Evolution of AF6-RAS association and its implications in mixed-lineage leukemia.

Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.

Mapping the Conformation Space of Wildtype and Mutant H-Ras with a Memetic, Cellular, and Multiscale Evolutionary Algorithm.

An important goal in molecular biology is to understand functional changes upon single-point mutations in proteins. Doing so through a detailed characterization of structure spaces and underlying energy landscapes is desirable but continues to challenge methods based on Molecular Dynamics. In this paper we propose a novel algorithm, SIfTER, which is based instead on stochastic optimization to circumvent the computational challenge of exploring the breadth of a protein's structure space. SIfTER is a data-driven evolutionary algorithm, leveraging experimentally-available structures of wildtype and variant sequences of a protein to define a reduced search space from where to efficiently draw samples corresponding to novel structures not directly observed in the wet laboratory. The main advantage of SIfTER is its ability to rapidly generate conformational ensembles, thus allowing mapping and juxtaposing landscapes of variant sequences and relating observed differences to functional changes. We apply SIfTER to variant sequences of the H-Ras catalytic domain, due to the prominent role of the Ras protein in signaling pathways that control cell proliferation, its well-studied conformational switching, and abundance of documented mutations in several human tumors. Many Ras mutations are oncogenic, but detailed energy landscapes have not been reported until now. Analysis of SIfTER-computed energy landscapes for the wildtype and two oncogenic variants, G12V and Q61L, suggests that these mutations cause constitutive activation through two different mechanisms. G12V directly affects binding specificity while leaving the energy landscape largely unchanged, whereas Q61L has pronounced, starker effects on the landscape. An implementation of SIfTER is made available at http://www.cs.gmu.edu/~ashehu/?q=OurTools. We believe SIfTER is useful to the community to answer the question of how sequence mutations affect the function of a protein, when there is an abundance of experimental structures that can be exploited to reconstruct an energy landscape that would be computationally impractical to do via Molecular Dynamics.

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects.

The crystal structure of RAS was first solved 25 years ago. In spite of tremendous and sustained efforts, there are still no drugs in the clinic that directly target this major driver of human cancers. Recent success in the discovery of compounds that bind RAS and inhibit signaling has fueled renewed enthusiasm, and in-depth understanding of the structure and function of RAS has opened new avenues for direct targeting. To succeed, we must focus on the molecular details of the RAS structure and understand at a high-resolution level how the oncogenic mutants impair function. Structural networks of intramolecular communication between the RAS active site and membrane-interacting regions on the G-domain are disrupted in oncogenic mutants. Although conserved across the isoforms, these networks are near hot spots of protein-ligand interactions with amino acid composition that varies among RAS proteins. These differences could have an effect on stabilization of conformational states of interest in attenuating signaling through RAS. The development of strategies to target these novel sites will add a fresh direction in the quest to conquer RAS-driven cancers. Clin Cancer Res; 21(8); 1810-8. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."

NMR-based functional profiling of RASopathies and oncogenic RAS mutations.

Defects in the RAS small G protein or its associated network of regulatory proteins that disrupt GTPase cycling are a major cause of cancer and developmental RASopathy disorders. Lack of robust functional assays has been a major hurdle in RAS pathway-targeted drug development. We used NMR to obtain detailed mechanistic data on RAS cycling defects conferred by oncogenic mutations, or full-length RASopathy-derived regulatory proteins. By monitoring the conformation of wild-type and oncogenic RAS in real-time, we show that opposing properties integrate with regulators to hyperactivate oncogenic RAS mutants. Q61L and G13D exhibited rapid nucleotide exchange and an unexpected susceptibility to GAP-mediated hydrolysis, in direct contrast with G12V, indicating different approaches must be taken to inhibit these oncoproteins. An NMR methodology was established to directly monitor RAS cycling by intact, multidomain proteins encoded by RASopathy genes in mammalian cell extracts. By measuring GAP activity from tumor cells, we demonstrate how loss of neurofibromatosis type 1 (NF1) increases RAS-GTP levels in NF1-derived cells. We further applied this methodology to profile Noonan Syndrome (NS)-derived SOS1 mutants. Combining NMR with cell-based assays allowed us to differentiate defects in catalysis, allosteric regulation, and membrane targeting of individual mutants, while revealing a membrane-dependent compensatory effect that attenuates dramatic increases in RAS activation shown by Y337C, L550P, and I252T. Our NMR method presents a precise and robust measure of RAS activity, providing mechanistic insights that facilitate discovery of therapeutics targeted against the RAS signaling network.

Characterization of the second ion-binding site in the G domain of H-Ras.

Ras is a small monomeric GTPase acting as molecular switch in multiple cellular processes. The N-terminal G domain of Ras binds GTP or GDP accompanied by a magnesium ion, which is strictly required for GTPase activity and performs a structural role. Another ion-binding site on the opposite face of the G domain has been recently observed to specifically associate with calcium acetate in the crystal [Buhrman, G., et al. (2010) Proc. Natl. Aacd. Sci. U.S.A. 107, 4931-4936]. In this article, we report thermodynamic measurements of the affinity and specificity of the remote ion-binding site in H-Ras as observed in solution. Using (15)N-(1)H nuclear magnetic resonance spectroscopy, we determined that, in contrast to the crystalline state, the remote site in solution is specific for a divalent cation, binding both calcium and magnesium with anions playing a minimal role. The affinity of the remote site for divalent cations is in the low millimolar range and remarkably different for GDP- and GppNHp-bound forms of the G domain, indicating that the GTP-binding pocket and the remote site are allosterically coupled through the distance of more than 25 Å. Considering that the remote site is oriented toward the membrane surface in vivo, we hypothesize that its cognate biological ligand might be a positively charged group extending from a lipid or an integral membrane protein.

Crystal structure and functional implication of the RUN domain of human NESCA.

NESCA, a newly discovered signaling adapter protein in the NGF-pathway, contains a RUN domain at its N-terminus. Here we report the crystal structure of the NESCA RUN domain determined at 2.0-Å resolution. The overall fold of the NESCA RUN domain comprises nine helices, resembling the RUN domain of RPIPx and the RUN1 domain of Rab6IP1. However, compared to the other RUN domains, the RUN domain of NESCA has significantly different surface electrostatic distributions at the putative GTPase-interacting interface. We demonstrate that the RUN domain of NESCA can bind H-Ras, a downstream signaling molecule of TrkA, with high affinity. Moreover, NESCA RUN can directly interact with TrkA. These results provide new insights into how NESCA participates in the NGF-TrkA signaling pathway.

Signal activation and inactivation by the Gα helical domain: a long-neglected partner in G protein signaling.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are positioned at the top of many signal transduction pathways. The G protein α subunit is composed of two domains, one that resembles Ras and another that is composed entirely of α helices. Historically most attention has focused on the Ras-like domain, but emerging evidence reveals that the helical domain is an active participant in G protein signaling.

Anti-cancer peptides from ras-p21 and p53 proteins.

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

Factors determining electrostatic fields in molecular dynamics simulations of the Ras/effector interface.

Using molecular dynamics simulations, we explore geometric and physical factors contributing to calculated electrostatic fields at the binding surface of the GTPase Ras with a spectroscopically labeled variant of a downstream effector, the Ras-binding domain of Ral guanine nucleotide dissociation stimulator (RalGDS). A related system (differing by mutation of one amino acid) has been studied in our group using vibrational Stark effect spectroscopy, a technique sensitive to electrostatic fields. Electrostatic fields were computed using the AMBER 2003 force field and averaged over snapshots from molecular dynamics simulation. We investigate geometric factors by exploring how the orientation of the spectroscopic probe changes on Ras-effector binding. In addition, we explore the physical origin of electrostatic fields at our spectroscopic probe by comparing contributions to the field from discrete components of the system, such as explicit solvent, residues on the Ras surface, and residues on the RalGDS surface. These models support our experimental hypothesis that vibrational Stark shifts are caused by Ras binding to its effector and not the structural rearrangements of the effector surface or probe reorientation on Ras-effector binding, for at least some of our experimental probes. These calculations provide physical insight into the origin, magnitude, and importance of electrostatic fields in protein-protein interactions and suggest new experiments to probe the field's role in protein docking.

A peptide from a ras effector-domain blocks ras-dependent cardiac hypertrophy in myocytes.

PNC-2 is a peptide corresponding to an effector domain (residues 96-110) of ras-p21 that strongly and specifically blocks mitogenic signal transduction by oncogenic but not activated, normally-expressed wild-type ras-p21 protein. Since myocardial hypertrophy can be induced both by oncogenic and overexpressed wild-type ras-p21, we investigated whether PNC-2 can block norepinephrine (NE)-induced, ras-dependent myocardial hypertrophy in cardiac myocytes. Since PNC-2 blocks oncogenic ras-p21-induced activation of JNK and ERK, we further determined whether this peptide blocks activation of these kinases in NE-treated myocytes. Using cultured neonatal rat ventricular myocytes (NRVM), we found that NE alone significantly increased NRVM surface area, (3)H-leucine uptake, protein/DNA ratio, and atrial nartiuretic factor (ANF) mRNA levels in these cells. However, pretreatment of the NRVM with PNC-2 linked on its carboxyl terminal end to a transmembrane-penetrating leader sequence (PNC-2-leader) resulted in strong inhibition of NE-mediated cell growth and (3)H-leucine uptake and in significantly lower protein/DNA ratios. Induction of ANF mRNA levels was likewise inhibited by PNC-2-leader. In contrast, no inhibition of any of these NE-induced events was observed with a negative control peptide, X13-leader. Western blot analysis showed that JNK and ERK1/2 activity, but not p38 activity, was increased in NRVM within 5 min of exposure to NE (2 microM). Pretreatment with PNC-2-leader decreased ERK1/2 and JNK activity to basal levels. We conclude that a synthetic peptide designed to block oncogenic ras can also counter the effects of NE-induced hypertrophy associated with overexpression of ras p21 by blocking JNK/ jun and ERK activation. PNC-2 may provide a prototype for novel therapy in cardiac conditions associated with activation of NE.

Protein farnesyltransferase-catalyzed isoprenoid transfer to peptide depends on lipid size and shape, not hydrophobicity.

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca(1)a(2)X motif of a range of proteins, including the oncoprotein H-Ras ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid) and the lipid chain interacts with, and forms part of the Ca(1)a(2)X peptide binding site. Previous studies have shown that H-Ras biological function is ablated when it is modified with lipids that are 3-5 orders of magnitude less hydrophobic than FPP. Here, we employed a library of anilinogeranyl diphosphate (AGPP) and phenoxygeranyl diphosphate (PGPP) derivatives with a range of polarities (log P (lipid alcohol) = 0.7-6.8, log P (farnesol) = 6.1) and shapes to examine whether FTase-catalyzed transfer to peptide is dependent on the hydrophobicity of the lipid. Analysis of steady-state transfer kinetics for analogues to dansyl-GCVLS peptide revealed that the efficiency of lipid transfer was highly dependent on both the shape and size, but was independent of the polarity of the analogue. These observations indicate that hydrophobic features of isoprenoids critical for their association with membranes and/or protein receptors are not required for efficient transfer to Ca(1)a(2)X peptides by FTase. Furthermore, the results of these studies indicate that the role played by the farnesyl lipid in the FTase mechanism is primarily structural. To explain these results we propose a model in which the FTase active site stabilizes a membrane interface-like environment.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

Two peptides derived from ras-p21 induce either phenotypic reversion or tumor cell necrosis of ras-transformed human cancer cells.

PURPOSE: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype. METHODS: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels. RESULTS: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides. CONCLUSION: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.

K-ras Asp12 mutant neither interacts with Raf, nor signals through Erk and is less tumorigenic than K-ras Val12.

Different mutant amino acids in the Ras proteins lead to distinct transforming capacities and different aggressiveness in human tumors. K-Ras Asp12 (K12D) is more prevalent in benign than in malignant human colorectal tumors, whereas K-Ras Val12 (K12V) associates with more advanced and metastatic carcinomas, higher recurrence and decreased survival. Here, we tested, in a nude mouse xenograft model, whether different human K-Ras oncogenes mutated at codon 12 to Val, Asp or Cys would confer NIH3T3 fibroblasts distinct oncogenic phenotypes. We studied tumor histology and growth, apoptotic and mitotic rates, activation of signal transduction pathways downstream of Ras and regulation of the cell cycle and apoptotic proteins in tumors derived from the implanted transformants. We found that the K12V oncogene induces a more aggressive tumorigenic phenotype than the K12D oncogene, whereas K12C does not induce tumors in this model. Thus, K12V mutant tumors proliferate about seven times faster, and have higher cellularity and mitotic rates than the K12D mutant tumors. A molecular analysis of the induced tumors shows that the K12V mutant protein interacts with Raf-1 and transduces signals mainly through the Erk pathway. Unexpectedly, in tumors induced by the K12D oncogene, the K-Ras mutant protein does not interact with Raf-1 nor activates the Erk canonical pathway. Instead, it transduces signals through the PI3K/Akt, JNK, p38 and FAK pathways. Finally, the higher growth rate of the K12V tumors associates with enhanced Rb phosphorylation, and PCNA and cyclin B upregulation, consistent with faster G1/S and G2/M transitions, without alteration of apoptotic regulation.

Structure of a transient intermediate for GTP hydrolysis by ras.

The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.

Naturally occurring T-cell response against mutated p21 ras oncoprotein in pancreatic cancer.

Mutated p21 ras proteins (muRas) are present in approximately 90% of pancreatic adenocarcinomas and express mutants which can function as cancer-specific antigens. To evaluate the frequency and magnitude of the natural T-cell response against muRas in 19 HLA-A2-positive patients with muRas-positive pancreatic carcinomas, antigen-experienced T lymphocytes in fresh peripheral blood mononuclear cells were shown by IFN-gamma enzyme-linked immunospot using muRas peptides (5-21) that encompass both HLA class I (HLA-A2)- and class II-restricted (HLA-DRB1) epitopes. Six of 19 patients (32%) were found to have a specific T-cell response against individual mutation-specific ras(5-21) but not against other ras mutations or wild-type ras. In contrast, none of 19 healthy subjects had T cells specifically secreting IFN-gamma (P = 0.004). The T-cell response consisted of both CD8(+) and CD4(+) T cells but was dominated by CD8 T cells in three of four patients. MuRas(5-14) and muRas(6-14) were shown to specifically induce CD8(+) T-cell mediated cytotoxicity against HLA-A2-positive, muRas-bearing pancreatic carcinoma cells. The T-cell response was not correlated with prognostic or clinical variables such as tumor-node-metastasis status, stage, or survival. In conclusion, a natural T-cell response against muRas proteins that could be exploited for immunostimulatory therapeutic approaches has been shown in a significant proportion of patients with pancreatic cancer.

Comparison of the predicted structures of loops in the ras-SOS protein bound to a single ras-p21 protein with the crystallographically determined structures in SOS bound to two ras-p21 proteins.

We have previously computed the structures of three loops, residues 591-596, 654-675 and 742-751, in the ras-p21 protein-binding domain (residues 568-1044) of the guanine nucleotide-exchange-promoting SOS protein that were crystallographically undefined when one molecule of ras-p21 (unbound to nucleotide) binds to SOS. Based on our computational results, we synthesized three peptides corresponding to sequences of each of these three loops and found that all three peptides strongly inhibit ras-p21 signaling. More recently, a new crystal structure of SOS has been determined in which this protein binds to two molecules of ras-p21, one unbound to GTP and one bound to GTP. In this structure, the 654-675 loop and residues 742-743 and 750-751 are now crystallographically defined. We have superimposed our energy-minimized structure of the ras-binding domain of SOS bound to one molecule of ras-p21 on the X-ray structure for SOS bound to two molecules of ras-p21. We find that, while the two structures are superimposable, there are large deviations of the residues 673 and 676 and 741 and 752, flanking the two loop segments. This suggests that the binding of the extra ras-p21 molecule, which is far from each of the three loops, induces conformational changes in these domains and further supports their role in signal transduction. In spite of these differences, we have superimposed our computed structures for the loop residues on those from the more recent X-ray structure. Our structure for the 654-675 segment is an anti-parallel beta-sheet with a reverse turn at residues 663-665; in the X-ray structure residues 655-662 adopt an alpha-helical conformation; on the other hand, our computed structure for residues 663-675 superimpose on the X-ray structure for these residues. We further find that our computed structures for residues 742-743 and 750-751 are superimposable on the X-ray structure for these residues.

Design, synthesis and biological evaluation of sugar-derived Ras inhibitors.

The design and synthesis of novel Ras inhibitors with a bicyclic scaffold derived from the natural sugar D-arabinose are presented. Molecular modelling showed that these ligands can bind Ras by accommodating the aromatic moieties and the phenylhydroxylamino group in a cavity near the Switch II region of the protein. All the synthetic compounds were active in inhibiting nucleotide exchange on p21 human Ras in vitro, and two of them selectively inhibited Ras-dependent cell growth in vivo.

Structure of the G60A mutant of Ras: implications for the dominant negative effect.

Substituting alanine for glycine at position 60 in v-H-Ras generated a dominant negative mutant that completely abolished the ability of v-H-Ras to transform NIH 3T3 cells and to induce germinal vesicle breakdown in Xenopus oocytes. The crystal structure of the GppNp-bound form of RasG60A unexpectedly shows that the switch regions adopt an open conformation reminiscent of the structure of the nucleotide-free form of Ras in complex with Sos. Critical residues that normally stabilize the guanine nucleotide and the Mg(2+) ion have moved considerably. Sos binds to RasG60A but is unable to catalyze nucleotide exchange. Our data suggest that the dominant negative effect observed for RasG60A.GTP could result from the sequestering of Sos in a non-productive Ras-GTP-guanine nucleotide exchange factor ternary complex.