We are using molecular engineering of a naturally occurring cellular structure called a vault to develop a flexible, targetable nano-scale capsule. Vaults are abundant cellular particles of unknown function found in nearly all eukaryotes (cells containing a nucleus). Cryo-electron microscopy combined with single particle reconstruction has provided overall dimensions of the vault at 42 x 75 nanometers (a nanometer is a millionth of a meter). These measurements indicate that the vault is larger in mass and size than many viruses. The overall structure of the intact vault is like a hollow barrel with two protruding caps and an indented waist with a very thin shell surrounding an internal cavity large enough to encompass several hundred proteins. Thus, the vault particle is a nanocapsule with incredible potential for compound encapsulation, protection, and delivery. Using a well-characterized insect virus into which a cloned gene can be easily inserted, it is possible to produce large quantities of a given protein in cultured insect cells. We have collaborated with a number of groups to use this system to produce large quantities of the major vault protein (MVP). Interestingly, the protein is able to self-assemble into vault-like particles. These MVP-only vaults are somewhat irregular, often containing distorted caps. However, we have demonstrated that co-production of all three vault proteins (MVP, TEP1 and VPARP) in insect cells results in self-assembly of particles that appear identical to naturally occurring vaults.
By using molecular genetic techniques to modify the gene encoding the major vault protein, vault proteins have been produced with chemically active peptides attached to their sequence. These modified proteins are incorporated into the inside of the vault particle without altering its basic structure. We propose to produce modified vault particles in order to test the concept that vaults can be bioengineered to allow their use in a wide variety of biological applications including drug delivery, biological sensors, enzyme delivery, controlled release, and eventually as parts for nano-electrical machines.
We are interested in the biogenesis and function of subcellular organelles. We have been concentrating on novel cytosolic ribonucleoprotein particles (RNPs) called vaults. Vaults were discovered in our laboratory and found to exist in most eukaryotic cells including Dictyostelium discoideum. They have an intricate shape composed of multiple arches reminiscent of cathedral vaults, hence their name. Vault size, shape and localization suggests that they may constitute a previously recognized component of the nuclear pore complex (NPC) called the nuclear pore plug. If vaults are NPC plugs, they may be involved in nucleo-cytoplasmic transport. Vaults have been shown to be upregulated in multidrug resistant (MDR) cancer cell lines and the particle may have a role in mediating drug resistance. The role of vaults in cancer may be related to the function of the vault RNA (vRNA) or one of the two high molecular weight vault proteins (TEP1 and VPARP). TEP1 is a vault protein that is shared by the nuclear RNP telomerase. In the cytoplasm as a vault component TEP1 binds the vault RNA, while in the nucleus as part of the telomerase complex TEP1 binds to the telomerase RNA. The other high molecular weight vault protein, VPARP, is a poly ADP ribose polymerase.
We are interested in elucidating the function of these unique structures and in manipulating their structure to give them new functions. We are using the baculovirus expression system to produce recombinant vaults in order to test the concept that vaults can have a broad nanosystems application as malleable nanocapsules. Toward this aim we are currently designing particles with encapsulated fluorescent probes and enzymatically active protein domains. In addition, a number of strategies are currently being considered to encapsulate chemically active small molecules into the vault particle. If successful, these vault nanocapsules can be bioengineered to allow their use in a wide variety of biological applications including drug delivery, biological sensors, enzyme delivery, controlled release, and nano-electrical machine (NEMS) applications.
Leonard H. Rome is a cell biologist and biochemist who has served on the UCLA School of Medicine faculty since he joined the Department of Biological Chemistry in 1979. He became a full professor in 1988 and wAs Senior Associate Dean for Research in the School of Medicine from 1997 - 2012. Dr. Rome earned his B.S. in Chemistry and M.S. and Ph.D. in Biological Chemistry at the University of Michigan, Ann Arbor. He was a postdoctoral fellow at the National Institutes of Health, where he worked on lysosome biogenesis. Dr. Rome has chaired the School of Medicine Faculty Executive Committee and is actively involved in graduate and medical education. He is a recipient of the School of Medicine Award for Excellence in Education. While Senior Associate Dean for Research, he organized a strategic plan for research in the School and spearheaded campus-wide efforts in genomics, proteomics, and computational biology. His laboratory research centers on a novel cellular organelle called a "vault" which was discovered in his laboratory. Dr. Rome is presently organizing a Nanoscience Interdisciplinnary Research Team, a collaboration of disciplines including cell biologists, engineers, chemists, and structural biologists who are engineering vaults so that they can be used in drug delivery.
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