Friday, 12 October 2018 17:55

Tales of the Vault: a Common, yet Mysterious Organelle

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Published in Microbial Sciences

2018.10.12 Vault 1Parts of a eukaryotic cell as you might see illustrated in a text book. Image created by Mariana Ruiz.

Do you remember learning about the organelles within eukaryotic cells? The mitochondrial powerhouse, the Golgi complex and its protein sorting and packaging, maybe the photosynthesis-housing chloroplast.  But did the vault ever make it onto your flashcards? You may be thinking “No, because there is no tiny safe floating around inside a cell.”

 

Vault organelles are so named because of their resemblance to vaulted ceilings, such as those found in gothic cathedrals, not because they act as microscopic lockboxes. These ribonucleoproteins, the largest of any eukaryotic ribonucleoprotein (3 times larger than eukaryotic ribosomes and 5 times larger than bacterial ribosomes), were first discovered in 1986. Originally, they were thought to be contamination from the process of isolating and preparing clathrin-coated vesicles from rat liver for TEM analysis. Subsequent analyses verified their existence and revealed their widespread existence across animal and fungal organisms including: mice, cows, rabbits, electric eels, and even slime molds. Additionally, homologs to the vault’s main protein (MVP, Major Vault Protein) have been found in a range of unicellular eukaryotes including Trypanosoma and Leishmania spp. However, no MVP homologues have yet been found in plants, yeast, insects, or worms. In all human cells examined thus far, vaults have been found ranging in number from several thousand to over 100,000 per cell.

 

2018.10.12 Vault 2The vaulted ceiling of BCE Place Gallaria in Toronto, Canada. This is the style of architecture that inspired Dr. Nancy Kedersha to name the newly discovered ribonucleoproteins "vaults." Photo by Luis Fernandes.

These barrel-shaped, ~13 MDa-sized particles are formed by the self-assembly of three proteins: MVP (Major Vault Protein), TEP1 (Telomerase-associated Protein 1), and VPARP or PARP4 (related to Poly-(ADP-ribose) Polymerase or PARP). Seventy-eight copies of MVP form the shell-like structure of vaults, whose expansive interior houses the VPARP polymerase and TEP1, which is bound to a small segment of untranslated RNA known as vRNA. Research has implicated vaults in a number of cellular processes such as innate immunity, cell signaling, and nuclear-cytoplasmic transport, however their exact functions in cells are as of yet unknown. Knockout mice are viable and show no phenotypic differences, indicating that if vaults do play a critical role, it is a redundant one. 2 of the 3 different MVPs encoded by Dictyostelium (slime mold) have been studied thus far, with single and combination knockouts revealing ~one-half to one-third slower growth than wildtype cells under nutrient stress.

 

While we may not know yet what exactly vaults normally do, researchers have already learned many things they *can* do. Or rather that they can be engineered to do. By editing their protein components, vaults can be modified to serve a number of functions in biotechnology such as cell targeting, drug delivery, vaccines and even a role in pollutant degradation.

 

Vaults Aid in Cell-Targeted Drug Delivery

 

Since the outer structure of a fully formed vault is composed of MVP, researchers can add tags to the C-terminus end of each MVP to direct the vault particles to specific cells. Kickhoefer and Han, et al. demonstrated this using vaults modified to target A431 Epithelial Cancer Cells in two different ways. In the first, vaults tagged with modified Epidermal Growth Factor bound directly to cells expressing Epidermal Growth Factor Receptor (EGFR), a commonly overexpressed membrane protein in many cancers. A second engineered set of vaults were tagged with an IgG-binding protein that enabled the vaults to bind indirectly to targeted cells via anti-EGFR antibodies. The success in targeting means that treatments to diseases such as cancer could be enhanced by specifically delivering therapeutic drugs directly to target cells.

 

2018.10.12 Vault 3The protein structure of a single vault nanoparticle form the Protein Data Bank. 78 copies of the Major Vault Protein (MVP), each shown as a different color here, assemble to form the barrel-like structure with an upper and lower half. The N-termini of the MVPs meet at the middle. Inside, VPARP proteins (not shown) bind to MVP through their INT domains and form 2 mirrored rings of VPARP within the upper and lower halves. This INT domain is what researchers use to package molecules inside the vault. The C-termini of the MVPs are a tthe far ends (top and bottom) of the vault particle, where engineered protein tags can help target the vault to specific sites. Deposited to the PDB by Kato K, Zhou Y, Tanaka H, Yao M, Yamashita E, Yoshimura M, and Tsukihara T.

Once bound to a targeted cell, vault uptake is not guaranteed, nor is avoidance of degradation inside the cell.  To address this problem, researchers borrowed a clever trick from viruses. The adenovirus viral protein VI (pVI) aids in host cell penetration, so researchers incorporated pVI into engineered vaults and observed an enhanced delivery of test molecules. When combined with cell targeting modifications, the efficiency of successful transfection of target cells was increased from requiring over 1,000,000 engineered vaults/cell to ~50,000 vaults/cell. Further modifications, including adding pVI directly to the N-terminus of MVP instead of packaging it within the vault, reduced this to ~500 vaults/cell necessary for successful biomolecule delivery. But how are medicines or other molecules packaged inside vaults?

 

To facilitate the loading of specific proteins inside of vaults, researchers take advantage of the MVP Interacting (or INT) Domain present on VPARP, through which VPARP binds to the N-terminus of MVP. VPARP proteins reside inside vaults forming 2 identical clusters, 1 in each of the upper and lower halves of the vault.

 

Researchers can add a modified INT Domain to protein(s) they wish to package inside vaults and the INT Domain acts as an anchor to the vault’s interior. The protein is then packaged inside the vault as it self-assembles or later during a moment when the vault ‘opens up’ on its own. Vaults are occasionally observed in half-vault form, and indeed vaults can be loaded by incubating fully formed ones with the INT-modified compound of interest.  Kickhoefer and Garcia, et al. provided a proof-of-concept by packaging luciferase in 2005.  Other research teams followed by packaging additional fluorescent molecules (mCherry and GFP) as well as the chemokine, CCL21, and other molecules relevant to infection.   

 

Vaults May Help Vaccine Delivery

 

Another exciting potential application of vaults is vaccine improvement. Rome and Kickhoefer highlight the features of vaults in comparison to those of an ‘ideal’ vaccine delivery vehicle. The small size of vaults (smaller than 100 nM) means that they can transit into the draining lymph nodes and elicit an immune response without removal by the liver or kidneys. The space inside vaults, combined with their stability, means they can securely package hundreds of protein copies. Engineered vaults can also be reproduced consistently and produced in large-scale quantities. Although purity and toxicity need to be determined in order to ensure patient safety, current research suggests that vaults are non-immunogenic. Just last year, in 2017, Jiang, et al. demonstrated the effectiveness of a Chlamydia-vault vaccine in mice through decreased bacterial loads when challenged with C. muridarum, lower inflammation at the site of infection, and higher numbers of antigen-specific CD4 cells. These results and the noted features of vaults make their use in vaccines very promising.

 

Vaults Increase Efficiency of Pollutant Degradation

 

But vaults aren’t only valuable to the medical community; one research team is using vaults for environmental cleanup. Many industrial waste sites contain high concentrations of toxic phenolic compounds. Eliminating these compounds enzymatically has proven challenging due to the limited stability of phenol-degrading enzymes, such as manganese peroxidase (MnP). Enter vault particles.

 

Dr. Shaily Mahendra’s team at UCLA looked to vaults as a means to enhance stability without compromising the clean-up goal. According to Dr. Mahendra, the best way to picture this is to imagine MnP as a bird in a cage (i.e. the vault). Harmful predators can’t get to the bird, but birdseed can easily fit through the bars. In the case of MnP, the vault protects against degradation and instability while allowing phenolic compounds through the ‘bars’ of MVP.

 

The Mahendra lab packaged MnP into vault particles and measured phenol degradation compared to unpackaged and enzyme-free controls. Their results showed that MnP packaged inside vaults remained stable and active longer than controls (including MnP by itself and unpackaged MnP-INT) for up to 3 hours under temperatures increasing up to 40°C. Moreover, in the span of 24 hours, the vault-stabilized MnP degraded phenol faster than these same controls, acting 3 times faster than the MnP-INT control. If vaults can be used to clean up contaminated sites in the future, they may help relieve concerns of enzyme stability and concerns associated with releasing non-native microbes into new environments.

 

The mysterious vault, named for lofty feats of architecture, is being increasingly recognized for enabling lofty feats of molecular biology and bioengineering. Its many biotech applications wouldn’t be possible without the basic research that identified vault proteins over 30 years ago. Imagine if vaults had been simply written off as foreign contaminants and never investigated further! The intertwined relationship between basic and applied studies of vaults will continue as scientists uncover the natural role of vault proteins and build on the many uses of these mysterious organelles.

 

Further Reading:

Benner, et al., 2017. Vault Nanoparticles: Chemical Modifications for Imaging and Enhanced Delivery. ACS Nano. 11: 872-881. https://pubs.acs.org/doi/10.1021/acsnano.6b07440 

Buehler, et al., 2014. Bioengineered Vaults: Self-Assembling Protein Shell— Lipophilic Core Nanoparticles for Drug Delivery. ACS Nano. 8: 7723-7732. https://pubs.acs.org/doi/abs/10.1021/nn5002694

Rome and Kickhoefer. 2013. Development of the Vault Particle as a Platform Technology. ACS Nano. 7: 889-902. https://pubs.acs.org/doi/abs/10.1021/nn3052082

Tripathi, et al., 2017. Biotechnological Advances for Restoring Degraded Land for Sustainable Development. Trends in Biotechnology. 35: 847-859. http://dx.doi.org/10.1016/j.tibtech.2017.05.001

 

Wang, et al., 2015. Vault Nanoparticles Packaged with Enzymes as an Efficient Pollutant Biodegradation Technology. ACS Nano. 9: 10931-10940. https://pubs.acs.org/doi/10.1021/acsnano.5b04073

 

Last modified on Friday, 12 October 2018 18:12
Janet Goins

Dr. Janet Goins is Assistant Director of UCLA's Undergraduate Research Center. She works to provide undergraduate students with research experiences that prepare them for future success in STEM-related careers. Previously, her research focused on the ecological impacts of algal host-virus interactions, the evolution of and molecular steps involved in host cell pathogen defense, and the biological factors that influence harmful algal blooms. She was awarded a 2012-2013 ASM-BWF Science Teaching Fellowship and completed its program. In 2015 she received the ASM Science Communication and Strategic Marketing Fellowship.

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