A Vaulted Mystery

A MINI MYSTERY: An electron micrograph taken by researchers 30 years ago reveals one of the first looks at the nanoscale structures now known as vaults. Abbreviations: large coated vesicle (LCV), vault (V), small coated vesicle (SCV)NANCY KEDERSHA AND LEONARD ROMEKedersha designed a way to purify the mystery particles, based on a procedure previously described in the literature for isolating coated vesicles, then stained and imaged what she’d collected using electron microscopy. The tiny structures had a complex but consistent barrel-shape morphology and measured 35 by 65 nanometers—much smaller than lysosomes, which range in diameter from 100 to more than 1,000 nanometers (1 micrometer), or mitochondria, which are 0.5 to 10 micrometers long. Kedersha also treated the particles with various proteases, as well as enzymes to digest RNA and DNA, to assess their constituent molecules, finding evidence of three major proteins and an RNA component. With a total mass of approximately 13 megadaltons, they appeared to be the largest eukaryotic ribonucleoprotein particles ever discovered. By comparison, ribosomes measure just 20 to 25 nanometers in diameter and weigh in at just over 3 megadaltons.

Kedersha dubbed the structures “vaults,” after the arched shape of the very first particle she and Rome observed, reminiscent of the vaulted ceilings of cathedrals.¹ To screen for these new nanostructures in other species, Kedersha developed an antibody against one of the vault proteins she’d discovered, and used it to purify vaults from species across the animal kingdom: the minibarrels were abundant in the cells of rabbits, mice, chickens, cows, bullfrogs, sea urchins, and several human cell lines—varying from 10,000 to 100,000 per cell. Remarkably, they all appeared to be similar in size, shape, and morphology to those Kedersha and Rome isolated from rat livers. Clearly, this was an important cellular structure, and there were no reports of anything like it in the literature.

The broad distribution and strong conservation of vaults in eukaryotic species suggest that their function is essential to cells, but that function remains unclear to this day. In fact, in the three decades that have passed since their discovery, vaults have gone largely unnoticed by the scientific community. But a handful of dedicated groups are making strides in understanding what vaults are and what they do, with clues emerging that hint at their roles in cargo transport, cellular motility, and drug resistance, among other possible functions.

Cracking the vault

Scientists have taken several approaches to deciphering the structure of the nanosize vaults, including cryo-electron and freeze-etch microscopy and three-dimensional image reconstruction. Such work has revealed a symmetrical central barrel with a cinched middle and a cap protruding from the barrel’s top and bottom. (See illustration.) Cross sections reveal a very thin shell surrounding a large, hollow interior. Interestingly, a vault’s interior is spacious enough to enclose molecules as large as ribosomal subunits, but researchers have not confirmed whether vaults ever house cellular cargo.

As Kedersha’s early analyses suggested, vaults are composed of multiple copies of at least four distinct components: three proteins and one RNA molecule. The major vault protein (MVP) accounts for some 75 percent of the particles’ mass, with each vault containing 78 copies of the protein. In fact, the expression of MVP in an insect cell line—insects themselves are one of the few eukaryotic organisms that don’t have vaults—results in the spontaneous formation of particles with morphologic characteristics similar to those of endogenous vaults.² Another protein typically found in vaults is vault poly(ADP-ribose) polymerase (VPARP). VPARP and MVP mRNA transcripts are expressed in similar patterns in the cell, and subcellular fractionation studies point to a strong binding between the two proteins.

Kedersha dubbed the structures “vaults,” after the arched shape of the very first particle she and Rome observed, reminiscent of the vaulted ceilings of cathedrals.

The third vault protein is TEP1, previously identified as the mammalian telomerase-associated protein 1, which binds RNA in the telomerase complex. TEP1-knockout mice exhibited no alterations in telomerase function, suggesting its role in the nucleus is redundant, but vaults purified from these animals revealed a complete absence of the fourth component of vaults: vault RNA (vRNA), a small untranslated RNA found at the tips of the particles. This work pointed to TEP1’s role in the recruitment and stabilization of vRNA.

The freeze-etching technique—which consists of physically breaking apart a frozen biological sample and then examining it with transmission electron microscopy—has revealed that vaults are not rigid, impermeable structures, but dynamic entities that are able to open and close, with a structure resembling a petaled flower.³ (See photograph below.) The “flowers” are usually seen in pairs, suggesting that an intact vault comprises two folded flowers with eight rectangular petals, each of which is connected to a central ring by a thin, short hook. (See illustration.)

The ability of vaults to open and close points to a possible function in cargo transport. At present, however, a definitive answer about the function of vaults remains elusive. In fact, in addition to cellular transport, more than a dozen roles for vaults have been proposed, including playing a part in multidrug resistance, cellular signaling, neuronal dysfunctions, and apoptosis and autophagy.

In search of function

THE STRUCTURE OF VAULTS: Vaults are hollow, barrel-shape structures, measuring 35 x 65 nanometers. They are symmetrical, with a crease along the outside of the barrel’s middle and smaller caps on either end.
See full infographic: JPG | PDF© LAURIE O'KEEFEVaults are found in the cytoplasm, so far appearing to be completely excluded from the nucleus (except in sea urchins⁴). Within the cytoplasm, however, they are not randomly dispersed: they colocalize and interact with cytoskeletal elements, such as actin stress fibers and microtubules, and are also abundant in highly motile cells such as macrophages, suggesting the structures may help cells move around.

Vaults’ interactions with cytoskeletal elements also lend support to the idea that these particles act as cytoplasmic cargo transporters. Researchers hypothesize that vaults open, encapsulate molecules, then close and travel across the cytoplasm along microtubules or actin fibers before releasing their contents into the desired subcellular compartment.

In addition to the now well-characterized flower pattern of vault opening, Rome and colleagues have proposed two alternative hypotheses for how vaults might open: by separating at the waist, splitting into two completely dissociated halves, or by the raising of opposing petals on the two vault halves, hinging from the caps to open at the waist.⁵ (See illustration.) The latter may avoid destroying the integrity of the whole particle, potentially allowing vaults to repeatedly transport and release cargos. More recently, researchers have found evidence that the vaults “breathe” in solution, taking up and releasing proteins without ever fully opening.

Vaults also seem to be closely associated with nuclear pore complexes (NPC), protein conglomerations that span the inner and outer membranes of the nuclear envelope. This raises the possibility that vaults shuttle contents between the cytoplasm and nucleus. Interestingly, some structural characteristics of vaults, such as mass, diameter, and shape, are very similar to those of the NPC, although research has not yet conclusively established whether vaults actually form some sort of plug to stop up the NPC.

Researchers have also proposed a role for vaults in cancer cells’ ability to resist the pharmaceuticals doctors throw at them. In 1993, immunologist and experimental pathologist Rik Scheper of VU University in Amsterdam and colleagues found that a non-small-cell lung cancer cell line could be selected for resistance to the chemotherapy drug doxorubicin.⁶ The resulting cells overexpressed a large protein initially named lung resistance-related protein (LRP). Two years later, the group discovered that LRP was nothing other than human MVP,⁷ and the literature soon blossomed with papers on the possible role of vaults in chemotherapeutic drug resistance.

Experiments have yielded several observations that exclude a direct participation of MVP in such resistance, however. Knockdown of MVP does not affect cell survival, for instance, and upregulation of MVP does not increase resistance to anticancer drugs.⁸ Thus, while many clinical studies recognize MVP as a negative prognostic factor for response to chemotherapy, it remains to be seen whether vaults play a direct role in drug resistance or whether they are merely markers of a drug-resistance phenotype.

Putting vaults to work

BLOOMING VAULTS: Splitting at the midsection, vaults appear to break open into two flower-shape structures (yellow arrows). Partially open vaults (orange arrows) are seen along the top of this electron micrograph image.JOHN HEUSERWhile many questions about vaults remain, including whether they serve as cargo transporters for the cell, their large, hollow interiors have led some scientists to see the nanobarrels as potential tools for the delivery of biomaterials. A variety of strategies for encapsulating biomaterials already exists, including viruses, liposomes, peptides, hydrogels, and synthetic and natural polymers, but the use of these materials is often limited by insufficient payload, immunogenicity, lack of targeting specificity, and the inability to control packaging and release. Vaults, on the other hand, possess all the features of an ideal delivery vehicle. These naturally occurring cellular nanostructures have a cavity large enough to sequester hundreds of proteins; they are homogeneous, regular, highly stable, and easy to engineer; and, most of all, they are nonimmunogenic and totally biocompatible.

But the actual packaging of foreign materials into vaults remains challenging. In 2005, Rome and long-time UCLA collaborator Valerie Kickhoefer discovered a particular region at the VPARP’s C-terminus, named major vault protein interaction domain (mINT), which is responsible for binding VPARP to MVP. The researchers hypothesized that mINT acts as a kind of zip code directing VPARP to the inside of the vault and speculated that any protein tagged with the mINT sequence at the C-terminus could be packaged into vaults just like VPARP. Fusing the sequence to luciferase, the enzyme that makes fireflies glow, and expressing the construct in an insect cell line, they successfully generated vaults with the engineered protein packaged inside the central barrel in the same two rings typically formed of VPARP.⁹

Rome and his colleagues have since demonstrated that the technique can successfully incorporate any number of proteins into the tiny cellular particles, and even discovered that they can make changes to vault proteins to alter such packaging. For example, the addition of extra amino acids at the N-terminus of MVP produces vaults with the engineered protein packaged exclusively at the waist. Conversely, the addition of extra amino acids at the MVP C-terminus produces two blobs of densely packed protein at the ends of vaults. Vaults can also be engineered to bind antibodies or express cancer cell ligands on their surface, allowing for the precise delivery of biomaterials to target cells. Researchers believe that, once inside the body, the engineered vaults act as slow-release particles for whatever protein is packaged inside.

Three decades after their chance dis­covery, vaults remain mysterious. But researchers are not waiting for all the questions
to be answered.

In collaboration with Rome, pathologist Kathleen Kelly’s group at UCLA is working to create a vault-based nasal spray that acts as a vaccine against Chlamydia infection.¹⁰ They engineered vaults to encase the major outer membrane protein (MOMP) of Chlamydia, which possesses highly immunogenic properties, then created a nasal spray to deliver the modified vaults to the nasal mucosa. After the immunization, they challenged female mice with a Chlamydia infection and found that the treatment significantly limited bacterial infection in mucosal tissue.

Vaults may also help fight cancer. The lymphoid chemokine CCL21 binds to the chemokine receptor CCR7 and serves as a chemoattractant for tumor-fighting cells of the immune system. Pulmonologist Steven Dubinett and immunologist Sherven Sharma of UCLA and their colleagues injected CCL21 into mice with a lung carcinoma, but because CCL21 is small, it rapidly dissipated out of the tumor and was relatively ineffective at drawing immune cells to the tumor. In collaboration with Rome’s group, the researchers tagged the chemokine with mINT to package it into vaults prior to injection, causing an increase in the number of leukocytic cells that infiltrated the tumor and, most importantly, leading to a significant decrease in tumor growth.¹¹ Rome and colleagues have since started a company to advance this vault-based therapy through human trials. (See “Opening the Medical Vault” below.)

Three decades after their chance discovery, vaults remain mysterious. But researchers are not waiting for all the questions to be answered. Vault-based therapies show promise in treating a variety of diseases, and the success of such applications could give these nanosize barrels a big dose of recognition. 

Eufemia S. Putortì recently completed her bachelor’s degree in medical and pharmaceutical biotechnology at the Vita-Salute San Raffaele University in Milan, Italy, with a thesis on the history and function of vaults. While an undergraduate, she interned in the lab of Massimo P. Crippa, a senior researcher at the institute, and is now working on her master’s degree in molecular and cellular medical biotechnology.

References

1. N.L. Kedersha, L.H. Rome, “Isolation and characterization of a novel ribonucleoprotein particle: Large structures contain a single species of small RNA,” J Cell Biol, 103:699-709, 1986.
2. A.G. Stephen et al., “Assembly of vault-like particles in insect cells expressing only the major vault protein,” J Biol Chem, 276:23217-20, 2001.
3. N.L. Kedersha et al., “Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry,” J Cell Biol, 112:225-35, 1991.
4. D.R. Hamill, K.A. Suprenant, “Characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport”, Dev Biol, 190:117-128, 1997.
5. M.J. Poderycki et al., “The vault exterior shell is a dynamic structure that allows incorporation of vault-associated proteins into its interior,” Biochemistry, 45:12184-93, 2006.
6. R.J. Scheper et al., “Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance,” Cancer Res, 53:1475-79, 1993.
7. G.L. Scheffer et al., “The drug resistance-related protein LRP is the human major vault protein,” Nat Med, 1:578-82, 1995.
8. K.E. Huffman, D.R. Corey, “Major vault protein does not play a role in chemoresistance or drug localization in a non-small cell lung cancer cell line,” Biochemistry, 44:2253-61, 2005.
9. V.A. Kickhoefer et al., “Engineering of vault nanocapsules with enzymatic and fluorescent properties,” PNAS, 102:4348-52, 2005.
10. C.I. Champion et al., “A vault nanoparticle vaccine induces protective mucosal immunity,” PLOS ONE, 4:e5409, 2009.
11. U.K. Kar et al., “Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth,” PLOS ONE, 6:e18758, 2011.
12. L.H. Rome, V.A. Kickhoefer, “Development of the vault particle as a platform technology,” ACS Nano, 7:889–902, 2013.