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Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

ABSTRACT: Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI.The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry.Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone.This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast.

SUBMITTER: Bartelle BB

PROVIDER: S-EPMC4470876 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

Bartelle Benjamin B BB Mana Miyeko D MD Suero-Abreu Giselle A GA Rodriguez Joe J JJ Turnbull Daniel H DH

Magnetic resonance in medicine 20141217 6

<h4>Purpose</h4>Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI.<h4>Methods</h4>The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus wh ...[more]

PMID: 25522343

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Project description:The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.

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Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

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Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

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