Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers.

The identification of small molecules that inhibit the sequencespecific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1A and HIF-1B proteins containing the basichelix-loop-helix and PAS domains. Expressed recombinant HIF-1A and HIF-1B proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a smallmolecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1A and HIF-1B proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-KB (NF-KB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-KB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters. (Cancer Res 2005; 65(19): 9047-55)

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes ϳ75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.Vaults are predominantly cytoplasmic ribonucleoprotein particles that have been conserved throughout evolution and are found in phylogenies as diverse as those of mammals, avians, amphibians, sea urchins, and slime molds (1). Many different roles including nucleocytoplasmic transport have been proposed for vaults since their first description in 1986 (2). However, their normal cellular function remains nebulous. Mammalian vaults comprise three proteins, the major vault protein (MVP) 1 (3), the vault poly(A)DP-ribose polymerase (VPARP) (4), and the telomerase-associated protein 1 (TEP1) (5) and one or more small untranslated RNAs (6). Vaults have been implicated in the phenomenon of multidrug resistance and as prognostic markers for cancer chemotherapy failure (7,8). One recent study has shown that the major vault protein is involved directly in the efflux of drugs from the nucleus (9). Although the majority of vaults are found in the cytoplasm, small amounts have been localized to the nuclear pore complexes (10). Recently a 31-Å resolution structure of the vault has been published indicating that vaults have a hollow interior consistent with a transport or sequestration function. Scanning transmission electron microscopic analysis has shown that the molecular mass of the vault is 12.9 Ϯ 1 MDa, and cryo-EM single-particle reconstruction has provided overall dimensions of 42 ϫ 75 nm (11). Freeze-etch images of the vault on polylysine-coated mica show that each half of the vault midsection can open into eight distinct "petals" (3), which has lead to the proposal that vaults may open and close in vivo. The MVP is presumed to be present in 96 copies/vault, based on the observed symmetry of the particle and the estimate that MVP accounts for ϳ75% of the total protein mass in the particle. In many way...

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ∼10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Vaults are large cytoplasmic ribonucleoprotein complexes of undetermined function. Mammalian vaults have two high molecular mass proteins of 193 and 240 kDa. We have identified a partial cDNA encoding the 240-kDa vault protein and determined it is identical to the mammalian telomerase-associated component, TEP1. TEP1 is the mammalian homolog of the Tetrahymena p80 telomerase protein and has been shown to interact specifically with mammalian telomerase RNA and the catalytic protein subunit hTERT. We show that while TEP1 is a component of the vault particle, vaults have no detectable telomerase activity. Using a yeast three-hybrid assay we demonstrate that several of the human vRNAs interact in a sequence-specific manner with TEP1. The presence of 16 WD40 repeats in the carboxyl terminus of the TEP1 protein is a convenient number for this protein to serve a structural or organizing role in the vault, a particle with eight-fold symmetry. The sharing of the TEP1 protein between vaults and telomerase suggests that TEP1 may play a common role in some aspect of ribonucleoprotein structure, function, or assembly.With a mass of 13 MDa, vaults are the largest ribonucleoprotein complexes known (1). Mammalian vaults are localized to the cytoplasm and are composed of a small RNA and three proteins of 100, 193, and 240 kDa in size (for review, see Refs. 2 and 3). The 100-kDa subunit, termed the major vault protein (MVP) 1 constitutes Ͼ70% of the particle mass. Its cDNA has been cloned from human, rat, Dictyostelium, and Discopyge ommata electric ray and their sequences are highly conserved both at the gene and protein level (4 -8). Likewise vault-associated RNA (vRNA) has been cloned from human, rat, mouse and bullfrog (9, 10). vRNAs exhibit species-specific length variation ranging from 86 to 141 bases; however, all of the vRNAs can be folded into a similar secondary structure. Furthermore, vRNA is not a structural component of the vault particle as it makes up Ͻ5% of the vault mass, and its degradation does not result in the alteration of vault structure (1). All mammalian vaults contain two higher molecular weight vault proteins, p193 and p240. Recently, we have identified the p193 by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis (11).Vaults have a unique barrel shape that is made up of two halves with each half vault capable of opening into a flower-like structure with eight petals surrounding a central ring. Each petal is formed by six copies of MVP, with 96 copies of MVP in the complete vault particle. Vaults are widely distributed throughout eukaryotes, and their morphology is highly conserved among these species, suggesting that their function is essential (12). Although the function of the vault particle has remained elusive, a portion of vaults have been localized to the cytoplasmic face of the nuclear membrane at or near nuclear pore complexes (13). Moreover, vault particle mass and symmetry are strikingly similar to the predicted mass of the put...

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experimentsshow that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.