Nucleotide Exchange on RAS Proteins Using Hydrolysable and Non-hydrolysable Nucleotides.

Biochemical and biophysical assays using recombinant RAS require the protein to be in either the active or inactive state. Here we describe methods to exchange the nucleotide present in the purified RAS protein with either GDPßS, GppNHp, or GTP depending on the assay requirement. In addition, we also describe the HPLC method used to validate the exchange process and provide information on the efficiency of the nucleotide exchange.

Comparative analysis of KRAS4a and KRAS4b splice variants reveals distinctive structural and functional properties.

KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Allosteric Regulation of Switch-II Domain Controls KRAS Oncogenicity.

RAS proteins are GTPases that regulate a wide range of cellular processes. RAS activity is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP). KRAS can be acetylated at lysine 104 (K104), and an acetylation-mimetic mutation of K104 to glutamine (K104Q) attenuates the in vitro-transforming capacity of oncogenic KRAS by interrupting GEF-induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR-Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional KrasLSL-G12D alleles. Homozygous animals for K104Q were viable, fertile, and arose at the expected Mendelian frequency, indicating that K104Q is not a complete loss-of-function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of KRASG12D was significantly attenuated by mutation at K104. Biochemical and structural analysis indicated that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic KRAS. Furthermore, K104 functioned in an allosteric network with M72, R73, and G75 on the α2 helix of the switch-II region. Intriguingly, point mutation of glycine 75 to alanine (G75A) also showed a strong negative regulatory effect on KRASG12D. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant KRAS and suggest that modulating the sites in its allosteric network may provide a unique therapeutic approach in cancers expressing mutant KRAS. SIGNIFICANCE: An allosteric network formed by interaction between lysine 104 and residues in the switch-II domain is required for KRAS oncogenicity, which could be exploited for developing inhibitors of the activated oncoprotein.

GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation.

Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein.

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a 'CAAX' box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

Structures of N-terminally processed KRAS provide insight into the role of N-acetylation.

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo.

PURPOSE: To successfully metastasize, tumor cells must respond appropriately to biological stressors encountered during metastatic progression. We sought to test the hypothesis that enhanced efficiency of mRNA translation during periods of metastatic stress is required for metastatic competence of osteosarcoma and that this metastasis-specific adaptation is amenable to therapeutic intervention. EXPERIMENTAL DESIGN: We employ novel reporter and proteomic systems that enable tracking of mRNA translation efficiency and output in metastatic osteosarcoma cells as they colonize the lungs. We test the potential to target mRNA translation as an antimetastatic therapeutic strategy through pharmacokinetic studies and preclinical assessment of the prototypic mTOR inhibitor, rapamycin, across multiple models of metastasis. RESULTS: Metastatic osteosarcoma cells translate mRNA more efficiently than nonmetastatic cells during critical stressful periods of metastatic colonization of the lung. Rapamycin inhibits translational output during periods of metastatic stress, mitigates lung colonization, and prolongs survival. mTOR-inhibiting exposures of rapamycin are achievable in mice using treatment schedules that correspond to human doses well below the MTDs defined in human patients, and as such are very likely to be tolerated over long exposures alone and in combination with other agents. CONCLUSIONS: Metastatic competence of osteosarcoma cells is dependent on efficient mRNA translation during stressful periods of metastatic progression, and the mTOR inhibitor, rapamycin, can mitigate this translation and inhibit metastasis in vivo Our data suggest that mTOR pathway inhibitors should be reconsidered in the clinic using rationally designed dosing schedules and clinical metrics related to metastatic progression. Clin Cancer Res; 22(24); 6129-41. ©2016 AACR.

Preparation of the low molecular weight serum proteome for mass spectrometry analysis.

The discovery of viable biomarkers or indicators of disease states is complicated by the inherent complexity of the chosen biological specimen. Every sample, whether it is serum, plasma, urine, tissue, cells, or a host of others, contains thousands of large and small components, each interacting in multiple ways. The need to concentrate on a group of these components to narrow the focus on a potential biomarker candidate becomes, out of necessity, a priority, especially in the search for immune-related low molecular weight serum biomarkers. One such method in the field of proteomics is to divide the sample proteome into groups based on the size of the protein, analyze each group, and mine the data for statistically significant items. This chapter details a portion of this method, concentrating on a method for fractionating and analyzing the low molecular weight proteome of human serum.

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae.

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Preparation of human cerebrospinal fluid for proteomics biomarker analysis.

The analysis of the cerebrospinal fluid (CSF) proteome in recent years has resulted in a valuable repository of data for targeting and diagnosing a variety of diseases, such as Parkinson's disease, Alzheimer's disease, traumatic brain injury, and amyotrophic lateral sclerosis. Human ventricular CSF contains numerous proteins that are unique to CSF due in part to the interaction of the biofluid with the brain. This allows researchers to obtain information from a region that would otherwise be inaccessible except through invasive surgery or during autopsy. Characterization of the CSF proteome requires that strict care be taken so that sample integrity and fidelity are maintained to ensure data reproducibility. Standardized methods in sample collection, storage, preparation, analysis, and data mining must be used for meaningful information to be obtained. The following method describes a simple and robust approach for preparing CSF samples for analysis via reversed-phase liquid chromatography (RPLC) and mass spectrometry (MS).

Preparation of human serum for prolactin measurement by multiple reaction monitoring mass spectrometry.

The measurement of the protein hormone prolactin (PRL) in biological samples has developed over the years into a routine clinical assay aiding the diagnosis of multiple medical conditions. PRL is known to exist in multiple isoforms circulating throughout the body. Current methodologies for measuring the PRL levels typically involve a variety of immunoassays. However, most of these tests are not capable of distinguishing between the different isoforms. To address this need, we have developed a highly specialized method employing multiple reaction monitoring mass spectrometry (MRM-MS) capable of monitoring seven distinct peptides from two of the most common prolactin isoforms (the 23 kDa PRL and its 16 kDa N-terminal cleavage product). Since serum is the main source of clinical specimen for the measurement of prolactin isoforms, the method described in this chapter is focused on the approach to processing whole serum samples for prolactin analysis via reversed-phase liquid chromatography (RPLC) and MRM-MS.

Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77.

PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.

Molecular profiling of the human nasal epithelium: A proteomics approach.

A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease.

Cancer biomarker discovery: Opportunities and pitfalls in analytical methods.

Many diseases result in specific and characteristic changes in the chemical and biochemical profiles of biological fluids and tissues prior to development of clinical symptoms. These changes are often useful diagnostic and prognostic biomarkers. Identifying biomarkers that can be used for the early detection of cancer will result in more efficient treatments, reduction in suffering, and lower mortality rates. An ideal screening test should be non-invasive with high sensitivity and specificity. Proteomic and metabolomic analyses of biological samples can reveal changes in abundance levels of metabolites and proteins that when validated and confirmed through clinical trials can function as clinical tests for early detection, diagnosis, monitoring disease progression, and predicting therapeutic response. While the past decade has seen great advancements in proteomics and metabolomics research producing potential biomarkers for cancer, most of the identified biomarkers have failed to replace existing clinical tests. To become a clinically approved test, a potential biomarker should be confirmed and validated using hundreds of specimens and should be reproducible, specific, and sensitive. A search of the scientific and medical literature indicates that many studies report the discovery of potential biomarkers without proper validation and/or they do not meet the above criteria. In this manuscript, we will discuss the successes and the pitfalls of biomarker research and comment on study and experimental design, which in most cases is lacking, resulting in suboptimal biomarkers.

Characterization of the human ventricular cerebrospinal fluid proteome obtained from hydrocephalic patients.

The continuing expansion of proteomic technology has been fueled by the potential for discovering novel biomarkers that may be used for the early detection of disease. It has been proposed that human cerebrospinal fluid (CSF), which surrounds and protects the brain and spinal cord from traumatic injury, may be a valuable target for the diagnosis of a variety of conditions such as Alzheimer's disease, traumatic brain injury, amyotrophic lateral sclerosis and Parkinson's disease. The immense complexity of biofluids, however, still requires that considerable development be made in the analytical techniques used so that comprehensive coverage of the proteins present in such samples is achieved. Using a simple separation strategy the protein complement of human ventricular cerebrospinal fluid obtained from patients with hydrocephalus was evaluated. The study resulted in the identification of over 1500 unique proteins that were found within all nine CSF samples that were analyzed. Comparison with the HUPO serum proteome database demonstrated that human ventricular CSF contains a large array of proteins that may be unique to CSF. This analysis greatly increases our knowledge of the protein content of this clinically important biofluid.

Analytical and statistical approaches to metabolomics research.

Metabolomics, the global profiling of metabolites in different living systems, has experienced a rekindling of interest partially due to the improved detection capabilities of the instrumental techniques currently being used in this area of biomedical research. The analytical methods of choice for the analysis of metabolites in search of disease biomarkers in biological specimens, and for the study of various low molecular weight metabolic pathways include NMR spectroscopy, GC/MS, CE/MS, and HPLC/MS. Global metabolite analysis and profiling of two different sets of data results in a plethora of data that is difficult to manage or interpret manually because of their subtle differences. Multivariate statistical methods and pattern-recognition programs were developed to handle the acquired data and to search for the discriminating features between data acquired from two sample sets, healthy and diseased. Metabolomics have been used in toxicology, plant physiology, and biomedical research. In this paper, we discuss various aspects of metabolomic research including sample collection, handling, storage, requirements for sample analysis, peak alignment, data interpretation using statistical approaches, metabolite identification, and finally recommendations for successful analysis.

Overcoming problems of compound storage in DMSO: solvent and process alternatives.

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration.