Proteostatic control of telomerase function through TRiC-mediated folding of TCAB1.

Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.

Enhancer of Rudimentary Cooperates with Conserved RNA-Processing Factors to Promote Meiotic mRNA Decay and Facultative Heterochromatin Assembly.

Erh1, the fission yeast homolog of Enhancer of rudimentary, is implicated in meiotic mRNA elimination during vegetative growth, but its function is poorly understood. We show that Erh1 and the RNA-binding protein Mmi1 form a stoichiometric complex, called the Erh1-Mmi1 complex (EMC), to promote meiotic mRNA decay and facultative heterochromatin assembly. To perform these functions, EMC associates with two distinct complexes, Mtl1-Red1 core (MTREC) and CCR4-NOT. Whereas MTREC facilitates assembly of heterochromatin islands coating meiotic genes silenced by the nuclear exosome, CCR4-NOT promotes RNAi-dependent heterochromatin domain (HOOD) formation at EMC-target loci. CCR4-NOT also assembles HOODs at retrotransposons and regulated genes containing cryptic introns. We find that CCR4-NOT facilitates HOOD assembly through its association with the conserved Pir2/ARS2 protein, and also maintains rDNA integrity and silencing by promoting heterochromatin formation. Our results reveal connections among Erh1, CCR4-NOT, Pir2/ARS2, and RNAi, which target heterochromatin to regulate gene expression and protect genome integrity.

Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly.

Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase biogenesis and suggest alternative approaches for inhibiting telomerase in cancer.

Transforming Growth Factor-ß (TGF-ß) Directly Activates the JAK1-STAT3 Axis to Induce Hepatic Fibrosis in Coordination with the SMAD Pathway.

Transforming growth factor-ß (TGF-ß) signals through both SMAD and non-SMAD pathways to elicit a wide array of biological effects. Existing data have shown the association and coordination between STATs and SMADs in mediating TGF-ß functions in hepatic cells, but it is not clear how STATs are activated under these circumstances. Here, we report that JAK1 is a constitutive TGFßRI binding protein and is absolutely required for phosphorylation of STATs in a SMAD-independent manner within minutes of TGF-ß stimulation. Following the activation of SMADs, TGF-ß also induces a second phase of STAT phosphorylation that requires SMADs, de novo protein synthesis, and contribution from JAK1. Our global gene expression profiling indicates that the non-SMAD JAK1/STAT pathway is essential for the expression of a subset of TGF-ß target genes in hepatic stellate cells, and the cooperation between the JAK1-STAT3 and SMAD pathways is critical to the roles of TGF-ß in liver fibrosis.

Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells.

The expression level of HLA class-I proteins is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences among people with regard to the steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult because of the tremendous polymorphism of HLA. We report relative expression levels of HLA-A, HLA-B, HLA-C, and HLA-E proteins for the specific haplotype A*02:01, B*44:02, C*05:01, which were characterized using two independent methods based on flow cytometry and mass spectrometry. PBLs from normal donors showed that HLA-A and HLA-B proteins are expressed at similar levels, which are 13-18 times higher than HLA-C by flow cytometry and 4-5 times higher than HLA-C by mass spectrometry; these differences may reflect variation in the conformation or location of proteins detected. HLA-E was detected at a level 25 times lower than that of HLA-C by mass spectrometry. Primary CD4(+) T cells infected with HIV in vitro were also studied because HIV downregulates selective HLA types. HLA-A and HLA-B were reduced on HIV-infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLA-A to be 1-3 times higher and HLA-B to be 2-5 times higher than HLA-C by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV-infected cells.

Microfilariae of Brugia malayi Inhibit the mTOR Pathway and Induce Autophagy in Human Dendritic Cells.

Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.

Casein kinase 1alpha governs antigen-receptor-induced NF-kappaB activation and human lymphoma cell survival.

The transcription factor NF-kappaB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-kappaB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-kappaB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1alpha (CK1alpha) as a bifunctional regulator of NF-kappaB. CK1alpha dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1alpha kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1alpha has thus a dual 'gating' function which first promotes and then terminates receptor-induced NF-kappaB. ABC DLBCL cells required CK1alpha for constitutive NF-kappaB activity, indicating that CK1alpha functions as a conditionally essential malignancy gene-a member of a new class of potential cancer therapeutic targets.

Telomerase modulates Wnt signalling by association with target gene chromatin.

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.

Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia.

Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as "hypothetical," the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well.

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima.

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.

A further insight into the sialome of the tropical bont tick, Amblyomma variegatum.

BACKGROUND: Ticks--vectors of medical and veterinary importance--are themselves also significant pests. Tick salivary proteins are the result of adaptation to blood feeding and contain inhibitors of blood clotting, platelet aggregation, and angiogenesis, as well as vasodilators and immunomodulators. A previous analysis of the sialotranscriptome (from the Greek sialo, saliva) of Amblyomma variegatum is revisited in light of recent advances in tick sialomes and provides a database to perform a proteomic study. RESULTS: The clusterized data set has been expertly curated in light of recent reviews on tick salivary proteins, identifying many new families of tick-exclusive proteins. A proteome study using salivary gland homogenates identified 19 putative secreted proteins within a total of 211 matches. CONCLUSIONS: The annotated sialome of A. variegatum allows its comparison to other tick sialomes, helping to consolidate an emerging pattern in the salivary composition of metastriate ticks; novel protein families were also identified. Because most of these proteins have no known function, the task of functional analysis of these proteins and the discovery of novel pharmacologically active compounds becomes possible.

15-deoxy-Delta12,14-prostaglandin J2 inhibits transcriptional activity of estrogen receptor-alpha via covalent modification of DNA-binding domain.

The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPARgamma)-dependent and PPARgamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ERalpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ(2) inhibits both 17beta-estradiol (E(2))-dependent and E(2)-independent ERalpha transcriptional activity by PPARgamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ERalpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ERalpha, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys(227) and Cys(240)) within the COOH-terminal zinc finger of ERalpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ(2). Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ERalpha and subsequent repression of ERalpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ERalpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERalpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERalpha transcriptional activity.

Cytokeratin-RNA cross-linking mediated by the antitumor aminoflavone, 5-amino-2,3-fluorophenyl-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one.

Aminoflavone (AF) is an anticancer drug in early clinical trials, and its antiproliferative activity involves the induction of DNA-protein cross-links. To identify the proteins cross-linked to nucleic acids, cesium chloride (CsCl) gradient centrifugation was used to isolate proteins tightly bound to nucleic acids in AF-treated human breast carcinoma MCF-7 cells. The identified proteins included structural proteins (several cytokeratins), transcription regulators, and stress response proteins. The identification of the cytokeratins was validated using direct immunoblotting of the high-density CsCl (nucleic acid) fractions isolated from AF-treated cells. Ribonuclease A pretreatment caused the cytokeratin signal in the heaviest CsCl fractions to disappear, suggesting that AF mediates RNA-cytokeratin cross-links. Additional experiments using radiolabeled AF showed that AF formed adducts with total RNA and mRNA with similar affinity to that of DNA. Moreover, 18S RNA was selectively pulled down using an anti-cytokeratin antibody after AF treatment. Consistent with the formation of these adducts, we found that AF inhibits RNA and protein synthesis in a dose- and time-dependent manner. This study provides evidence for the formation of AF-mediated cytokeratin-RNA cross-links and the presence of cytokeratin-RNA complexes. Thus, in addition to its anticancer activity, AF might be a useful molecular probe to study the potential role of cytokeratins in the subcellular localization and metabolism of RNA.

An insight into the sialome of the soft tick, Ornithodorus parkeri.

While hard ticks (Ixodidae) take several days to feed on their hosts, soft ticks (Argasidae) feed faster, usually taking less than 1h per meal. Saliva assists in the feeding process by providing a cocktail of anti-hemostatic, anti-inflammatory and immunomodullatory compounds. Saliva of hard ticks has been shown to contain several families of genes each having multiple members, while those of soft ticks are relatively unexplored. Analysis of the salivary transcriptome of the soft tick Ornithodorus parkeri, the vector of the relapsing fever agent Borrelia parkeri, indicates that gene duplication events have led to a large expansion of the lipocalin family, as well as of several genes containing Kunitz domains indicative of serine protease inhibitors, and several other gene families also found in hard ticks. Novel protein families with sequence homology to insulin growth factor-binding protein (prostacyclin-stimulating factor), adrenomedulin, serum amyloid A protein precursor and similar to HIV envelope protein were also characterized for the first time in the salivary gland of a blood-sucking arthropod. The sialotranscriptome of O. parkeri confirms that gene duplication events are an important driving force in the creation of salivary cocktails of blood-feeding arthropods, as was observed with hard ticks and mosquitoes. Most of the genes coding for expanded families are homologous to those found in hard ticks, indicating a strong common evolutionary path between the two families. As happens to all genera of blood-sucking arthropods, several new proteins were also found, indicating the process of adaptation to blood feeding still continues to recent times.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.

Identification and Validation of Loa loa Microfilaria-Specific Biomarkers: a Rational Design Approach Using Proteomics and Novel Immunoassays.

UNLABELLED: Immunoassays are currently needed to quantify Loa loa microfilariae (mf). To address this need, we have conducted proteomic and bioinformatic analyses of proteins present in the urine of a Loa mf-infected patient and used this information to identify putative biomarkers produced by L. loa mf. In total, 70 of the 15,444 described putative L. loa proteins were identified. Of these 70, 18 were L. loa mf specific, and 2 of these 18 (LOAG_16297 and LOAG_17808) were biologically immunogenic. We developed novel reverse luciferase immunoprecipitation system (LIPS) immunoassays to quantify these 2 proteins in individual plasma samples. Levels of these 2 proteins in microfilaremic L. loa-infected patients were positively correlated to mf densities in the corresponding blood samples (r = 0.71 and P < 0.0001 for LOAG_16297 and r = 0.61 and P = 0.0002 for LOAG_17808). For LOAG_16297, the levels in plasma were significantly higher in Loa-infected (geometric mean [GM], 0.045 µg/ml) than in uninfected (P < 0.0001), Wuchereria bancrofti-infected (P = 0.0005), and Onchocerca volvulus-infected (P < 0.0001) individuals, whereas for LOAG_17808 protein, they were not significantly different between Loa-infected (GM, 0.123 µg/ml) and uninfected (P = 0.06) and W. bancrofti-infected (P = 0.32) individuals. Moreover, only LOAG_16297 showed clear discriminative ability between L. loa and the other potentially coendemic filariae. Indeed, the specificity of the LOAG_16297 reverse LIPS assay was 96% (with a sensitivity of 77%). Thus, LOAG_16297 is a very promising biomarker that will be exploited in a quantitative point-of-care immunoassay for determination of L. loa mf densities. IMPORTANCE: Loa loa, the causative agent of loiasis, is a parasitic nematode transmitted to humans by the tabanid Chrysops fly. Some individuals infected with L. loa microfilariae (mf) in high densities are known to experience post-ivermectin severe adverse events (SAEs [encephalopathy, coma, or death]). Thus, ivermectin-based mass drug administration (MDA) programs for onchocerciasis and for lymphatic filariasis control have been interrupted in parts of Africa where these filarial infections coexist with L. loa. To allow for implementation of MDA for onchocerciasis and lymphatic filariasis, tools that can accurately identify people at risk of developing post-ivermectin SAEs are needed. Our study, using host-based proteomics in combination with novel immunoassays, identified a single Loa-specific antigen (LOAG_16297) that can be used as a biomarker for the prediction of L. loa mf levels in the blood of infected patients. Therefore, the use of such biomarker could be important in the point-of-care assessment of L. loa mf densities.

Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry.

Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C(18) microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.

A Proteomic Analysis of the Body Wall, Digestive Tract, and Reproductive Tract of Brugia malayi.

Filarial worms are parasitic nematodes that cause devastating diseases such as lymphatic filariasis (LF) and onchocerciasis. Filariae are nematodes with complex anatomy including fully developed digestive tracts and reproductive organs. To better understand the basic biology of filarial parasites and to provide insights into drug targets and vaccine design, we conducted a proteomic analysis of different anatomic fractions of Brugia malayi, a causative agent of LF. Approximately 500 adult female B. malayi worms were dissected, and three anatomical fractions (body wall, digestive tract, and reproductive tract) were obtained. Proteins from each anatomical fraction were extracted, desalted, trypsinized, and analyzed by microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry. In total, we identified 4,785 B. malayi proteins. While 1,894 were identified in all three anatomic fractions, 396 were positively identified only within the digestive tract, 114 only within the body wall, and 1,011 only within the reproductive tract. Gene set enrichment analysis revealed a bias for transporters to be present within the digestive tract, suggesting that the intestine of adult filariae is functional and important for nutrient uptake or waste removal. As expected, the body wall exhibited increased frequencies of cytoskeletal proteins, and the reproductive tract had increased frequencies of proteins involved in nuclear regulation and transcription. In assessing for possible vaccine candidates, we focused on proteins sequestered within the digestive tract, as these could possibly represent "hidden antigens" with low risk of prior allergic sensitization. We identified 106 proteins that are enriched in the digestive tract and are predicted to localize to the surface of cells in the the digestive tract. It is possible that some of these proteins are on the luminal surface and may be accessible by antibodies ingested by the worm. A subset of 27 of these proteins appear especially promising vaccine candidates as they contain significant non-cytoplasmic domains, only 1-2 transmembrane domains, and a high degree of homology to W. bancrofti and/or O. volvulus.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

The use of mass spectrometry in genomics.

Recent advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the near routine analysis of oligonucleotides and intact nucleic acids. These developments have led to the use of mass spectrometry (MS) as a detection platform for genomics studies. Among the various uses of mass spectrometry in genomics, applications focused on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) are particularly well-suited to MALDI or ESI-based analysis. It is predicted that continued developments in methodology and instrumentation will further improve the capabilities of mass spectrometry for nucleic acid analysis.