1.

<p>(a) A post-translational modification of NCAM1 is missing in cells expressing no or low levels of PrP. Western blot analysis of selected NMuMG cell extracts revealed increased total levels of NCAM1 in all cell clones upon 48 h TGFB1 exposure. Whereas cells expressing wt levels of PrP give rise to a continuous pattern of NCAM1 signals, PrP-deficient cells exhibit more distinct NCAM1 bands, whose masses correspond to the expected masses of the three predominant NCAM1 isoforms. Note that the PrP^C band pattern observed in NMuMG cells tends to be more complex than the corresponding pattern in, for example, the Neuro2a cell model, possibly reflecting a greater heterogeneity of its N-glycans in these cells. (b) Screening of a larger number of stable PrP shRNA NMuMG clones further corroborated a direct correlation between PrP expression levels and post-translationally modified NCAM1 isoforms. Stable PrP shRNA clone 1, which exhibited no reduction in post-translationally-modified NCAM1 signals, turned out to express near wild-type levels of PrP, thereby establishing this clone as a false negative shRNA control. (c) Stable PrP-deficiency impairs polysialylation of NCAM1 at N-glycan acceptor sites. To characterize the post-translational NCAM1 modification lacking in PrP-deficient cells, extracts from wt or stable PrP kd NMuMG cells, which had been treated with TGFB1 for 48 h, were subjected to enzymatic digestion with glycosylases known to remove terminating sialic acids (exo-N), cut polysialic acid chains (endo-N) or hydrolyze the linkage of N-glycan groups to asparagine side-chains within ‘NxS/T’ acceptor sites (PNGase F). Note that complete removal of N-glycans abolishes the discriminating NCAM1 modification. (d) Interpretative panel of western blot bands observed in subpanel c. Red lines indicate expected cleavage sites for treatment conditions shown.</p

PrP^C expression is transcriptionally upregulated during EMT.

<p>(a) Double-immunofluorescence analyses of NMuMG cells before and after 48 h exposure to TGFB1, depicting the changes to cell shape and actin cytoskeleton that accompany EMT in this cell model. (b) Western blot analysis of E-cadherin and PrP^C protein levels in NMuMG cell extracts during 72 h of exposure to TGFB1. (c) Profound upregulation of <i>Prnp</i> gene transcription accounts for increased PrP^C protein levels during EMT based on a time-course RT-PCR analysis of PrP transcripts in NMuMG cells following addition of TGFB1 to the cell culture medium. (d) Comparison of E-cadherin and PrP protein levels in wt NMuMG cells and PrP-deficient derivative cell clones obtained by CRISRP-Cas9-based PrP knockout or stable shRNA-based kd. The ‘negative control’ represents a cell clone which had been subjected to identical CRISPR-Cas9-based <i>Prnp</i> knockout procedures but did not result in a PrP knockout. (e) Immunofluorescence analysis of E-cadherin and F-actin in wt or PrP-deficient cells before and after TGFB1 exposure. Disorganized E-cadherin distribution at cell-cell junctions and failure of PrP-deficient cells to exhibit directional alignment following TGFB1 exposure.</p

PrP deficiency prevents EMT-dependent NCAM1 polysialylation by inhibiting transcriptional activation of ST8SIA2 gene.

<p>(a) PrP is not required for NCAM1 polysialylation in N2a cells made to express ST8SIA2 from a heterologous expression plasmid. Note that higher levels of PSA-NCAM1 in CRISPR-Cas9-based PrP knockout N2a cells reflect higher levels of NCAM1 substrate in the PrP ko cell clones. (b) CRISPR-Cas9-based knockout of PrP in muscle C2C12 myocytes has little effect on total NCAM1 levels but causes profound upregulation of NCAM1 polysialylation before or throughout myotube differentiation. The control represents a C2C12 cell clone, which underwent all steps of CRISPR-Cas9 manipulation as the positive PrP ko clone but did not give rise to PrP ablation. FBS, cells grown in fetal bovine serum; HS, cells grown in horse serum (known to induce myotube formation). (c) Stable PrP ko or kd, but not transient PrP kd, impairs upregulation of ST8SIA2 transcripts in NMuMG cells in response to TGFB1 exposure. Note the different ordinate scales of subpanels. (d) ST8SIA2 is the polyST primarily responsible for EMT-dependent NCAM1 polysialylation in the NMuMG cell model. Transient kd of ST8SIA2 in NMuMG cells mimics stable PrP-deficiency with regard to its inhibition of NCAM1 polysialylation. Note that in contrast to the stable PrP knockout or kd, transient PrP-kd does not interfere with NCAM1 polysialylation.</p