BIOTECHNOLOGYStructural Stability of Vault Particles
Section snippets
INTRODUCTION
Some 20 years ago, Kedersha and Rome purified a large ribonucleoprotein particle from rat liver homogenates and named it the “vault” particle on the basis of its morphological resemblance to vaulted ceilings in medieval cathedrals.1., 2. Cryoelectron microscopy (CryoEM) single particle reconstructions and X-ray crystallography show vaults to posses a hollow, barrellike structure with two protruding caps and an invaginated waist.3., 4. With a molecular weight of 12.9 ± 1 MDa and dimensions of ~ 420 Å
Vault Purification
Recombinant vaults formed from CP-MVP were purified from baculovirus infected Sf9 cells, as previously described.5., 13. A 12-residue cysteine rich motif (MAGCGCPCGCGA) on the N-terminus of CP-MVP was previously shown to help stabilize the recombinant vaults.4., 13. Purified vault particles were resuspended in 20 mM citrate-phosphate buffer pH 6.5.
Sample Preparation
Vault solutions at different pH values were prepared in 20 mM isotonic citrate/phosphate buffer by dialyzing stock solutions into buffers ranging from
Far-UV Circular Dichroism (CD) Spectroscopy
Alterations in vault secondary structure were studied by monitoring the changes in the CD signal over a wide range of pH (3–8) when subjected to thermal gradients from 10 to 90°C. The CD signal is expressed in units of mean residue ellipticity which are independent of molecular weight, since the average molecular weight of vault particles in solution vary across the pH range due to conformational alterations. Using MALLS, Goldsmith et al.14 showed that the average molecular weight of CP-MVP
DISCUSSION
Employing a variety of techniques, we have extensively characterized the behavior of vaults over a wide range of solution variables (i.e., pH and temperature). The pH range examined covers the extent of physiological pH in different cellular compartments and organs and should therefore provide a basis for further interpretation of vault behavior in vivo. In addition, the combination of pH and temperature effects on the structural integrity of vaults should aid in formulation of these
CONCLUSION
Understanding the effects of pH and temperature on vault stability should aid in the ultimate goal of utilizing these particles as potential drug delivery devices. Our studies show that vault conformation was altered as a function of decreasing pH in which intact particles at neutral pH open into flowerlike structures at acidic pH. Opening of intact vaults could be potentially developed as an intracellular controlled-release device for drug delivery.
With the identification of at least 10
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Cited by (31)
Vault nanocapsule-mediated biomimetic silicification for efficient and robust immobilization of proteins in silica composites
2021, Chemical Engineering JournalCitation Excerpt :Vault/silica composites only formed in the weakly acidic pH range (5.5–6.5), while no silica precipitate was observed when the pH was increased to 7 or 7.5 (Fig. 1e). Lower pHs were not tested because vaults lose secondary structural integrity, disassociate into halves, and form aggregates at pH 5 or lower [23,24]. The progress of biomimetic silicification relies on the interaction between biomolecules and silica precursors as well as the bio-surfaces displaying functional groups that are able to catalyze silica deposition [21,25,26].
Bioengineered protein-based nanocage for drug delivery
2016, Advanced Drug Delivery ReviewsVault particles: A new generation of delivery nanodevices
2012, Current Opinion in BiotechnologyCitation Excerpt :Another function for vaults was proposed by Herlevsen et al. [24] who found that MVP knockdown disrupted the lysosomal compartment. Interestingly, an independent set of experiments demonstrated vault dissociation at low pH [25,26]. The former authors proposed that the acidic nature of the lysosomes may serve as an excellent microenvironment with which to trigger vault dissociation.
Endogenous Vaults and Bioengineered Vault Nanoparticles for Treatment of Glioblastomas. Implications for Future Targeted Therapies
2012, Neurosurgery Clinics of North AmericaCitation Excerpt :To analyze their potential therapeutic potential, Esfandiary and colleagues investigated the bioengineered vault nanoparticle structural changes in response to various pH and temperature conditions. This analysis found that bioengineered vault nanoparticles remained the most stable below 40°C and that the flowerlike petal structures open at an acidic pH. This opening could serve as a potential mechanism to release therapeutic contents from inside the bioengineered vault nanoparticle.89 Investigating a different potential mechanism of delivery, Lai and colleagues incorporated the membrane lytic domain of adenovirus protein VI (pVI) into vault interiors, where its activity was maintained.
Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development
2011, Advanced Drug Delivery ReviewsCitation Excerpt :The ability to stabilize live, attenuated vaccines is important for developing effective, cheap, robust, stable formulations capable of long term storage in diverse climates including underdeveloped countries that may lack cold storage and transportation capabilities. In this section, select examples are discussed while the interested reader is referred to the following references for additional examples of high throughput formulation screening to stabilize attenuated virus and virus-like particle vaccines [178,193–202]. In one study, Kissmann et al. combined the use of dynamic light scattering, static light scattering, circular dichroism, fluorescence spectroscopy, and optical density measurements to screen the effect of solution pH and over 25 different excipients on the stability of virus like particles (VLPs) containing influenza antigens [193].
Multidimensional methods for the formulation of biopharmaceuticals and vaccines
2011, Journal of Pharmaceutical Sciences