fully-processed-recombinant-kras4b-isolating-characterizing

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 ...

Frederick National Laboratory for Cancer Research

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 cerebrospinal fluid for proteomics biomarker analysis.

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.

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

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.

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

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. 

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

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.

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

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 Mg-free structures of KRAS with flexible N-termini. In the Mg-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 Mg-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg 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 Mg binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

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

Neurofibromin is a tumor suppressor encoded by the NF1 gene which is mutated in the Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS-mitogen-activated protein kinase (MAPK) 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-multi angle 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 electron microscopy 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 coexpression 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.

Timothy Waybright

NCI RAS Initiative,
Cancer Research Technology Program,
NCI RAS Initiative, Cancer Research Technology Program

Frederick National Laboratory for Cancer Research

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

Timothy Waybright

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Frederick National Laboratory for Cancer Research

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

NCI RAS Initiative,
Cancer Research Technology Program,
NCI RAS Initiative, Cancer Research Technology Program